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diabetes is the leading cause of chronic kidney disease ( ckd ) in developed countries , including the u.s . diabetic kidney disease accounts for 40% of prevalent ckd and 50% of incident end - stage renal disease , and it has increased in direct proportion to the increasing prevalence of diabetes ( 24 ) . people with diabetes often suffer from microvascular and macrovascular complications , including retinopathy , nephropathy , coronary artery disease , peripheral arterial disease , and stroke , as well as early mortality ( 5,6 ) . compared with persons with diabetes and preserved kidney function , those with diabetes and ckd face even higher risks of morbidity and mortality . indeed , both reduced kidney function and albuminuria are independent predictors for cardiovascular disease as well as all - cause mortality ( 7,8 ) . accurate estimation of glomerular filtration rate ( gfr ) and identification of ckd are important . in clinical practice glomerular filtration rate estimated using serum creatinine ( egfrcr ) is the most common approach ; however , creatinine is influenced by age , muscle mass , sex , and race ( 9 ) . given these limitations , serum cystatin c has been proposed as an alternative filtration marker ( 10 ) . cystatin c , an endogenous protein believed to be produced by all nucleated cells , is less affected by age , race , and muscle mass and , in the general population , associates more strongly with all - cause and cardiovascular mortality than does serum creatinine ( 11,12 ) . however , bmi , diabetes , and inflammation may affect cystatin c levels independent of kidney function ( 13 ) . given the high prevalence of obesity in the population with diabetes , as well as the suggestion that cystatin may perform differently in patients with diabetes , there is controversy as to whether cystatin c based or creatinine - based estimated gfr ( egfr ) equations should be used to estimate kidney function in this population ( 14,15 ) . furthermore , it is unknown whether the associations of diabetes complications with kidney function estimated using cystatin c ( egfrcys ) are similar to those observed using egfrcr . using nationally representative data from the 19992002 national health and nutrition examination survey ( nhanes ) , we estimated the prevalence of reduced kidney function ( egfr < 60 ml / min/1.73 m ) among persons with diabetes using the 2012 ckd - epi cystatin c ( 16 ) and 2009 ckd - epi creatinine ( 17 ) equations and investigated the discordance in ckd classification by the two filtration markers . we also compared the associations of egfrcr and egfrcys with prevalent complications of diabetes , including albuminuria , peripheral arterial disease , retinopathy , and coronary artery disease , as well as incident all - cause and cardiovascular mortality . nhanes is an ongoing cross - sectional , multistage , stratified , clustered probability sample of the u.s . cystatin c concentrations were measured in a subsample of the nhanes 19992002 participants aged 12 years and older who were not missing serum creatinine ( 18 ) . for the current study , we included all participants in the cystatin c subsample aged 20 years or older ( n = 4,457 ; 778 of whom had diabetes ) . the ankle - brachial index ( abi ) , used to define peripheral arterial disease , was measured only in persons aged 40 years or older ( n = 556 of the 778 participants with diabetes , 10 of whom with abi > 1.5 were excluded owing to concern for calcified atherosclerosis ) ( 19,20 ) . we classified persons as having diabetes if they reported a physician diagnosis of diabetes , took antidiabetes pills or insulin injections , or had a glycated hemoglobin ( hba1c ) value of 6.5% . no distinction preserved / reduced kidney function was defined as egfr 60 ml / min/1.73 m / egfr < 60 ml / min/1.73 m , estimated using standardized creatinine and cystatin c values and the ckd - epi 2009 and 2012 equations , respectively ( 16,17 ) . the term advanced ckd was used to indicate ckd stage 4 or 5 ( egfr < 30 ml / min/1.73 m ) . creatinine values from nhanes 19992000 were standardized [ standard creatinine ( mg / dl ) = 0.147 + 1.013 ( nhanes 19992000 uncalibrated serum creatinine [ mg / dl ] ) , whereas no correction to the creatinine values in the 20012002 survey was needed ( 21,22 ) . cystatin c values were recalibrated and standardized to the international federation of clinical chemistry and laboratory medicine ( ifcc ) standard : ifcc standard cystatin c ( mg / l ) = 1.12 ( cystatin c [ mg / l ] 0.12 ) ( 22 ) . hypertension was defined as mean systolic pressure 140 mmhg , mean diastolic pressure 90 mmhg , self - reported hypertension , or the use of an antihypertensive medication . smoking status was determined using answers to the questions , have you smoked at least 100 cigarettes in your life ? and do you now smoke cigarettes ? coronary artery disease was defined on the basis of a self - reported history of coronary heart disease , angina , or previous heart attack . albuminuria was defined as a urinary albumin - to - creatinine ratio ( acr ) 30 mg / g . peripheral arterial disease was defined by an abi < 0.90 in either leg ( 23 ) . diabetic retinopathy was self - reported ( has a doctor ever told you that diabetes has affected your eyes or that you had retinopathy ? ) . information on all - cause and cardiovascular mortality was obtained using the linkage of nhanes data to death certificate data from the national death index ( 24 ) . outcomes of interest included all - cause and cardiovascular ( icd-10 code i00i78 ) mortality . length of follow - up for each participant was calculated as the date of the nhanes examination to date of death or 31 december 2006 ( whichever occurred first ) . all statistical analyses incorporated modified sampling weights , primary sampling units , and strata specific to the sample with available cystatin c in order to generate nationally representative estimates of the u.s . kidney function was analyzed both as a continuous measure using restricted cubic splines and as a categorical measure according to k / doqi classification ( egfr < 15 , 1529 , 3059 , 6089 , and 90200 ml / min/1.73 individuals with egfr values > 200 ml / min/1.73 m were reassigned a value of 200 ml / min/1.73 m ( two persons with egfrcr > 200 ml / min/1.73 m ) . modified poisson regression models were used to examine the relationship of egfr with the prevalence of coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy ( 25 ) . the proportional hazards assumption was tested using log - log plots by category of egfr . all multivariable models were adjusted for age ( years ) , female sex ( yes , no ) , and race ( non - hispanic black , non - hispanic white , hispanic , other ) as well as current smoking status ( current , former , never ) , bmi ( measured as weight in kilograms divided by the square of height in meters ) , hypercholesterolemia ( yes , no ) , low hdl ( yes , no ) , hypertension ( yes , no ) , coronary artery disease ( yes , no ) , and albuminuria ( yes , no ; only in analyses where albuminuria was not the outcome ) . given the possible relationship between cystatin c and obesity , interactions between bmi , egfrcys , and adverse outcomes were tested in multivariable models . all analyses were conducted using stata , version 12 ( stata , college station , tx ) . nhanes is an ongoing cross - sectional , multistage , stratified , clustered probability sample of the u.s . cystatin c concentrations were measured in a subsample of the nhanes 19992002 participants aged 12 years and older who were not missing serum creatinine ( 18 ) . for the current study , we included all participants in the cystatin c subsample aged 20 years or older ( n = 4,457 ; 778 of whom had diabetes ) . the ankle - brachial index ( abi ) , used to define peripheral arterial disease , was measured only in persons aged 40 years or older ( n = 556 of the 778 participants with diabetes , 10 of whom with abi > 1.5 were excluded owing to concern for calcified atherosclerosis ) ( 19,20 ) . we classified persons as having diabetes if they reported a physician diagnosis of diabetes , took antidiabetes pills or insulin injections , or had a glycated hemoglobin ( hba1c ) value of 6.5% . no distinction preserved / reduced kidney function was defined as egfr 60 ml / min/1.73 m / egfr < 60 ml / min/1.73 m , estimated using standardized creatinine and cystatin c values and the ckd - epi 2009 and 2012 equations , respectively ( 16,17 ) . the term advanced ckd was used to indicate ckd stage 4 or 5 ( egfr < 30 ml / min/1.73 m ) . creatinine values from nhanes 19992000 were standardized [ standard creatinine ( mg / dl ) = 0.147 + 1.013 ( nhanes 19992000 uncalibrated serum creatinine [ mg / dl ] ) , whereas no correction to the creatinine values in the 20012002 survey was needed ( 21,22 ) . cystatin c values were recalibrated and standardized to the international federation of clinical chemistry and laboratory medicine ( ifcc ) standard : ifcc standard cystatin c ( mg / l ) = 1.12 ( cystatin c [ mg / l ] 0.12 ) ( 22 ) . hypertension was defined as mean systolic pressure 140 mmhg , mean diastolic pressure 90 mmhg , self - reported hypertension , or the use of an antihypertensive medication . smoking status was determined using answers to the questions , have you smoked at least 100 cigarettes in your life ? and do you now smoke cigarettes ? coronary artery disease was defined on the basis of a self - reported history of coronary heart disease , angina , or previous heart attack . albuminuria was defined as a urinary albumin - to - creatinine ratio ( acr ) 30 mg / g . peripheral arterial disease was defined by an abi < 0.90 in either leg ( 23 ) . diabetic retinopathy was self - reported ( has a doctor ever told you that diabetes has affected your eyes or that you had retinopathy ? ) . information on all - cause and cardiovascular mortality was obtained using the linkage of nhanes data to death certificate data from the national death index ( 24 ) . outcomes of interest included all - cause and cardiovascular ( icd-10 code i00i78 ) mortality . length of follow - up for each participant was calculated as the date of the nhanes examination to date of death or 31 december 2006 ( whichever occurred first ) . all statistical analyses incorporated modified sampling weights , primary sampling units , and strata specific to the sample with available cystatin c in order to generate nationally representative estimates of the u.s . kidney function was analyzed both as a continuous measure using restricted cubic splines and as a categorical measure according to k / doqi classification ( egfr < 15 , 1529 , 3059 , 6089 , and 90200 ml / min/1.73 m ) for both egfrcr and egfrcys . individuals with egfr values > 200 ml / min/1.73 m were reassigned a value of 200 ml / min/1.73 m ( two persons with egfrcr > 200 ml / min/1.73 m ) . modified poisson regression models were used to examine the relationship of egfr with the prevalence of coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy ( 25 ) . the proportional hazards assumption was tested using log - log plots by category of egfr . all multivariable models were adjusted for age ( years ) , female sex ( yes , no ) , and race ( non - hispanic black , non - hispanic white , hispanic , other ) as well as current smoking status ( current , former , never ) , bmi ( measured as weight in kilograms divided by the square of height in meters ) , hypercholesterolemia ( yes , no ) , low hdl ( yes , no ) , hypertension ( yes , no ) , coronary artery disease ( yes , no ) , and albuminuria ( yes , no ; only in analyses where albuminuria was not the outcome ) . given the possible relationship between cystatin c and obesity , interactions between bmi , egfrcys , and adverse outcomes were tested in multivariable models . all analyses were conducted using stata , version 12 ( stata , college station , tx ) . persons with diabetes were older , more likely to be black , and more likely to be male than those without diabetes ( table 1 ) . two - thirds of persons with diabetes had hypertension , which was nearly twice as prevalent compared with those without diabetes ( 63.8% vs. 33.4% ) . there was also a substantial difference in the distribution of bmi : 50% of the u.s . population with diabetes had a bmi > 30 kg / m compared with 28% without diabetes , and 28% of persons with diabetes had a bmi 35 kg / m compared with 12% of those without diabetes . the prevalence of reduced kidney function was almost three times higher in persons with diabetes compared with those without diabetes ( egfrcr 16.5% vs. 5.8% , egfrcys 22.0% vs. 7.9% ) . adults aged 20 years by diabetes status : nhanes 19992002 cystatin c subsample ( n = 4,457 ) the trend of higher prevalence of reduced kidney function by egfrcys persisted across subgroups of sex , race , age , and bmi ( supplementary fig . the absolute difference in reduced kidney function prevalence estimated by cystatin c versus creatinine was 6.9% in those aged 6080 years and 10.3% in those aged 80 years and older . similarly , the absolute difference was larger among persons with bmi > 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 20.0% vs. 13.1% ) than among those with bmi < 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 21.3% vs. 16.9% ) . discordance between egfrcr and egfrcys in the classification of reduced kidney function was 11.8% in persons with diabetes and 4.7% in persons without diabetes . in the population with diabetes , 10.4% of those classified as having preserved kidney function by egfrcr ( 8.7% of the 83.6% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . in the population without diabetes , 3.6% of those classified as having preserved kidney function by egfrcr ( 3.4% of the 94.2% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . within the overall population , diabetes status was significantly associated with reclassification from preserved kidney function by egfrcr to reduced kidney function by egfrcys ( odds ratio [ or ] 3.1 [ 95% ci 1.94.9 ] , p < 0.001 ) ( supplementary table 2 ) . this association was attenuated but still significant after sequential adjustment for egfrcr and bmi but not after further adjustment for age . in contrast , egfrcr , bmi , age , and albuminuria were all significantly associated with reclassification by egfrcys in multivariable regression ( egfrcr , or 0.9 per 1 ml / min/1.73 m increase , p < 0.001 ; bmi , or 1.5 per 5 kg / m increase , p < 0.001 ; age , or 1.8 per decade increase , p < 0.001 ; acr > 30 mg / g , or 2.2 , p = 0.01 ) . in the population with diabetes , the prevalence of albuminuria , retinopathy , coronary artery disease , and peripheral arterial disease was high , even among those with preserved kidney function ( fig . the most common of these complications was albuminuria , present in 27.0% of those with egfrcr 60 ml / min/1.73 m and 55.9% of those with egfrcr < 60 ml / min/1.73 m. the prevalence of coronary artery disease , retinopathy , and peripheral artery disease was also two to three times higher among those with reduced kidney function than those with preserved kidney function . in general , the probability of microvascular and macrovascular complications increased with lower egfr ; above egfr 90 ml / min/1.73 m , relationships between egfr and vascular complications were more variable . the unadjusted relationships between egfr , coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy were similar using egfrcr and egfrcys , although there was suggestion of a u - shape ( with higher risk at both higher and lower levels of gfr ) in the association between albuminuria , retinopathy , and egfrcr but not egfrcys ( supplementary fig . adults with diabetes according to the presence or absence of reduced kidney function ( estimated using creatinine and cystatin c ) . after adjustment for demographic and traditional cardiovascular risk factors , the prevalence ratios for vascular complications ( coronary artery disease , peripheral arterial disease , albuminuria , retinopathy ) by egfr category were similar using creatinine or cystatin c ( table 2 ) . compared with a reference group of egfr 6090 ml / min/1.73 m , persons with advanced ckd ( egfr 1530 ml / min/1.73 m ) had a higher prevalence of coronary artery disease , albuminuria , and retinopathy , regardless of filtration marker used . the adjusted relationships between egfr category and peripheral arterial disease were similar in magnitude but not statistically significant . adjusted prevalence ratios ( 95% ci ) for categories of kidney function with complications of diabetes , by filtration marker , among u.s . adults with diabetes ( n = 778 ) * there were 153 deaths ( 63 from a cardiovascular cause ) during a median follow - up of 5.3 years among the 778 participants with diabetes . the risk of both all - cause and cardiovascular mortality increased with lower egfr , regardless of filtration marker used ( table 3 ) . compared with the reference group of egfr 6090 ml / min/1.73 m , persons with egfr 1530 ml / min/1.73 m had a significantly higher risk of all - cause mortality ( egfrcys , hazard ratio [ hr ] 3.8 , p = 0.007 ; egfrcr , hr 2.6 , p = 0.04 ) . m was significantly associated with increased cardiovascular mortality when estimated by cystatin c ( hr 5.3 , p = 0.007 ) but not by creatinine ( hr 2.0 , p = 0.3 ) . there were no significant interactions between bmi , egfrcys , and either all - cause or cardiovascular mortality . adjusted hazard ratios ( 95% ci ) for categories of kidney function with all - cause and cardiovascular mortality , by filtration marker , among u.s . persons with diabetes were older , more likely to be black , and more likely to be male than those without diabetes ( table 1 ) . two - thirds of persons with diabetes had hypertension , which was nearly twice as prevalent compared with those without diabetes ( 63.8% vs. 33.4% ) . there was also a substantial difference in the distribution of bmi : 50% of the u.s . population with diabetes had a bmi > 30 kg / m compared with 28% without diabetes , and 28% of persons with diabetes had a bmi 35 kg / m compared with 12% of those without diabetes . the prevalence of reduced kidney function was almost three times higher in persons with diabetes compared with those without diabetes ( egfrcr 16.5% vs. 5.8% , egfrcys 22.0% vs. 7.9% ) . adults aged 20 years by diabetes status : nhanes 19992002 cystatin c subsample ( n = 4,457 ) the trend of higher prevalence of reduced kidney function by egfrcys persisted across subgroups of sex , race , age , and bmi ( supplementary fig . the absolute difference in reduced kidney function prevalence estimated by cystatin c versus creatinine was 6.9% in those aged 6080 years and 10.3% in those aged 80 years and older . similarly , the absolute difference was larger among persons with bmi > 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 20.0% vs. 13.1% ) than among those with bmi < 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 21.3% vs. 16.9% ) . discordance between egfrcr and egfrcys in the classification of reduced kidney function was 11.8% in persons with diabetes and 4.7% in persons without diabetes . in the population with diabetes , 10.4% of those classified as having preserved kidney function by egfrcr ( 8.7% of the 83.6% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . in the population without diabetes , 3.6% of those classified as having preserved kidney function by egfrcr ( 3.4% of the 94.2% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . within the overall population , diabetes status was significantly associated with reclassification from preserved kidney function by egfrcr to reduced kidney function by egfrcys ( odds ratio [ or ] 3.1 [ 95% ci 1.94.9 ] , p < 0.001 ) ( supplementary table 2 ) . this association was attenuated but still significant after sequential adjustment for egfrcr and bmi but not after further adjustment for age . in contrast , egfrcr , bmi , age , and albuminuria were all significantly associated with reclassification by egfrcys in multivariable regression ( egfrcr , or 0.9 per 1 ml / min/1.73 m increase , p < 0.001 ; bmi , or 1.5 per 5 kg / m increase , p < 0.001 ; age , or 1.8 per decade increase , p < 0.001 ; acr > 30 mg / g , or 2.2 , p = 0.01 ) . in the population with diabetes , the prevalence of albuminuria , retinopathy , coronary artery disease , and peripheral arterial disease was high , even among those with preserved kidney function ( fig . the most common of these complications was albuminuria , present in 27.0% of those with egfrcr 60 ml / min/1.73 m and 55.9% of those with egfrcr < 60 ml / min/1.73 m. the prevalence of coronary artery disease , retinopathy , and peripheral artery disease was also two to three times higher among those with reduced kidney function than those with preserved kidney function . in general , the probability of microvascular and macrovascular complications increased with lower egfr ; above egfr 90 ml / min/1.73 m , relationships between egfr and vascular complications were more variable . the unadjusted relationships between egfr , coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy were similar using egfrcr and egfrcys , although there was suggestion of a ( with higher risk at both higher and lower levels of gfr ) in the association between albuminuria , retinopathy , and egfrcr but not egfrcys ( supplementary fig . adults with diabetes according to the presence or absence of reduced kidney function ( estimated using creatinine and cystatin c ) . after adjustment for demographic and traditional cardiovascular risk factors , the prevalence ratios for vascular complications ( coronary artery disease , peripheral arterial disease , albuminuria , retinopathy ) by egfr category were similar using creatinine or cystatin c ( table 2 ) . compared with a reference group of egfr 6090 ml / min/1.73 m , persons with advanced ckd ( egfr 1530 ml / min/1.73 m ) had a higher prevalence of coronary artery disease , albuminuria , and retinopathy , regardless of filtration marker used . the adjusted relationships between egfr category and peripheral arterial disease were similar in magnitude but not statistically significant . adjusted prevalence ratios ( 95% ci ) for categories of kidney function with complications of diabetes , by filtration marker , among u.s . there were 153 deaths ( 63 from a cardiovascular cause ) during a median follow - up of 5.3 years among the 778 participants with diabetes . the risk of both all - cause and cardiovascular mortality increased with lower egfr , regardless of filtration marker used ( table 3 ) . compared with the reference group of egfr 6090 ml / min/1.73 m , persons with egfr 1530 ml / min/1.73 m had a significantly higher risk of all - cause mortality ( egfrcys , hazard ratio [ hr ] 3.8 , p = 0.007 ; egfrcr , hr 2.6 , p = 0.04 ) . by contrast , egfr 1530 ml / min/1.73 m was significantly associated with increased cardiovascular mortality when estimated by cystatin c ( hr 5.3 , p = 0.007 ) but not by creatinine ( hr 2.0 , p = 0.3 ) . there were no significant interactions between bmi , egfrcys , and either all - cause or cardiovascular mortality . adjusted hazard ratios ( 95% ci ) for categories of kidney function with all - cause and cardiovascular mortality , by filtration marker , among u.s . this nationally representative study of persons with diabetes suggests that the use of cystatin c to estimate kidney function would result in a higher prevalence of reduced kidney function than would estimates using serum creatinine . reclassification from preserved kidney function using creatinine to reduced kidney function using cystatin c occurred more commonly among persons with diabetes than those without , but this observation was explained by differences in the distributions of egfr , bmi , age , and albuminuria between the two populations : reclassification was significantly associated with lower egfrcr , higher bmi , older age , and acr > 30 mg / g . lower egfr as determined by either creatinine or cystatin was associated with higher odds of prevalent vascular complications ; however , the shape of the relationship with albuminuria and retinopathy at higher levels of egfr differed slightly by filtration marker in unadjusted analysis . similarly , while low egfr was robustly associated with all - cause mortality , only egfrcys showed significant association with cardiovascular mortality . differences in egfrcr and egfrcys have been noted previously in the general population ( 26,27 ) . in the u.s . noninstitutionalized civilian population , kidney function estimated using cystatin c resulted in a reduced kidney function prevalence of 8.7% compared with an estimated 6.5% using creatinine ( 26 ) . in a cross - sectional study of 1,360 inhabitants of the alpine region in europe , pattaro et al . ( 28 ) noted that the lin concordance correlation coefficient of egfrcr and egfrcys was 0.56 , with significant differences by age ( 0.57 in those 65 years old vs. 0.38 in those < 65 years old ) but not by diabetes status . our results differ somewhat from those of this prior study ; the presence of diabetes differentially affected kidney disease classification by egfrcr and egfrcys , at least in univariable analysis an observation that may be attributable to our larger sample size and distinct american population . neither creatinine nor cystatin c is a perfect marker of glomerular filtration ; each has non - gfr determinants . some have argued that neither marker can adequately estimate true gfr in persons with diabetes ; however , this concern is primarily relevant for those with high gfr not reduced gfr as in the current study ( 29,30 ) . the strong relationship between reclassification to reduced kidney function by egfrcys and age may be due to inherent properties of the filtration markers . in the case of creatinine , muscle mass and diet older persons may be sicker than their younger peers ; thus , kidney function estimated using serum creatinine may be confounded by cachexia and muscle wasting . a similar explanation could apply to the differences seen with albuminuria ( i.e. , those with albuminuria are sicker than their peers without albuminuria ) . to our knowledge , the observation that reclassification by cystatin c occurs more frequently among those with albuminuria is novel ; however , it is fully consistent with a recent study demonstrating that the decrement in egfrcys associated with 24-h albuminuria > 30 mg was greater than that of egfrcr or measured gfr ( 27 ) . in the population with diabetes , both serum creatinine and cystatin serum creatinine may poorly estimate kidney function given the tendency of persons with diabetes to have a lower than average muscle mass . cystatin c may be directly affected by both bmi and diabetes ( 13,32,33 ) . in obese individuals , cystatin c levels are higher , and egfrcys significantly underestimates true kidney function ( 27,31 ) . indeed , our study suggests that much of the association between diabetes and cystatin c is driven by differences in the distribution of age and bmi . additional work is needed to determine whether an approach using cystatin c or both filtration markers ( the latter of which better approximates measured gfr in the overall population ) would improve kidney function estimates in the population with diabetes ( 16 ) . conventional wisdom is that , among persons with diabetes , kidney function decline and vascular complications go hand in hand . certainly , our results support the association of prevalent complications with very low egfr whether estimated by creatinine or cystatin c. interestingly , in the upper ranges of preserved kidney function , the association between egfrcr and retinopathy reversed , with higher levels of egfrcr conferring increased odds of retinopathy , although this was not statistically significant in adjusted analysis . these observations are consistent with previous studies demonstrating weaker - than - expected correlation between egfrcr , albuminuria , and retinopathy ( 34 ) . because of the more monotonic relationship seen between egfrcys , albuminuria , and retinopathy , it is possible that kidney function based on cystatin c may prove a better predictor of diabetes complications than that based on creatinine . however , this may be more useful in defining a low - risk group than a high - risk group , given the larger differences in the upper ranges of egfr . we also observed that the relationship between egfrcys and all - cause and cardiovascular mortality was stronger than the corresponding relationship with egfrcr , similar to findings in the general population ( 11,12,35 ) and previous studies of persons with diabetes ( 36 ) . additional prospective studies are needed to determine whether egfrcys provides better risk stratification for subsequent diabetes complications . we relied on a single measurement of creatinine and cystatin ; gfr is estimated from these filtration markers and not measured directly . as such , we can not assess which filtration marker most closely approximates true kidney function . additional research is needed to determine whether the confounding by age and bmi ( or other unmeasured confounders such as thyroid disease ) in diabetes favors egfrcr , egfrcys , or perhaps a combination of the two . next , despite being nationally representative , the subset of nhanes with diabetes was relatively small , with a short duration of follow - up for mortality outcomes . some of the vascular complications were self - reported , and insofar as reporting may vary by level of egfr , this may lead to bias . finally , persons with more severe kidney disease are likely underrepresented in nhanes , thus limiting accuracy in the very low ranges of gfr . in summary , this study demonstrates that using cystatin c to classify kidney function among persons with diabetes results in a higher prevalence of reduced kidney function yet the same or stronger associations with vascular complications and mortality . future studies are needed to determine whether incorporating cystatin c measurement into clinical care and kidney function estimation would improve outcomes in persons with diabetes .

objectiveserum cystatin c is an alternative to serum creatinine for estimating glomerular filtration rate ( gfr ) , since cystatin c is less influenced by age and muscle mass . among persons with diabetes , we compared the performance of gfr estimated using cystatin c ( egfrcys ) with that using creatinine ( egfrcr ) for the identification of reduced kidney function and its association with diabetes complications.research design and methodswe analyzed data from adult participants from the 19992002 national health and nutrition examination survey with available cystatin c ( n = 4,457 ) . kidney function was dichotomized as preserved ( egfr 60 ml / min/1.73 m2 ) or reduced ( egfr < 60 ml / min/1.73 m2 ) using the 2012 chronic kidney disease epidemiology collaboration ( ckd - epi ) cystatin c and the 2009 ckd - epi creatinine equations.resultsamong 778 persons with diabetes , the prevalence of reduced kidney function was 16.5% using egfrcr and 22.0% using egfrcys . more persons with diabetes were reclassified from preserved kidney function by egfrcr to reduced kidney function by egfrcys than persons without diabetes ( odds ratio 3.1 [ 95% ci 1.94.9 ] , p < 0.001 ) . the associations between lower egfr and higher prevalence of albuminuria , retinopathy , peripheral arterial disease , and coronary artery disease were robust regardless of filtration marker . similarly , the risk of all - cause mortality increased with lower egfrcr and egfrcys . only lower egfrcys was significantly associated with cardiovascular mortality.conclusionsmore persons with diabetes had reduced kidney function by egfrcys than by egfrcr , and lower egfrcys was strongly associated with diabetes complications . whether egfrcys is superior to egfrcr in approximating true kidney function in a diabetic population requires additional study .

Introduction Research Design and Methods Study Population Assessment of Diabetes and Kidney Function Other Variables of Interest Prevalent Micro- and Macrovascular Outcomes Mortality Follow-up Statistical Analyses Results Study Population Prevalence of Reduced Kidney Function in Persons With Diabetes by Filtration Marker Reclassification of Reduced Kidney Function by Filtration Marker Association of Kidney Function With Micro- and Macrovascular Complications by Filtration Marker All-Cause and Cardiovascular Mortality in Those With and Without Reduced Kidney Function Conclusions Supplementary Material

people with diabetes often suffer from microvascular and macrovascular complications , including retinopathy , nephropathy , coronary artery disease , peripheral arterial disease , and stroke , as well as early mortality ( 5,6 ) . indeed , both reduced kidney function and albuminuria are independent predictors for cardiovascular disease as well as all - cause mortality ( 7,8 ) . accurate estimation of glomerular filtration rate ( gfr ) and identification of ckd are important . in clinical practice glomerular filtration rate estimated using serum creatinine ( egfrcr ) is the most common approach ; however , creatinine is influenced by age , muscle mass , sex , and race ( 9 ) . cystatin c , an endogenous protein believed to be produced by all nucleated cells , is less affected by age , race , and muscle mass and , in the general population , associates more strongly with all - cause and cardiovascular mortality than does serum creatinine ( 11,12 ) . given the high prevalence of obesity in the population with diabetes , as well as the suggestion that cystatin may perform differently in patients with diabetes , there is controversy as to whether cystatin c based or creatinine - based estimated gfr ( egfr ) equations should be used to estimate kidney function in this population ( 14,15 ) . furthermore , it is unknown whether the associations of diabetes complications with kidney function estimated using cystatin c ( egfrcys ) are similar to those observed using egfrcr . using nationally representative data from the 19992002 national health and nutrition examination survey ( nhanes ) , we estimated the prevalence of reduced kidney function ( egfr < 60 ml / min/1.73 m ) among persons with diabetes using the 2012 ckd - epi cystatin c ( 16 ) and 2009 ckd - epi creatinine ( 17 ) equations and investigated the discordance in ckd classification by the two filtration markers . we also compared the associations of egfrcr and egfrcys with prevalent complications of diabetes , including albuminuria , peripheral arterial disease , retinopathy , and coronary artery disease , as well as incident all - cause and cardiovascular mortality . for the current study , we included all participants in the cystatin c subsample aged 20 years or older ( n = 4,457 ; 778 of whom had diabetes ) . the ankle - brachial index ( abi ) , used to define peripheral arterial disease , was measured only in persons aged 40 years or older ( n = 556 of the 778 participants with diabetes , 10 of whom with abi > 1.5 were excluded owing to concern for calcified atherosclerosis ) ( 19,20 ) . no distinction preserved / reduced kidney function was defined as egfr 60 ml / min/1.73 m / egfr < 60 ml / min/1.73 m , estimated using standardized creatinine and cystatin c values and the ckd - epi 2009 and 2012 equations , respectively ( 16,17 ) . information on all - cause and cardiovascular mortality was obtained using the linkage of nhanes data to death certificate data from the national death index ( 24 ) . kidney function was analyzed both as a continuous measure using restricted cubic splines and as a categorical measure according to k / doqi classification ( egfr < 15 , 1529 , 3059 , 6089 , and 90200 ml / min/1.73 individuals with egfr values > 200 ml / min/1.73 m were reassigned a value of 200 ml / min/1.73 m ( two persons with egfrcr > 200 ml / min/1.73 m ) . modified poisson regression models were used to examine the relationship of egfr with the prevalence of coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy ( 25 ) . for the current study , we included all participants in the cystatin c subsample aged 20 years or older ( n = 4,457 ; 778 of whom had diabetes ) . the ankle - brachial index ( abi ) , used to define peripheral arterial disease , was measured only in persons aged 40 years or older ( n = 556 of the 778 participants with diabetes , 10 of whom with abi > 1.5 were excluded owing to concern for calcified atherosclerosis ) ( 19,20 ) . no distinction preserved / reduced kidney function was defined as egfr 60 ml / min/1.73 m / egfr < 60 ml / min/1.73 m , estimated using standardized creatinine and cystatin c values and the ckd - epi 2009 and 2012 equations , respectively ( 16,17 ) . information on all - cause and cardiovascular mortality was obtained using the linkage of nhanes data to death certificate data from the national death index ( 24 ) . kidney function was analyzed both as a continuous measure using restricted cubic splines and as a categorical measure according to k / doqi classification ( egfr < 15 , 1529 , 3059 , 6089 , and 90200 ml / min/1.73 m ) for both egfrcr and egfrcys . modified poisson regression models were used to examine the relationship of egfr with the prevalence of coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy ( 25 ) . persons with diabetes were older , more likely to be black , and more likely to be male than those without diabetes ( table 1 ) . population with diabetes had a bmi > 30 kg / m compared with 28% without diabetes , and 28% of persons with diabetes had a bmi 35 kg / m compared with 12% of those without diabetes . the prevalence of reduced kidney function was almost three times higher in persons with diabetes compared with those without diabetes ( egfrcr 16.5% vs. 5.8% , egfrcys 22.0% vs. 7.9% ) . adults aged 20 years by diabetes status : nhanes 19992002 cystatin c subsample ( n = 4,457 ) the trend of higher prevalence of reduced kidney function by egfrcys persisted across subgroups of sex , race , age , and bmi ( supplementary fig . similarly , the absolute difference was larger among persons with bmi > 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 20.0% vs. 13.1% ) than among those with bmi < 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 21.3% vs. 16.9% ) . discordance between egfrcr and egfrcys in the classification of reduced kidney function was 11.8% in persons with diabetes and 4.7% in persons without diabetes . in the population with diabetes , 10.4% of those classified as having preserved kidney function by egfrcr ( 8.7% of the 83.6% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . in the population without diabetes , 3.6% of those classified as having preserved kidney function by egfrcr ( 3.4% of the 94.2% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . within the overall population , diabetes status was significantly associated with reclassification from preserved kidney function by egfrcr to reduced kidney function by egfrcys ( odds ratio [ or ] 3.1 [ 95% ci 1.94.9 ] , p < 0.001 ) ( supplementary table 2 ) . in contrast , egfrcr , bmi , age , and albuminuria were all significantly associated with reclassification by egfrcys in multivariable regression ( egfrcr , or 0.9 per 1 ml / min/1.73 m increase , p < 0.001 ; bmi , or 1.5 per 5 kg / m increase , p < 0.001 ; age , or 1.8 per decade increase , p < 0.001 ; acr > 30 mg / g , or 2.2 , p = 0.01 ) . in the population with diabetes , the prevalence of albuminuria , retinopathy , coronary artery disease , and peripheral arterial disease was high , even among those with preserved kidney function ( fig . the most common of these complications was albuminuria , present in 27.0% of those with egfrcr 60 ml / min/1.73 m and 55.9% of those with egfrcr < 60 ml / min/1.73 m. the prevalence of coronary artery disease , retinopathy , and peripheral artery disease was also two to three times higher among those with reduced kidney function than those with preserved kidney function . in general , the probability of microvascular and macrovascular complications increased with lower egfr ; above egfr 90 ml / min/1.73 m , relationships between egfr and vascular complications were more variable . the unadjusted relationships between egfr , coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy were similar using egfrcr and egfrcys , although there was suggestion of a u - shape ( with higher risk at both higher and lower levels of gfr ) in the association between albuminuria , retinopathy , and egfrcr but not egfrcys ( supplementary fig . adults with diabetes according to the presence or absence of reduced kidney function ( estimated using creatinine and cystatin c ) . after adjustment for demographic and traditional cardiovascular risk factors , the prevalence ratios for vascular complications ( coronary artery disease , peripheral arterial disease , albuminuria , retinopathy ) by egfr category were similar using creatinine or cystatin c ( table 2 ) . compared with a reference group of egfr 6090 ml / min/1.73 m , persons with advanced ckd ( egfr 1530 ml / min/1.73 m ) had a higher prevalence of coronary artery disease , albuminuria , and retinopathy , regardless of filtration marker used . the risk of both all - cause and cardiovascular mortality increased with lower egfr , regardless of filtration marker used ( table 3 ) . compared with the reference group of egfr 6090 ml / min/1.73 m , persons with egfr 1530 ml / min/1.73 m had a significantly higher risk of all - cause mortality ( egfrcys , hazard ratio [ hr ] 3.8 , p = 0.007 ; egfrcr , hr 2.6 , p = 0.04 ) . m was significantly associated with increased cardiovascular mortality when estimated by cystatin c ( hr 5.3 , p = 0.007 ) but not by creatinine ( hr 2.0 , p = 0.3 ) . adjusted hazard ratios ( 95% ci ) for categories of kidney function with all - cause and cardiovascular mortality , by filtration marker , among u.s . persons with diabetes were older , more likely to be black , and more likely to be male than those without diabetes ( table 1 ) . population with diabetes had a bmi > 30 kg / m compared with 28% without diabetes , and 28% of persons with diabetes had a bmi 35 kg / m compared with 12% of those without diabetes . the prevalence of reduced kidney function was almost three times higher in persons with diabetes compared with those without diabetes ( egfrcr 16.5% vs. 5.8% , egfrcys 22.0% vs. 7.9% ) . adults aged 20 years by diabetes status : nhanes 19992002 cystatin c subsample ( n = 4,457 ) the trend of higher prevalence of reduced kidney function by egfrcys persisted across subgroups of sex , race , age , and bmi ( supplementary fig . similarly , the absolute difference was larger among persons with bmi > 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 20.0% vs. 13.1% ) than among those with bmi < 30 kg / m ( reduced kidney function by egfrcys vs. egfrcr : 21.3% vs. 16.9% ) . discordance between egfrcr and egfrcys in the classification of reduced kidney function was 11.8% in persons with diabetes and 4.7% in persons without diabetes . in the population with diabetes , 10.4% of those classified as having preserved kidney function by egfrcr ( 8.7% of the 83.6% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . in the population without diabetes , 3.6% of those classified as having preserved kidney function by egfrcr ( 3.4% of the 94.2% with egfrcr 60 ml / min/1.73 m ) were reclassified as having reduced kidney function by egfrcys ( supplementary table 1 ) . within the overall population , diabetes status was significantly associated with reclassification from preserved kidney function by egfrcr to reduced kidney function by egfrcys ( odds ratio [ or ] 3.1 [ 95% ci 1.94.9 ] , p < 0.001 ) ( supplementary table 2 ) . in contrast , egfrcr , bmi , age , and albuminuria were all significantly associated with reclassification by egfrcys in multivariable regression ( egfrcr , or 0.9 per 1 ml / min/1.73 m increase , p < 0.001 ; bmi , or 1.5 per 5 kg / m increase , p < 0.001 ; age , or 1.8 per decade increase , p < 0.001 ; acr > 30 mg / g , or 2.2 , p = 0.01 ) . in the population with diabetes , the prevalence of albuminuria , retinopathy , coronary artery disease , and peripheral arterial disease was high , even among those with preserved kidney function ( fig . the most common of these complications was albuminuria , present in 27.0% of those with egfrcr 60 ml / min/1.73 m and 55.9% of those with egfrcr < 60 ml / min/1.73 m. the prevalence of coronary artery disease , retinopathy , and peripheral artery disease was also two to three times higher among those with reduced kidney function than those with preserved kidney function . in general , the probability of microvascular and macrovascular complications increased with lower egfr ; above egfr 90 ml / min/1.73 m , relationships between egfr and vascular complications were more variable . the unadjusted relationships between egfr , coronary artery disease , peripheral arterial disease , albuminuria , and retinopathy were similar using egfrcr and egfrcys , although there was suggestion of a ( with higher risk at both higher and lower levels of gfr ) in the association between albuminuria , retinopathy , and egfrcr but not egfrcys ( supplementary fig . adults with diabetes according to the presence or absence of reduced kidney function ( estimated using creatinine and cystatin c ) . after adjustment for demographic and traditional cardiovascular risk factors , the prevalence ratios for vascular complications ( coronary artery disease , peripheral arterial disease , albuminuria , retinopathy ) by egfr category were similar using creatinine or cystatin c ( table 2 ) . compared with a reference group of egfr 6090 ml / min/1.73 m , persons with advanced ckd ( egfr 1530 ml / min/1.73 m ) had a higher prevalence of coronary artery disease , albuminuria , and retinopathy , regardless of filtration marker used . the risk of both all - cause and cardiovascular mortality increased with lower egfr , regardless of filtration marker used ( table 3 ) . compared with the reference group of egfr 6090 ml / min/1.73 m , persons with egfr 1530 ml / min/1.73 m had a significantly higher risk of all - cause mortality ( egfrcys , hazard ratio [ hr ] 3.8 , p = 0.007 ; egfrcr , hr 2.6 , p = 0.04 ) . by contrast , egfr 1530 ml / min/1.73 m was significantly associated with increased cardiovascular mortality when estimated by cystatin c ( hr 5.3 , p = 0.007 ) but not by creatinine ( hr 2.0 , p = 0.3 ) . adjusted hazard ratios ( 95% ci ) for categories of kidney function with all - cause and cardiovascular mortality , by filtration marker , among u.s . this nationally representative study of persons with diabetes suggests that the use of cystatin c to estimate kidney function would result in a higher prevalence of reduced kidney function than would estimates using serum creatinine . reclassification from preserved kidney function using creatinine to reduced kidney function using cystatin c occurred more commonly among persons with diabetes than those without , but this observation was explained by differences in the distributions of egfr , bmi , age , and albuminuria between the two populations : reclassification was significantly associated with lower egfrcr , higher bmi , older age , and acr > 30 mg / g . similarly , while low egfr was robustly associated with all - cause mortality , only egfrcys showed significant association with cardiovascular mortality . noninstitutionalized civilian population , kidney function estimated using cystatin c resulted in a reduced kidney function prevalence of 8.7% compared with an estimated 6.5% using creatinine ( 26 ) . in the case of creatinine , muscle mass and diet older persons may be sicker than their younger peers ; thus , kidney function estimated using serum creatinine may be confounded by cachexia and muscle wasting . in the population with diabetes , both serum creatinine and cystatin serum creatinine may poorly estimate kidney function given the tendency of persons with diabetes to have a lower than average muscle mass . certainly , our results support the association of prevalent complications with very low egfr whether estimated by creatinine or cystatin c. interestingly , in the upper ranges of preserved kidney function , the association between egfrcr and retinopathy reversed , with higher levels of egfrcr conferring increased odds of retinopathy , although this was not statistically significant in adjusted analysis . because of the more monotonic relationship seen between egfrcys , albuminuria , and retinopathy , it is possible that kidney function based on cystatin c may prove a better predictor of diabetes complications than that based on creatinine . we also observed that the relationship between egfrcys and all - cause and cardiovascular mortality was stronger than the corresponding relationship with egfrcr , similar to findings in the general population ( 11,12,35 ) and previous studies of persons with diabetes ( 36 ) . in summary , this study demonstrates that using cystatin c to classify kidney function among persons with diabetes results in a higher prevalence of reduced kidney function yet the same or stronger associations with vascular complications and mortality .

in general , physical activity or exercise improves health and reduces the risk of developing several diseases , like cardiovascular disease and type ii diabetes . these health - promoting effects result from the immediate and long - term influence of exercise on many of the human body 's systems , including the cardiovascular system , the energy systems , and the immune system . in a healthy population , these effects are related to both the duration and intensity of the physical activity , with high - intensity exercise yielding the most profound effects [ 1 , 2 ] . paradoxically , one bout of physical activity has a temporary suppressive effect on the function of both innate and adaptive immune cells , for example , neutrophils , monocytes [ 4 , 5 ] , macrophages , natural killer cells , t - cells , and b - cells . the effect of acute exercise on patients with immune - mediated health conditions , such as multiple sclerosis ( ms ) , is much less studied . ms is a chronic demyelinating , inflammatory disease of the central nervous system ( cns ) , predominantly affecting young adults in their most productive years . based on previous efforts focusing on the role of the adaptive immune system in the pathogenesis of ms , it is currently well established that autoreactive t helper type 1 ( th1 ) and th17 cells mediate the inflammatory processes in the cns [ 11 , 12 ] . recent evidence also suggests involvement of innate immunity , including dendritic cells ( dc ) , in the initiation and maintenance as well as progression of ms [ 13 , 14 ] . in human blood , two major subsets of dc have been identified , namely myeloid or conventional dc ( cdc ) and plasmacytoid dc ( pdc ) . they are characterized by a difference in the expression profile of cytokine receptors and cytokines [ 1619 ] , of migratory markers and migration potential and of toll - like receptors ( tlr ) . since dc have the unique capacity to polarize the differentiation of t - cells , they are central in regulating the balance between inflammation and tolerance . for this , dc continuously capture antigens from the environment , process them , and present them on the cell - surface complexed to major histocompatibility ( mhc ) molecules , for example , human leukocyte antigen- ( hla- ) dr . together with context - dependent expression of costimulatory molecules , such as cd86 , and secretion of cytokines , dc can induce either effector t - cells or regulatory t - cells ( treg ) . previously , we demonstrated that significantly lower percentages of circulating dc are found in the peripheral blood of ms patients carrying ms - associated genetic risk factors . these reduced levels of dc may reflect enhanced trafficking from the blood towards the cns . indeed , whereas the capacity of dc to migrate to the sites of inflammation is regulated by the expression of chemokines and chemokine receptors , we have reported increased proportions of circulating cdc and pdc positive for the migratory molecule c - c chemokine receptor 5 ( ccr5 ) in ms patients . although this suggests that dc may drive the inflammatory response in ms , additional processes are likely to be involved , as indicated by the fact that circulating dc subsets were still significantly lower when comparing ms patients and healthy controls both carrying the same ms - associated genetic risk factor . the objective of current study was to investigate whether one exercise bout could increase and mobilize dc rapidly in the peripheral blood in healthy controls and ms patients . moreover , the function of dc following acute physical activity was evaluated in both healthy controls and ms patients . a total of 22 ms patients , diagnosed according to the revised mcdonald criteria and aged > 18 years , were recruited . in addition , 9 control subjects that were matched for gender , age , and body mass index ( bmi ) were included in the study ( table 1 ) . subjects were excluded if they had an expanded disability status scale ( edss ) score > 6 , that is , not being able to walk 100 m without walking aid , a diagnosis of diabetes mellitus type ii , other autoimmune diseases ( diabetes mellitus type i and/or rheumatoid arthritis ) , and other chronic diseases ( cardiovascular , pulmonary , and/or renal ) , were pregnant , participated in another study , had contraindications to perform physical activity , received corticosteroid treatment 3 months prior to the start of the study , or had an acute ms exacerbation 6 months prior to the start of the study . patient and control characteristics and medication use are depicted in supplementary table 1 ( in supplementary material available online at http://dx.doi.org/10.1155/2015/158956 ) . all subjects gave informed consent in accordance with the declaration of helsinki and the protocol was approved by the local ethics committees of hasselt university and of the antwerp university hospital . all patients and controls performed an acute physical exercise test using technogym training equipment ( lj capelle aan den ijssel , the netherlands ) . the exercise bout started with a moderate - to - high - intensity endurance exercise , including 15 min of cycle ergometry followed by a 15 min walking session during which study subjects must have reached their individually calculated heart rate ( hr ) = [ resting hr + 65% ( maximum hr resting hr ) ] . next , the study subjects performed a moderate - to - high - intensity resistance exercise , including unilateral leg strength training ( leg press , leg extension , and leg curl ) and bilateral arm strength training ( chest press , latissimus pull , arm curl ) consisting of 3 10 repetition sets that were interspersed by 2 min rest intervals , during which study subjects trained at 70% of 1 repetition maximum ( rm ) . venous blood was collected before the exercise bout , immediately after the bout and 2 hours after the bout in both heparin and serum - separating tubes ( bd biosciences , erembodegem , belgium ) . leukocyte cell counts were measured using an automated cell counter ( abx micros 60 , horiba , deurne , belgium ) . next , leukocytes were isolated using density gradient purification ( ficoll paque plus , ge healthcare , chalfont st giles , uk ) for ex vivo flow cytometric analysis of dc and treg subsets . for evaluation of treg numbers using intracellular cytokine staining , leukocytes were additionally treated with 10 g / ml brefeldin a ( life technologies , merelbeke , belgium ) for 1618 hours . subsequently , leukocytes were fixed and permeabilized using a fixation and permeabilization concentrate and diluent , according to the manufacturer 's instructions ( ebioscience , vienna , austria ) . simultaneously , 1 ml of peripheral blood was ( i ) stimulated overnight with 2 g / ml lipopolysaccharide ( lps ) , a tlr4 ligand ( invivogen , toulouse , france ) , and 50 g / ml interferon- ( ifn- ) ( immunotools , friesoythe , germany ) or ( ii ) stimulated overnight with 10 g / ml imiquimod ( iq ) , a tlr7 ligand ( invivogen ) , or ( iii ) left untreated as a control . plasma was collected and stored at 20c for batch analysis of tlr - mediated cytokine production . next , leukocytes were enriched after red blood cell lysis ( 0.155 m nh4cl , 0.01 m khco3 , and 0.1 mm na2-edta ) for evaluation of dc activation state and chemokine responsiveness . immunophenotyping of dc was done by direct immunofluorescence staining using the following fluorochrome - labeled mouse anti - human monoclonal antibodies : anti - blood dendritic cell antigen-1 ( bdca-1 ) phycoerythrin ( pe ) ( miltenyi biotec , leiden , the netherlands ) , anti - bdca-2 allophycocyanin ( apc ) ( miltenyi biotec ) , anti - lineage i ( lin i ; anti - cd3 , anti - cd14 , anti - cd16 , anti - cd19 , anti - cd20 , and anti - cd56 ) fluorescein isothiocyanate ( fitc ) ( bd biosciences ) , anti - cd62l pe - cyanine 7 ( pe - cy7 ) ( ebioscience ) , anti - cd86 v450 ( bd biosciences ) , anti - ccr5 pe - cy7 ( bd biosciences ) , and anti - hla - dr apc - h7 ( bd biosciences ) antibodies . treg subsets were characterized using the following fluorochrome - labeled mouse anti - human monoclonal antibodies : anti - cd3 peridinin chlorophyll protein cy5.5 ( percp cy5.5 ) ( bd biosciences ) , anti - cd4 apc - h7 ( bd biosciences ) , anti - cd8 pacific blue ( pb ) ( life technologies ) , anti - cd25 pe - cy7 ( bd biosciences ) , anti - il-10 apc ( bd biosciences ) , anti - transforming growth factor- ( tgf- ) ( iq products , groningen , the netherlands ) , and anti - forkhead box p3 ( foxp3 ) alexa 488 ( bd biosciences ) . in all flow cytometric assays , dead cells were excluded by addition of violet live / dead stain ( life technologies ) to the antibody mixture . fluorescence minus one in combination with nonreactive isotype - matched antibodies was used as control . for analytical flow cytometry , at least 10 events were measured using a cyflow ml flow cytometer ( partec , mnster , germany ) . serum levels of matrix metalloproteinase-9 ( mmp-9 ) ( meso scale discovery , rockville , md , usa ) , macrophage inflammatory protein-1 ( mip-1 ) ( ebioscience ) , and fms - related tyrosine kinase 3 ligand ( flt3l , r&d , minneapolis , mn , usa ) were quantified using elisa according to manufacturer 's instructions . for quantitative detection of the cytokines secreted following stimulation of peripheral blood , collected plasma samples were analyzed using the following commercially available elisa kits : il-1 , il-6 , il-12p70 , tnf- , ifn- ( pbl interferonsource , piscataway , nj , usa ) , mmp-9 , and caspase-1 ( r&d ) , according to manufacturer 's instructions . a model was built stepwise per outcome variable , starting from a univariate model with time as the only fixed effect . new models were constructed , step by step , by adding other fixed effect variables including ms type , gender , age , bmi , edss , and ms - specific medication . the variable ms type included relapsing - remitting ( rr ) and chronically progressive ( cp ) ms patients . the variable ms - specific medication included untreated patients , 1st - line treatment , and 2nd - line treatment . since the variables edss , ms type , and ms - specific medication have no value in healthy individuals , we added an indicator variable to discriminate between ms patients and healthy controls . a p value < 0.10 ( f test ) was used as the threshold for retaining a fixed effect during model building to decrease the chance of missing a significant effect in the final model . subsequently , possible interaction effects between the retained fixed effects were assessed and also retained if p < 0.10 . for interpretation of the fixed effects in the final model , the threshold for statistical significance was set at p < 0.05 . since we aim to investigate temporal effects , we only report main and interaction effects of time , that is , the effect of one exercise bout . models consisting of more than 12 parameters were not interpreted because of the risk of overfitting . when of interest , post hoc analyses were performed using contrast and subgroup analyses , that is , applying the scheff and the bonferroni correction , respectively . diagnostics were based on the studentized residuals and response variables were logarithmically transformed when necessary . graphs were generated in graphpad version 5 software ( prism , la jolla , ca , usa ) . all data are presented as mean standard error of the mean ( sem ) . since it was previously demonstrated by others that acute physical activity alters the number and function of circulating cells of the immune system , we first investigated the absolute number of circulating leukocytes and leukocyte subsets following one exercise bout in both ms patients ( n = 22 ) and healthy controls ( n = 9 ) . both study groups showed a rapid and immediate increase in absolute leukocyte numbers upon one exercise bout ( p < 0.001 ) that normalized after two hours of recovery ( p = 0.020 , figure 1 ) . an immediate increase in the absolute number of lymphocytes ( p < 0.001 ) was found upon one exercise bout , which did not recover after a 2-hour resting phase ( p = 0.020 , figure 1 ) . a distinct response between patients and controls was found for the absolute number of monocytes ( p < 0.001 ) and granulocytes ( p < 0.001 ) following the exercise bout ( figure 1 ) . more specifically , whereas patients display an immediate increase in the monocyte count ( p < 0.001 ) , which recovers after the 2-hour resting phase ( p < 0.001 ) , no response to one exercise bout was observed in healthy controls . patients and controls showed an immediate increase in the absolute granulocyte number after one exercise bout ( ms : p < 0.001 . controls : p < 0.001 ) , although this response was significantly higher in healthy controls as compared to ms patients . furthermore , granulocyte numbers did not recover after the 2-hour recovery period in any of the study groups ( ms : p < 0.001 . noteworthy , the higher the patient 's edss score , the less pronounced the increase in leukocytes ( p < 0.001 ) and monocyte numbers next , we investigated the absolute number of circulating dc subsets , namely cdc ( lin bdca-1 ) and pdc ( lin bdca-2 ) . in both patients and controls , the cdc and pdc count increased rapidly and immediately after one exercise bout ( cdc : p < 0.001 , pdc : p < 0.001 , figure 1 ) . following a 2-hour recovery period , pdc counts normalized to steady - state levels ( p < 0.001 ) , whereas no significant drop to steady - state levels could be demonstrated for the cdc number . also the number of naturally occurring cd25foxp3 treg ( p < 0.001 ) and antigen - induced il-10-producing type 1 treg ( tr1 ) ( p = 0.031 ) increased immediately after one exercise bout in both patients and controls ( figure 1 ) . the number of tr1 remained increased up to 2 hours after the exercise bout ( p < 0.001 ) . in addition , a significantly different response for the number of tgf--producing t helper 3 ( th3 ) cells was observed between patients and controls ( p = 0.037 , figure 1 ) . whereas an immediate increase in the number of th3 cells following one exercise bout was observed in healthy controls ( p = 0.006 ) , which remained high up to two hours after exercise ( p = 0.010 ) , no effect of the exercise bout on the th3 cell count in patients could be demonstrated . previous studies demonstrated that migration of leukocyte subsets , including dc , from and towards the peripheral blood is regulated by distinct signals , chemokines , and chemokine receptors . here , we observed that the absolute number of cdc and pdc expressing the cell adhesion molecule cd62 ligand ( cd62l ) increased immediately after one exercise bout in patients and controls ( cdc : p = 0.017 ; pdc : p = 0.002 , figure 2 ) . after two hours of rest , the number of cd62l cdc did not significantly recover to steady - state values , while the number of cd62l pdc did ( p < 0.001 ) . furthermore , the absolute number of cdc and pdc expressing ccr5 increased immediately after one exercise bout in patients and controls ( cdc : p = 0.024 ; pdc : p < 0.001 ; figure 2 ) but failed to significantly return to steady - state values after two hours of recovery . given the pronounced effect of one exercise bout on dc expressing migratory markers together with the fact that mip-1 , the ligand for ccr5 , induces mobilization of dc precursors , we quantified the serum levels of signals involved in cell migration . in our hands , we were not able to detect any mip-1 in the serum of patients and controls ( data not shown ) . however , the serum level of flt3l , a growth factor and a key regulator of dc homeostasis , immediately increased following one exercise bout in ms patients and healthy controls ( p < 0.001 , figure 3 ) . following two hours of rest , flt3l concentration significantly normalized to steady - state values ( p < 0.001 ) . in addition , patients and controls responded differently to one exercise bout with regard to the serum level of mmp-9 ( p = 0.008 , figure 3 ) , a cell migration - associated proteinase . while the mmp-9 serum concentration immediately increased upon acute physical activity in controls ( p = 0.009 ) and remained high up to two hours after the exercise bout ( p = 0.026 ) , patients only showed a slight , but significant , increase after the 2-hour recovery period ( p = 0.028 ) . in order to assess the effect of one exercise bout on the activation state and chemokine receptor responsiveness of circulating dc in an inflammatory microenvironment , blood samples were stimulated with a tlr4 ligand , lps , in combination with ifn- , or a tlr7 ligand , iq . both ligands are known to activate cdc and pdc , respectively [ 30 , 31 ] . using flow cytometry , we measured the fold change in the expression level of ccr5 , cd86 , and hla - dr on cdc and pdc upon tlr stimulation . in both patients and controls , cd86 expression is upregulated on cdc following stimulation with lps and ifn-. however , the upregulation of this costimulatory marker was significantly less pronounced two hours after the exercise bout as compared to steady - state values ( p = 0.009 , figure 4 ) . on pdc , the upregulation of the expression of hla - dr following iq stimulation was significantly less pronounced immediately after the exercise bout in patients and controls ( p = 0.011 , figure 4 ) , whereas it normalized again after a 2-hour recovery period ( p = 0.043 ) . no effect of acute exercise following tlr stimulation could be demonstrated for the expression levels of ccr5 and hla - dr on cdc and of ccr5 and cd86 on pdc . we observed significantly larger amounts of il-12p70 released upon lps and ifn- stimulation two hours after the exercise bout in patients and controls ( figure 5 ) as compared to steady - state secretion levels ( p < 0.001 ) and to secretion levels immediately after the exercise bout ( p < 0.001 ) , whereas no effect on il-12p70 secretion in patients and controls was found immediately after the exercise bout . no effect on tnf- secretion upon lps and ifn- stimulation could be demonstrated immediately after acute exercise in both patients and controls . tnf- secretion upon lps and ifn- stimulation was significantly influenced by the treatment regimen of the study groups ( p = 0.005 ) . two hours after the exercise bout , a significant increase in the secretion of tnf- was found following lps and ifn- stimulation as compared to steady - state secretion levels ( p < 0.001 ) and to secretion levels immediately after the exercise bout ( p = 0.007 ) in patients with 2nd - line treatment , while this could not be demonstrated in untreated patients and patients with 1st - line treatment . also in controls a trend towards an increase in tnf- secretion upon lps and ifn- stimulation two hours after the exercise bout was observed as compared to secretion levels immediately after the exercise bout ( p = 0.073 ) . mmp-9 secretion upon lps and ifn- stimulation , on the other hand , rapidly and immediately increased following acute physical activity ( p = 0.016 ) and remained highly inducible after the 2-hour resting phase in patients and controls ( p = 0.017 , figure 5 ) . no effect of acute exercise on il-1 , il-6 , ifn- , and caspase-1 secretion following lps and ifn- stimulation could be demonstrated . similarly , no effect of acute exercise was found on the secretion of il-1 , il-6 , il-12p70 , tnf- , ifn- , mmp-9 , and caspase-1 following iq stimulation . in this study , we have demonstrated a rapid and immediate increase in absolute leukocyte number , including lymphocytes and granulocytes , upon one exercise bout in ms patients and healthy controls , which is in line with previous findings by others . interestingly , the number of lymphocytes and granulocytes remained high after a 2-hour recovery period , although the total number of leukocytes normalized again . furthermore , in ms patients also the monocyte count increased immediately after the exercise bout and recovered to steady - state values after two hours of rest , while healthy controls failed to show a significant response in monocyte numbers to one exercise bout . interestingly , the higher the patient 's edss score , the less pronounced the total leukocyte and monocyte response , which is possibly a consequence of a less intensive exercise due to greater immobility in these patients [ 32 , 33 ] . furthermore , also the absolute number of leukocyte subsets with well - described immunoregulatory functions , namely , treg and cdc and pdc , was immediately elevated upon one exercise bout in ms patients and healthy controls , in agreement with other studies [ 3436 ] . since we and others previously reported that dc may play an important role in the immunopathogenesis of ms [ 13 , 14 , 23 ] , we aimed here to better understand the mechanisms that may underlie dc accumulation in the peripheral blood following acute physical activity . interestingly , increased levels of flt3l , which specifically recruits steady - state cdc and pdc towards the circulation [ 3739 ] , were found in patients and controls upon one exercise bout . flt3l - mobilized blood dc were previously shown to migrate towards ccr5 ligands . in accordance with this , increased numbers of cdc and pdc expressing ccr5 upon one exercise bout in patients and controls were found in this study . in addition , increased numbers of cdc and pdc expressing the cell adhesion molecule cd62l were found upon acute exercise in patients and controls . furthermore , the mmp-9 serum level increased immediately upon acute exercise in controls , as demonstrated by others [ 41 , 42 ] , while patients only show an increase after two hours of recovery . the increased secretion of mmp-9 immediately after acute exercise in patients and controls upon lps and ifn- stimulation suggests that mmp-9 found in serum is , at least in part , produced by stimulated dc . others previously demonstrated exercise - induced leukocyte mobilization from the marginal pool towards the circulation . here , we suggest that circulating flt3l might specifically mobilize steady - state ccr5-expressing cdc and pdc from the marginal pool upon acute exercise , as indicated by migration of flt3l - mobilized blood dc towards ccr5 ligands . given its function in transendothelial migration , also cd62l expression on cdc and pdc may contribute to their mobilization towards the circulation driven by mmp-9-dependent remodelling of the vascular endothelium comprising the marginal pool . indeed , mmp-9 was previously shown to be involved in stem cell mobilization from the bone marrow by remodelling of the matrix and basement membranes , as well as in vascular remodelling in murine models . overall , we propose that rapid dc cell recruitment from the marginal pool upon physical exercise involves cell - surface expression of the migratory molecules , ccr5 and cd62l , and is mediated by migration - promoting mediators , such as flt3l and mmp-9 . furthermore , our results suggest that cdc and pdc from ms patients and healthy controls are less responsive to tlr stimulation after one exercise bout . indeed , upregulation of cd86 expression on cdc following tlr stimulation was less pronounced in both patients and controls after the 2-hour recovery phase as compared to steady - state values . in addition , upregulation of hla - dr expression on pdc following tlr stimulation was less pronounced in both patients and controls immediately after one exercise bout . similarly , lancaster et al . showed lower upregulation of cd80 , cd86 , and mhc class ii expression on monocytes from healthy volunteers following tlr activation in samples obtained immediately after and following 2 hours of recovery from a 1.5-hour strenuous exercise in comparison with samples obtained at rest . on the other hand , we observed increased secretion of il-12p70 and tnf- immediately after acute exercise in patients and controls upon lps and ifn- stimulation . since tnf- and il-12p70 can be secreted by cdc [ 23 , 49 ] , we propose here that the observed increase in the number of cdc , at least in part , contributes to the increased secretion of inflammatory mediators upon lps and ifn- stimulation after acute exercise , rather than increased secretion per individual cdc , which is in line with the above - mentioned reduced upregulation of costimulatory markers . this hypothesis is supported by findings of others demonstrating that an absolute increase in monocyte number was responsible for the increased tlr - mediated proinflammatory cytokine production after physical exercise . in fact , these monocytes produced less cytokine per cell [ 4 , 5 , 50 ] . future studies are , however , needed to support tlr - mediated secretion of inflammatory cytokines using intracellular flow cytometry in order to characterize the identity of the cytokine - producing cell types as well as the amount of cytokine produced per cell after one exercise bout . in conclusion , our results indicate accumulation of dc in the peripheral blood after one exercise bout which , at least in part , is mediated by a flt3l- and mmp-9-mediated process and involves cell - surface expression of the migratory molecules , ccr5 and cd62l . further elucidation of the mechanisms involved may shed light on current knowledge regarding the pathologic role of dc in various inflammatory diseases . moreover , our results demonstrate a reduced tlr responsiveness of cdc and pdc after acute physical activity , indicating that dc are less prone to drive inflammatory processes following exercise [ 1 , 2 ] . together with the observed increase of treg numbers upon one exercise bout , our findings may present a negative feedback mechanism for the immune system 's ability to induce tissue damage and inflammation following exercise . ultimately , this may provide a tool to modulate the underlying disease pathogenesis of ms contributing to the long - term health benefits of regular exercise .

in healthy individuals , one exercise bout induces a substantial increase in the number of circulating leukocytes , while their function is transiently suppressed . the effect of one exercise bout in multiple sclerosis ( ms ) is less studied . since recent evidence suggests a role of dendritic cells ( dc ) in the pathogenesis of ms , we investigated the effect of one combined endurance / resistance exercise bout on the number and function of dc in ms patients and healthy controls . our results show a rapid increase in the number of dc in response to physical exercise in both ms patients and controls . further investigation revealed that in particular dc expressing the migratory molecules ccr5 and cd62l were increased upon acute physical activity . this may be mediated by flt3l- and mmp-9-dependent mobilization of dc , as demonstrated by increased circulating levels of flt3l and mmp-9 following one exercise bout . circulating dc display reduced tlr responsiveness after acute exercise , as evidenced by a less pronounced upregulation of activation markers , hla - dr and cd86 , on plasmacytoid dc and conventional dc , respectively . our results indicate mobilization of dc , which may be less prone to drive inflammatory processes , following exercise . this may present a negative feedback mechanism for exercise - induced tissue damage and inflammation .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

paradoxically , one bout of physical activity has a temporary suppressive effect on the function of both innate and adaptive immune cells , for example , neutrophils , monocytes [ 4 , 5 ] , macrophages , natural killer cells , t - cells , and b - cells . the effect of acute exercise on patients with immune - mediated health conditions , such as multiple sclerosis ( ms ) , is much less studied . based on previous efforts focusing on the role of the adaptive immune system in the pathogenesis of ms , it is currently well established that autoreactive t helper type 1 ( th1 ) and th17 cells mediate the inflammatory processes in the cns [ 11 , 12 ] . recent evidence also suggests involvement of innate immunity , including dendritic cells ( dc ) , in the initiation and maintenance as well as progression of ms [ 13 , 14 ] . in human blood , two major subsets of dc have been identified , namely myeloid or conventional dc ( cdc ) and plasmacytoid dc ( pdc ) . they are characterized by a difference in the expression profile of cytokine receptors and cytokines [ 1619 ] , of migratory markers and migration potential and of toll - like receptors ( tlr ) . together with context - dependent expression of costimulatory molecules , such as cd86 , and secretion of cytokines , dc can induce either effector t - cells or regulatory t - cells ( treg ) . previously , we demonstrated that significantly lower percentages of circulating dc are found in the peripheral blood of ms patients carrying ms - associated genetic risk factors . these reduced levels of dc may reflect enhanced trafficking from the blood towards the cns . indeed , whereas the capacity of dc to migrate to the sites of inflammation is regulated by the expression of chemokines and chemokine receptors , we have reported increased proportions of circulating cdc and pdc positive for the migratory molecule c - c chemokine receptor 5 ( ccr5 ) in ms patients . although this suggests that dc may drive the inflammatory response in ms , additional processes are likely to be involved , as indicated by the fact that circulating dc subsets were still significantly lower when comparing ms patients and healthy controls both carrying the same ms - associated genetic risk factor . the objective of current study was to investigate whether one exercise bout could increase and mobilize dc rapidly in the peripheral blood in healthy controls and ms patients . moreover , the function of dc following acute physical activity was evaluated in both healthy controls and ms patients . all patients and controls performed an acute physical exercise test using technogym training equipment ( lj capelle aan den ijssel , the netherlands ) . the exercise bout started with a moderate - to - high - intensity endurance exercise , including 15 min of cycle ergometry followed by a 15 min walking session during which study subjects must have reached their individually calculated heart rate ( hr ) = [ resting hr + 65% ( maximum hr resting hr ) ] . next , the study subjects performed a moderate - to - high - intensity resistance exercise , including unilateral leg strength training ( leg press , leg extension , and leg curl ) and bilateral arm strength training ( chest press , latissimus pull , arm curl ) consisting of 3 10 repetition sets that were interspersed by 2 min rest intervals , during which study subjects trained at 70% of 1 repetition maximum ( rm ) . venous blood was collected before the exercise bout , immediately after the bout and 2 hours after the bout in both heparin and serum - separating tubes ( bd biosciences , erembodegem , belgium ) . next , leukocytes were isolated using density gradient purification ( ficoll paque plus , ge healthcare , chalfont st giles , uk ) for ex vivo flow cytometric analysis of dc and treg subsets . immunophenotyping of dc was done by direct immunofluorescence staining using the following fluorochrome - labeled mouse anti - human monoclonal antibodies : anti - blood dendritic cell antigen-1 ( bdca-1 ) phycoerythrin ( pe ) ( miltenyi biotec , leiden , the netherlands ) , anti - bdca-2 allophycocyanin ( apc ) ( miltenyi biotec ) , anti - lineage i ( lin i ; anti - cd3 , anti - cd14 , anti - cd16 , anti - cd19 , anti - cd20 , and anti - cd56 ) fluorescein isothiocyanate ( fitc ) ( bd biosciences ) , anti - cd62l pe - cyanine 7 ( pe - cy7 ) ( ebioscience ) , anti - cd86 v450 ( bd biosciences ) , anti - ccr5 pe - cy7 ( bd biosciences ) , and anti - hla - dr apc - h7 ( bd biosciences ) antibodies . serum levels of matrix metalloproteinase-9 ( mmp-9 ) ( meso scale discovery , rockville , md , usa ) , macrophage inflammatory protein-1 ( mip-1 ) ( ebioscience ) , and fms - related tyrosine kinase 3 ligand ( flt3l , r&d , minneapolis , mn , usa ) were quantified using elisa according to manufacturer 's instructions . since the variables edss , ms type , and ms - specific medication have no value in healthy individuals , we added an indicator variable to discriminate between ms patients and healthy controls . since we aim to investigate temporal effects , we only report main and interaction effects of time , that is , the effect of one exercise bout . since it was previously demonstrated by others that acute physical activity alters the number and function of circulating cells of the immune system , we first investigated the absolute number of circulating leukocytes and leukocyte subsets following one exercise bout in both ms patients ( n = 22 ) and healthy controls ( n = 9 ) . both study groups showed a rapid and immediate increase in absolute leukocyte numbers upon one exercise bout ( p < 0.001 ) that normalized after two hours of recovery ( p = 0.020 , figure 1 ) . an immediate increase in the absolute number of lymphocytes ( p < 0.001 ) was found upon one exercise bout , which did not recover after a 2-hour resting phase ( p = 0.020 , figure 1 ) . a distinct response between patients and controls was found for the absolute number of monocytes ( p < 0.001 ) and granulocytes ( p < 0.001 ) following the exercise bout ( figure 1 ) . more specifically , whereas patients display an immediate increase in the monocyte count ( p < 0.001 ) , which recovers after the 2-hour resting phase ( p < 0.001 ) , no response to one exercise bout was observed in healthy controls . patients and controls showed an immediate increase in the absolute granulocyte number after one exercise bout ( ms : p < 0.001 . controls : p < 0.001 ) , although this response was significantly higher in healthy controls as compared to ms patients . furthermore , granulocyte numbers did not recover after the 2-hour recovery period in any of the study groups ( ms : p < 0.001 . noteworthy , the higher the patient 's edss score , the less pronounced the increase in leukocytes ( p < 0.001 ) and monocyte numbers next , we investigated the absolute number of circulating dc subsets , namely cdc ( lin bdca-1 ) and pdc ( lin bdca-2 ) . in both patients and controls , the cdc and pdc count increased rapidly and immediately after one exercise bout ( cdc : p < 0.001 , pdc : p < 0.001 , figure 1 ) . also the number of naturally occurring cd25foxp3 treg ( p < 0.001 ) and antigen - induced il-10-producing type 1 treg ( tr1 ) ( p = 0.031 ) increased immediately after one exercise bout in both patients and controls ( figure 1 ) . the number of tr1 remained increased up to 2 hours after the exercise bout ( p < 0.001 ) . in addition , a significantly different response for the number of tgf--producing t helper 3 ( th3 ) cells was observed between patients and controls ( p = 0.037 , figure 1 ) . whereas an immediate increase in the number of th3 cells following one exercise bout was observed in healthy controls ( p = 0.006 ) , which remained high up to two hours after exercise ( p = 0.010 ) , no effect of the exercise bout on the th3 cell count in patients could be demonstrated . here , we observed that the absolute number of cdc and pdc expressing the cell adhesion molecule cd62 ligand ( cd62l ) increased immediately after one exercise bout in patients and controls ( cdc : p = 0.017 ; pdc : p = 0.002 , figure 2 ) . after two hours of rest , the number of cd62l cdc did not significantly recover to steady - state values , while the number of cd62l pdc did ( p < 0.001 ) . furthermore , the absolute number of cdc and pdc expressing ccr5 increased immediately after one exercise bout in patients and controls ( cdc : p = 0.024 ; pdc : p < 0.001 ; figure 2 ) but failed to significantly return to steady - state values after two hours of recovery . given the pronounced effect of one exercise bout on dc expressing migratory markers together with the fact that mip-1 , the ligand for ccr5 , induces mobilization of dc precursors , we quantified the serum levels of signals involved in cell migration . in our hands , we were not able to detect any mip-1 in the serum of patients and controls ( data not shown ) . however , the serum level of flt3l , a growth factor and a key regulator of dc homeostasis , immediately increased following one exercise bout in ms patients and healthy controls ( p < 0.001 , figure 3 ) . in addition , patients and controls responded differently to one exercise bout with regard to the serum level of mmp-9 ( p = 0.008 , figure 3 ) , a cell migration - associated proteinase . while the mmp-9 serum concentration immediately increased upon acute physical activity in controls ( p = 0.009 ) and remained high up to two hours after the exercise bout ( p = 0.026 ) , patients only showed a slight , but significant , increase after the 2-hour recovery period ( p = 0.028 ) . in order to assess the effect of one exercise bout on the activation state and chemokine receptor responsiveness of circulating dc in an inflammatory microenvironment , blood samples were stimulated with a tlr4 ligand , lps , in combination with ifn- , or a tlr7 ligand , iq . using flow cytometry , we measured the fold change in the expression level of ccr5 , cd86 , and hla - dr on cdc and pdc upon tlr stimulation . in both patients and controls , cd86 expression is upregulated on cdc following stimulation with lps and ifn-. however , the upregulation of this costimulatory marker was significantly less pronounced two hours after the exercise bout as compared to steady - state values ( p = 0.009 , figure 4 ) . on pdc , the upregulation of the expression of hla - dr following iq stimulation was significantly less pronounced immediately after the exercise bout in patients and controls ( p = 0.011 , figure 4 ) , whereas it normalized again after a 2-hour recovery period ( p = 0.043 ) . no effect of acute exercise following tlr stimulation could be demonstrated for the expression levels of ccr5 and hla - dr on cdc and of ccr5 and cd86 on pdc . we observed significantly larger amounts of il-12p70 released upon lps and ifn- stimulation two hours after the exercise bout in patients and controls ( figure 5 ) as compared to steady - state secretion levels ( p < 0.001 ) and to secretion levels immediately after the exercise bout ( p < 0.001 ) , whereas no effect on il-12p70 secretion in patients and controls was found immediately after the exercise bout . no effect on tnf- secretion upon lps and ifn- stimulation could be demonstrated immediately after acute exercise in both patients and controls . two hours after the exercise bout , a significant increase in the secretion of tnf- was found following lps and ifn- stimulation as compared to steady - state secretion levels ( p < 0.001 ) and to secretion levels immediately after the exercise bout ( p = 0.007 ) in patients with 2nd - line treatment , while this could not be demonstrated in untreated patients and patients with 1st - line treatment . also in controls a trend towards an increase in tnf- secretion upon lps and ifn- stimulation two hours after the exercise bout was observed as compared to secretion levels immediately after the exercise bout ( p = 0.073 ) . mmp-9 secretion upon lps and ifn- stimulation , on the other hand , rapidly and immediately increased following acute physical activity ( p = 0.016 ) and remained highly inducible after the 2-hour resting phase in patients and controls ( p = 0.017 , figure 5 ) . no effect of acute exercise on il-1 , il-6 , ifn- , and caspase-1 secretion following lps and ifn- stimulation could be demonstrated . similarly , no effect of acute exercise was found on the secretion of il-1 , il-6 , il-12p70 , tnf- , ifn- , mmp-9 , and caspase-1 following iq stimulation . in this study , we have demonstrated a rapid and immediate increase in absolute leukocyte number , including lymphocytes and granulocytes , upon one exercise bout in ms patients and healthy controls , which is in line with previous findings by others . interestingly , the number of lymphocytes and granulocytes remained high after a 2-hour recovery period , although the total number of leukocytes normalized again . furthermore , in ms patients also the monocyte count increased immediately after the exercise bout and recovered to steady - state values after two hours of rest , while healthy controls failed to show a significant response in monocyte numbers to one exercise bout . interestingly , the higher the patient 's edss score , the less pronounced the total leukocyte and monocyte response , which is possibly a consequence of a less intensive exercise due to greater immobility in these patients [ 32 , 33 ] . furthermore , also the absolute number of leukocyte subsets with well - described immunoregulatory functions , namely , treg and cdc and pdc , was immediately elevated upon one exercise bout in ms patients and healthy controls , in agreement with other studies [ 3436 ] . since we and others previously reported that dc may play an important role in the immunopathogenesis of ms [ 13 , 14 , 23 ] , we aimed here to better understand the mechanisms that may underlie dc accumulation in the peripheral blood following acute physical activity . interestingly , increased levels of flt3l , which specifically recruits steady - state cdc and pdc towards the circulation [ 3739 ] , were found in patients and controls upon one exercise bout . in accordance with this , increased numbers of cdc and pdc expressing ccr5 upon one exercise bout in patients and controls were found in this study . in addition , increased numbers of cdc and pdc expressing the cell adhesion molecule cd62l were found upon acute exercise in patients and controls . furthermore , the mmp-9 serum level increased immediately upon acute exercise in controls , as demonstrated by others [ 41 , 42 ] , while patients only show an increase after two hours of recovery . the increased secretion of mmp-9 immediately after acute exercise in patients and controls upon lps and ifn- stimulation suggests that mmp-9 found in serum is , at least in part , produced by stimulated dc . others previously demonstrated exercise - induced leukocyte mobilization from the marginal pool towards the circulation . here , we suggest that circulating flt3l might specifically mobilize steady - state ccr5-expressing cdc and pdc from the marginal pool upon acute exercise , as indicated by migration of flt3l - mobilized blood dc towards ccr5 ligands . overall , we propose that rapid dc cell recruitment from the marginal pool upon physical exercise involves cell - surface expression of the migratory molecules , ccr5 and cd62l , and is mediated by migration - promoting mediators , such as flt3l and mmp-9 . furthermore , our results suggest that cdc and pdc from ms patients and healthy controls are less responsive to tlr stimulation after one exercise bout . indeed , upregulation of cd86 expression on cdc following tlr stimulation was less pronounced in both patients and controls after the 2-hour recovery phase as compared to steady - state values . in addition , upregulation of hla - dr expression on pdc following tlr stimulation was less pronounced in both patients and controls immediately after one exercise bout . showed lower upregulation of cd80 , cd86 , and mhc class ii expression on monocytes from healthy volunteers following tlr activation in samples obtained immediately after and following 2 hours of recovery from a 1.5-hour strenuous exercise in comparison with samples obtained at rest . on the other hand , we observed increased secretion of il-12p70 and tnf- immediately after acute exercise in patients and controls upon lps and ifn- stimulation . since tnf- and il-12p70 can be secreted by cdc [ 23 , 49 ] , we propose here that the observed increase in the number of cdc , at least in part , contributes to the increased secretion of inflammatory mediators upon lps and ifn- stimulation after acute exercise , rather than increased secretion per individual cdc , which is in line with the above - mentioned reduced upregulation of costimulatory markers . this hypothesis is supported by findings of others demonstrating that an absolute increase in monocyte number was responsible for the increased tlr - mediated proinflammatory cytokine production after physical exercise . future studies are , however , needed to support tlr - mediated secretion of inflammatory cytokines using intracellular flow cytometry in order to characterize the identity of the cytokine - producing cell types as well as the amount of cytokine produced per cell after one exercise bout . in conclusion , our results indicate accumulation of dc in the peripheral blood after one exercise bout which , at least in part , is mediated by a flt3l- and mmp-9-mediated process and involves cell - surface expression of the migratory molecules , ccr5 and cd62l . further elucidation of the mechanisms involved may shed light on current knowledge regarding the pathologic role of dc in various inflammatory diseases . moreover , our results demonstrate a reduced tlr responsiveness of cdc and pdc after acute physical activity , indicating that dc are less prone to drive inflammatory processes following exercise [ 1 , 2 ] . together with the observed increase of treg numbers upon one exercise bout , our findings may present a negative feedback mechanism for the immune system 's ability to induce tissue damage and inflammation following exercise . ultimately , this may provide a tool to modulate the underlying disease pathogenesis of ms contributing to the long - term health benefits of regular exercise .

a large number of studies has clearly shown the existence of a strong inverse correlation between plasma high - density lipoprotein cholesterol ( hdl - c ) concentrations and the incidence of coronary heart disease ( chd ) , but the significance of such association has been recently questioned . intervention clinical trials carried out with agents efficient in raising hdl - c levels , including niacin and cholesteryl ester transfer protein ( cetp ) inhibitors , have failed in showing a reduction in cardiovascular events . in addition , mendelian randomization studies have shown that increased hdl - c levels caused by common variants in hdl related genes are not necessarily associated with reduced cardiovascular risk . one possible explanation for this discrepancy is that the plasma hdl - c concentration does not reflect the very complex hdl system , involving different hdl particles and a number of receptors , transporters , enzyme , and transfer proteins . moreover , cholesterol is not the active component of hdl and there is convincing evidence that at least some of the atheroprotective functions of hdl relate to specific hdl components or subclasses , which concentration in plasma may be totally unrelated to the hdl - c level . hdl are a highly heterogeneous lipoprotein family composed by several subclasses with different density , shape , and size . the density of the hdl particles is inversely related to their size , reflecting the relative contents of low density non - polar core lipid , and high density surface protein . most part of plasma hdl has a globular shape , the central core is composed by non - polar lipids ( triglycerides and cholesteryl esters ) surrounded by a monolayer of polar lipids ( phospholipids and unesterified cholesterol ) and apolipoproteins . a minor fraction of plasma hdl has a non - spherical structure and they consist in discoidal bilayer of polar lipids , in which non - polar core is lacking ; apolipoproteins run from side to side of the disk , with polar residues facing the aqueous phase and non - polar residues facing the acyl chains of the lipid bilayer . the protein component of hdl is formed mainly by apolipoprotein a - i ( apoa - i ) , for the 70% , and apolipoprotein a - ii ( apoa - ii ) , for the 20% . two major particle subclasses have been identified on the basis of major apolipoprotein composition : particles containing only apoa - i ( lpa - i ) , and particles containing both apoa - i and apoa - ii ( lpa - i : a - ii ) . recent shotgun proteomic analysis showed that hdl contain 48 or more proteins , among these apoa - iv , apocs , apoe , lecithin : cholesterol acyltransferase ( lcat ) , cetp , phospholipid transfer protein ( pltp ) , paraoxonase ( pon ) , and platelet - activating factor acetylhydrolase ( paf - ah ) circulate in plasma bound to hdl . most of the proteins carried by hdl are not apolipoproteins , and represent very minor components of these particles . it is also possible to classify hdl on the basis of density ( hdl2 , with density of 1.063 to 1.120 g / ml , and hdl3 , 1.120 to 1.210 according to charge , hdl can be divided into - and pre--migrating particles on agarose gel , and combining charge and size these two subclass - es can be divided into 12 distinct apoa - i - containing particles , referred to as pre- ( pre-1 and pre-2 ) , ( 1 , 2 , and 3 ) and pre- ( pre-1 , pre-2 , and pre-3 ) on the basis of mobility that is slower or faster than albumin , respectively , and decreasing size . due to its highly dynamic nature apoa - i and apoa - ii are synthe - sized mainly by the liver and , to a lesser extent , by the small intestine and are secreted as components of triglyceride - rich lipoproteins . in circulation , pltp promotes the transfer of surface components ( phospholipids , cholesterol , and apolipoproteins ) from triglyceride - rich lipoproteins to hdl . the regulatory role of pltp is achieved through two main functions , phospholipid transfer activity and the capability to modulate hdl size and composition in a process called hdl conversion . hepatocytes are able to secrete apoa - i in lipid - free or lipid - poor and lipidated forms . apoa - i is secreted as pro - apoa - i and converted to a mature form by a metalloprotease in plasma . there are three potential sources of lipid - poor apoa - i in plasma : it may be released as lipid - poor protein after its synthesis in the liver and intestine , it may be released from triglyceride - rich lipoproteins that are undergoing lipolysis by lipoprotein lipase , and it may be generated in the circulation during the remodeling of mature , spherical hdl particles . lipid free - apoa - i acquires phospholipids and cholesterol through the interaction with the atp binding cassette transporter a1 ( abca1 ) , to form pre--hdl , a pathway dependent on abca1 expression . once in the circulation , pre--hdl are the preferential substrate of lcat ( fig . 1 ) , that converts lecithin and cholesterol into lysolecithin and cholesteryl esters , using apoa - i as cofactor . the cholesterol esters generated by lcat are more hydrophobic than free cholesterol and thus migrate into the hydrophobic core of the lipoprotein , with the resulting conversion of small , discoidal pre--hdl into mature , spherical , -migrating hdl ( -hdl ) . lcat thus plays a central role in intravascular hdl metabolism and in the determination of plasma hdl level . esterification of cholesterol in plasma by lcat is also necessary for cholesterol uptake from the liver , either directly through the scavenger receptor class b member 1 ( sr - bi ) or indirectly through cetp . the -hdl produced by lcat ( hdl3 ) interact in the plasma with cetp , that exchanges cholesteryl esters for triglycerides between hdl and triglyceride - rich lipoproteins , generating large cholesteryl ester - poor and triglyceride - rich hdl particles ( hdl2 ) . mature , large -hdl particles can be converted back to pre--hdl through the action of pltp and the endothelial and hepatic lipases , that hydrolyze triglycerides and phospholipids on hdl ( fig . plasma half - life of pre--hdl is short , they are rapidly cleared through the kidney , while mature -hdl have a slower turnover . hdl components are catabolized in different ways ; the major sites of catabolism of the protein components are liver and kidney . the kidney , according to hydrophobicity , filters lipid - free apolipoproteins ; apoa - i and apoa - ii can be reabsorbed through cubulin receptors in the kidney proximal tubules . when reabsorption is impaired , hydrophilic apolipoproteins ( apoa - i and apoa - iv , but no apoa - ii ) can be excreted into urine . glomerular filtration barrier prevents access of mature hdl particles to the proximal tubules ; however , cubulin may bind filtered lipid - poor hdl . hdl particles can entirely be removed by holoparticles hdl receptors . in the liver , holo - hdl particles accumulated in endosomal compartments can be transferred to lysosomes for degradation or in a small proportion , they can be resecreted into the circulation . one of the most important function of hdl is to promote the removal of cholesterol from peripheral cells , including macrophages within the arterial wall , and shuttle it to the liver for excretion through the bile and feces in a process called reverse cholesterol transport ( rct ) . it results in a net mass transport of cholesterol from the arterial wall into the bile . this pathway is described as an anti - atherogenic process by preventing arterial cholesterol accumulation , plaque destabilization , and development of acute cardiovascular events . cell cholesterol efflux is the first and limiting step in rct and consists in the exchange of unesterified cholesterol between cells and extracellular acceptors . this exchange can occur by several processes : via aqueous diffusion , which occurs according to the direction of cholesterol gradient , or through three main distinct and protein - mediated pathways . lipid - free / lipid - poor apolipoproteins , mainly apoa - i , represent the principal cholesterol acceptors via abca1 . all plasma hdl subclasses , including mature -hdl particles and discoidal pre--hdl , are efficient cholesterol acceptors via the abcg1 pathway , while sr - bi promotes cell cholesterol efflux only to mature , large -hdl . the cholesterol accumulated in hdl is esterified in plasma by lcat with the resulting formation of cholesteryl esters . then , hydrophobic cholesteryl esters move to the core while unesterified cholesterol is removed from the surface of hdl , leading to the progressive enlargement of these particles . for long time , lcat has been considered necessary for efficient rct by keeping unesterified cholesterol gradient from cells to hdl , but recent data suggest that even if functional lcat is not present , macrophage cholesterol efflux and rct can occur . large amount of the cholesteryl esters formed by the lcat are exchanged with triglycerides through cetp - mediated process into apob containing lipoproteins that are finally catabolized by the liver . alternatively , hdl - cholesteryl esters are taken - up by the liver through sr - bi . atheroprotection mediated by hdl is not only through their major role in rct , but also through other relevant functions ; one of the most important and well studied is the ability of hdl to maintain endothelial cell homeostasis and integrity . hdl have potent antioxidant properties , mediated by molecules carried by hdl ( pon-1 , paf - ah , and lcat ) or by apoa - i and apoa - ii , as well as anti - inflammatory , antithrombotic , cytoprotective , vasodilatory , anti - infectious activities and the capacity to enhance insulin secretion . this wide spectrum of biological activities likely reflects the heterogeneity of hdl particles ; however , the hdl - protective activities can be lost in some pathological conditions and hdl can even acquire proatherogenic properties . hdl are a highly heterogeneous lipoprotein family composed by several subclasses with different density , shape , and size . the density of the hdl particles is inversely related to their size , reflecting the relative contents of low density non - polar core lipid , and high density surface protein . most part of plasma hdl has a globular shape , the central core is composed by non - polar lipids ( triglycerides and cholesteryl esters ) surrounded by a monolayer of polar lipids ( phospholipids and unesterified cholesterol ) and apolipoproteins . a minor fraction of plasma hdl has a non - spherical structure and they consist in discoidal bilayer of polar lipids , in which non - polar core is lacking ; apolipoproteins run from side to side of the disk , with polar residues facing the aqueous phase and non - polar residues facing the acyl chains of the lipid bilayer . the protein component of hdl is formed mainly by apolipoprotein a - i ( apoa - i ) , for the 70% , and apolipoprotein a - ii ( apoa - ii ) , for the 20% . two major particle subclasses have been identified on the basis of major apolipoprotein composition : particles containing only apoa - i ( lpa - i ) , and particles containing both apoa - i and apoa - ii ( lpa - i : a - ii ) . recent shotgun proteomic analysis showed that hdl contain 48 or more proteins , among these apoa - iv , apocs , apoe , lecithin : cholesterol acyltransferase ( lcat ) , cetp , phospholipid transfer protein ( pltp ) , paraoxonase ( pon ) , and platelet - activating factor acetylhydrolase ( paf - ah ) circulate in plasma bound to hdl . most of the proteins carried by hdl are not apolipoproteins , and represent very minor components of these particles . it is also possible to classify hdl on the basis of density ( hdl2 , with density of 1.063 to 1.120 g / ml , and hdl3 , 1.120 to 1.210 according to charge , hdl can be divided into - and pre--migrating particles on agarose gel , and combining charge and size these two subclass - es can be divided into 12 distinct apoa - i - containing particles , referred to as pre- ( pre-1 and pre-2 ) , ( 1 , 2 , and 3 ) and pre- ( pre-1 , pre-2 , and pre-3 ) on the basis of mobility that is slower or faster than albumin , respectively , and decreasing size . due to its highly dynamic nature , apoa - i is involved in major pathways of hdl metabolism . apoa - i and apoa - ii are synthe - sized mainly by the liver and , to a lesser extent , by the small intestine and are secreted as components of triglyceride - rich lipoproteins . in circulation , pltp promotes the transfer of surface components ( phospholipids , cholesterol , and apolipoproteins ) from triglyceride - rich lipoproteins to hdl . the regulatory role of pltp is achieved through two main functions , phospholipid transfer activity and the capability to modulate hdl size and composition in a process called hdl conversion . hepatocytes are able to secrete apoa - i in lipid - free or lipid - poor and lipidated forms . apoa - i is secreted as pro - apoa - i and converted to a mature form by a metalloprotease in plasma . there are three potential sources of lipid - poor apoa - i in plasma : it may be released as lipid - poor protein after its synthesis in the liver and intestine , it may be released from triglyceride - rich lipoproteins that are undergoing lipolysis by lipoprotein lipase , and it may be generated in the circulation during the remodeling of mature , spherical hdl particles . lipid free - apoa - i acquires phospholipids and cholesterol through the interaction with the atp binding cassette transporter a1 ( abca1 ) , to form pre--hdl , a pathway dependent on abca1 expression . 1 ) , that converts lecithin and cholesterol into lysolecithin and cholesteryl esters , using apoa - i as cofactor . the cholesterol esters generated by lcat are more hydrophobic than free cholesterol and thus migrate into the hydrophobic core of the lipoprotein , with the resulting conversion of small , discoidal pre--hdl into mature , spherical , -migrating hdl ( -hdl ) . lcat thus plays a central role in intravascular hdl metabolism and in the determination of plasma hdl level . esterification of cholesterol in plasma by lcat is also necessary for cholesterol uptake from the liver , either directly through the scavenger receptor class b member 1 ( sr - bi ) or indirectly through cetp . the -hdl produced by lcat ( hdl3 ) interact in the plasma with cetp , that exchanges cholesteryl esters for triglycerides between hdl and triglyceride - rich lipoproteins , generating large cholesteryl ester - poor and triglyceride - rich hdl particles ( hdl2 ) . mature , large -hdl particles can be converted back to pre--hdl through the action of pltp and the endothelial and hepatic lipases , that hydrolyze triglycerides and phospholipids on hdl ( fig . plasma half - life of pre--hdl is short , they are rapidly cleared through the kidney , while mature -hdl have a slower turnover . hdl components are catabolized in different ways ; the major sites of catabolism of the protein components are liver and kidney . the kidney , according to hydrophobicity , filters lipid - free apolipoproteins ; apoa - i and apoa - ii can be reabsorbed through cubulin receptors in the kidney proximal tubules . when reabsorption is impaired , hydrophilic apolipoproteins ( apoa - i and apoa - iv , but no apoa - ii ) can be excreted into urine . glomerular filtration barrier prevents access of mature hdl particles to the proximal tubules ; however , cubulin may bind filtered lipid - poor hdl . hdl particles can entirely be removed by holoparticles hdl receptors . in the liver , holo - hdl particles accumulated in endosomal compartments can be transferred to lysosomes for degradation or in a small proportion , they can be resecreted into the circulation . one of the most important function of hdl is to promote the removal of cholesterol from peripheral cells , including macrophages within the arterial wall , and shuttle it to the liver for excretion through the bile and feces in a process called reverse cholesterol transport ( rct ) . it results in a net mass transport of cholesterol from the arterial wall into the bile . this pathway is described as an anti - atherogenic process by preventing arterial cholesterol accumulation , plaque destabilization , and development of acute cardiovascular events . cell cholesterol efflux is the first and limiting step in rct and consists in the exchange of unesterified cholesterol between cells and extracellular acceptors . this exchange can occur by several processes : via aqueous diffusion , which occurs according to the direction of cholesterol gradient , or through three main distinct and protein - mediated pathways . lipid - free / lipid - poor apolipoproteins , mainly apoa - i , represent the principal cholesterol acceptors via abca1 . all plasma hdl subclasses , including mature -hdl particles and discoidal pre--hdl , are efficient cholesterol acceptors via the abcg1 pathway , while sr - bi promotes cell cholesterol efflux only to mature , large -hdl . the cholesterol accumulated in hdl is esterified in plasma by lcat with the resulting formation of cholesteryl esters . then , hydrophobic cholesteryl esters move to the core while unesterified cholesterol is removed from the surface of hdl , leading to the progressive enlargement of these particles . for long time , lcat has been considered necessary for efficient rct by keeping unesterified cholesterol gradient from cells to hdl , but recent data suggest that even if functional lcat is not present , macrophage cholesterol efflux and rct can occur . large amount of the cholesteryl esters formed by the lcat are exchanged with triglycerides through cetp - mediated process into apob containing lipoproteins that are finally catabolized by the liver . alternatively , hdl - cholesteryl esters are taken - up by the liver through sr - bi . atheroprotection mediated by hdl is not only through their major role in rct , but also through other relevant functions ; one of the most important and well studied is the ability of hdl to maintain endothelial cell homeostasis and integrity . hdl have potent antioxidant properties , mediated by molecules carried by hdl ( pon-1 , paf - ah , and lcat ) or by apoa - i and apoa - ii , as well as anti - inflammatory , antithrombotic , cytoprotective , vasodilatory , anti - infectious activities and the capacity to enhance insulin secretion . this wide spectrum of biological activities likely reflects the heterogeneity of hdl particles ; however , the hdl - protective activities can be lost in some pathological conditions and hdl can even acquire proatherogenic properties . lcat is principally synthetized in the liver and in little amount in other tissues , such as brain and testes , and circulates in plasma compartment at concentration of 5 mg / l mainly bound to hdl but also to ldl . lcat converts phosphatidylcholine and cholesterol into cholesteryl ester and lysophosphatidylcholine in plasma and other biological fluids . in rct pathway , lcat plays a key role and it is thought to help facilitate this process by leading formation of large and mature hdl . furthermore , it is reported that the majority of cholesteryl esters formed by lcat are removed by the liver . without lcat , hdl - c , apoa - i and apoa - ii levels in the plasma are drastically reduced for the lacking formation of mature and spherical shaped hdl and for the rapid catabolism of discoidal hdl by the kidney . on the basis of these evidences , variations in lcat activity seem to be naturally implicated in atherosclerosis prevention or development . to elucidate the role of lcat in atherosclerosis , a large number of studies have been performed in both animal models and humans ( table 1 ) . studies carried out in animal models led to controversial results , often dependent on species utilized . studies performed in mice in which lcat was overexpressed or downregulated suggest that activity of lcat is not associated to atheroprotection and the lack of enzyme is not associated to increased atherosclerosis , even if the hdl - c levels in plasma are very low . the increased atherosclerosis in mice with lcat overexpression is probably due to the accumulation in plasma of dysfunctional large apoe - rich hdl , which were shown to be defective in the delivery of cholesterol to the liver through sr - bi . when the lcat gene was overexpressed in rabbits , opposite results were obtained : aortic lesions were reduced after atherogenic diet , even if large hdl particles containing apoe were detected . the contradictory results obtained in the studies on animal models do not clarify the role of lcat in atherosclerosis , allowing for further consideration . the role of lcat in atherosclerosis was also explored in humans , both in general population and in subjects at high cardiovascular risk . as observed in animal studies , the epic - norfolk was the first prospective study investigating the correlation of lcat plasma levels and atherosclerosis carried out in general population in more than 2,700 subjects . one - third of enrolled subjects developed coronary artery diseases ( cads ) , but no associations between plasma lcat levels and risk to develop future cad was observed . when individuals were divided according to gender , increased lcat levels correlated with lower risk of cad only in men , while in women was the opposite . reduction of lcat concentration / activity associated with absence of cad was described in the copenhagen city heart study , that enrolled more than 10,000 participants , and in the copenhagen general population study , in which more than 50,000 subjects are involved . the variants s208 t found in the coding region of lcat gene was associated with reduction in hdl - c and apoa - i levels , but not with increased risk of myocardial infarction , ischemic heart disease , and ischemic cerebrovascular disease . in agreement with the results obtained in the general population , an observational study carried out in 540 subjects at high cardiovascular risk showed that low plasma lcat levels are not associated with higher carotid intima - media thickness ( imt ) , a marker of preclinical atherosclerosis . consistent with these results , in various studies it was demonstrated that an increased lcat concentration is associated to cad . increased levels of lcat activity was associated with increased imt in 74 subjects with metabolic syndrome , as well as in the control subjects of the study . in another study from the same group , a recent study analyzed the relationship between lcat activity and triglyceride metabolism and ldl particle size in 550 patients at high cardiovascular risk . increased lcat activity was associated with formation of small ldl particles that are more atherogenic than large particles , but no parameters of subclinical atherosclerosis were analyzed . on other side , some studies affirm the opposite : decreased lcat activity is associated with cad . early studies supporting this evidence were carried out in 1973 in subjects at high cardiovascular risk . few years later , in 100 subjects divided according to the degree of atherosclerotic disease , lcat activity was found positively correlated with the severity of coronary atherosclerosis . lower levels of lcat activity were also observed in patients with ischemic heart disease , and in a study on patients with acute myocardial infarction . while epidemiological studies have repeatedly shown a strong and inverse correlation between plasma hdl - c concentrations and the incidence of chd , the significance of such association for chd development has been recently questioned , and clinical trials with various drugs able to increase hdl - c levels did not show the expected benefits . hdl metabolism is regulated by a large number of factors that modify plasma levels of circulating hdl , and plasma hdl - c levels are remarkably susceptible to variations in these factors which also affect hdl shape , size , density , and lipid and apolipoprotein composition , and as a consequence hdl function . investigations of factors involved in hdl metabolism thus represent a good way to understand the relationship between hdl and chd , and will likely translate in the development of innovative therapeutic approaches to chd prevention and treatment specifically affecting hdl function independent of plasma hdl - c levels .

epidemiological data clearly show the existence of a strong inverse correlation between plasma high - density lipoprotein cholesterol ( hdl - c ) concentrations and the incidence of coronary heart disease . this relation is explained by a number of atheroprotective properties of hdl , first of all the ability to promote macrophage cholesterol transport . hdl are highly heterogeneous and are continuously remodeled in plasma thanks to the action of a number of proteins and enzymes . among them , lecithin : cholesterol acyltransferase ( lcat ) plays a crucial role , being the only enzyme able to esterify cholesterol within lipoproteins . lcat is synthetized by the liver and it has been thought to play a major role in reverse cholesterol transport and in atheroprotection . however , data from animal studies , as well as human studies , have shown contradictory results . increased lcat concentrations are associated with increased hdl - c levels but not necessarily with atheroprotection . on the other side , decreased lcat concentration and activity are associated with decreased hdl - c levels but not with increased atherosclerosis . these contradictory results confirm that hdl - c levels per se do not represent the functionality of the hdl system .

INTRODUCTION HDL METABOLISM AND FUNCTION HDL heterogeneity HDL metabolism HDL functions LCAT AND ATHEROSCLEROSIS CONCLUSIONS

a large number of studies has clearly shown the existence of a strong inverse correlation between plasma high - density lipoprotein cholesterol ( hdl - c ) concentrations and the incidence of coronary heart disease ( chd ) , but the significance of such association has been recently questioned . intervention clinical trials carried out with agents efficient in raising hdl - c levels , including niacin and cholesteryl ester transfer protein ( cetp ) inhibitors , have failed in showing a reduction in cardiovascular events . in addition , mendelian randomization studies have shown that increased hdl - c levels caused by common variants in hdl related genes are not necessarily associated with reduced cardiovascular risk . one possible explanation for this discrepancy is that the plasma hdl - c concentration does not reflect the very complex hdl system , involving different hdl particles and a number of receptors , transporters , enzyme , and transfer proteins . moreover , cholesterol is not the active component of hdl and there is convincing evidence that at least some of the atheroprotective functions of hdl relate to specific hdl components or subclasses , which concentration in plasma may be totally unrelated to the hdl - c level . hdl are a highly heterogeneous lipoprotein family composed by several subclasses with different density , shape , and size . the density of the hdl particles is inversely related to their size , reflecting the relative contents of low density non - polar core lipid , and high density surface protein . most part of plasma hdl has a globular shape , the central core is composed by non - polar lipids ( triglycerides and cholesteryl esters ) surrounded by a monolayer of polar lipids ( phospholipids and unesterified cholesterol ) and apolipoproteins . a minor fraction of plasma hdl has a non - spherical structure and they consist in discoidal bilayer of polar lipids , in which non - polar core is lacking ; apolipoproteins run from side to side of the disk , with polar residues facing the aqueous phase and non - polar residues facing the acyl chains of the lipid bilayer . two major particle subclasses have been identified on the basis of major apolipoprotein composition : particles containing only apoa - i ( lpa - i ) , and particles containing both apoa - i and apoa - ii ( lpa - i : a - ii ) . recent shotgun proteomic analysis showed that hdl contain 48 or more proteins , among these apoa - iv , apocs , apoe , lecithin : cholesterol acyltransferase ( lcat ) , cetp , phospholipid transfer protein ( pltp ) , paraoxonase ( pon ) , and platelet - activating factor acetylhydrolase ( paf - ah ) circulate in plasma bound to hdl . most of the proteins carried by hdl are not apolipoproteins , and represent very minor components of these particles . due to its highly dynamic nature apoa - i and apoa - ii are synthe - sized mainly by the liver and , to a lesser extent , by the small intestine and are secreted as components of triglyceride - rich lipoproteins . the regulatory role of pltp is achieved through two main functions , phospholipid transfer activity and the capability to modulate hdl size and composition in a process called hdl conversion . apoa - i is secreted as pro - apoa - i and converted to a mature form by a metalloprotease in plasma . there are three potential sources of lipid - poor apoa - i in plasma : it may be released as lipid - poor protein after its synthesis in the liver and intestine , it may be released from triglyceride - rich lipoproteins that are undergoing lipolysis by lipoprotein lipase , and it may be generated in the circulation during the remodeling of mature , spherical hdl particles . lcat thus plays a central role in intravascular hdl metabolism and in the determination of plasma hdl level . esterification of cholesterol in plasma by lcat is also necessary for cholesterol uptake from the liver , either directly through the scavenger receptor class b member 1 ( sr - bi ) or indirectly through cetp . mature , large -hdl particles can be converted back to pre--hdl through the action of pltp and the endothelial and hepatic lipases , that hydrolyze triglycerides and phospholipids on hdl ( fig . hdl components are catabolized in different ways ; the major sites of catabolism of the protein components are liver and kidney . glomerular filtration barrier prevents access of mature hdl particles to the proximal tubules ; however , cubulin may bind filtered lipid - poor hdl . in the liver , holo - hdl particles accumulated in endosomal compartments can be transferred to lysosomes for degradation or in a small proportion , they can be resecreted into the circulation . one of the most important function of hdl is to promote the removal of cholesterol from peripheral cells , including macrophages within the arterial wall , and shuttle it to the liver for excretion through the bile and feces in a process called reverse cholesterol transport ( rct ) . this exchange can occur by several processes : via aqueous diffusion , which occurs according to the direction of cholesterol gradient , or through three main distinct and protein - mediated pathways . lipid - free / lipid - poor apolipoproteins , mainly apoa - i , represent the principal cholesterol acceptors via abca1 . the cholesterol accumulated in hdl is esterified in plasma by lcat with the resulting formation of cholesteryl esters . then , hydrophobic cholesteryl esters move to the core while unesterified cholesterol is removed from the surface of hdl , leading to the progressive enlargement of these particles . for long time , lcat has been considered necessary for efficient rct by keeping unesterified cholesterol gradient from cells to hdl , but recent data suggest that even if functional lcat is not present , macrophage cholesterol efflux and rct can occur . large amount of the cholesteryl esters formed by the lcat are exchanged with triglycerides through cetp - mediated process into apob containing lipoproteins that are finally catabolized by the liver . alternatively , hdl - cholesteryl esters are taken - up by the liver through sr - bi . atheroprotection mediated by hdl is not only through their major role in rct , but also through other relevant functions ; one of the most important and well studied is the ability of hdl to maintain endothelial cell homeostasis and integrity . hdl have potent antioxidant properties , mediated by molecules carried by hdl ( pon-1 , paf - ah , and lcat ) or by apoa - i and apoa - ii , as well as anti - inflammatory , antithrombotic , cytoprotective , vasodilatory , anti - infectious activities and the capacity to enhance insulin secretion . this wide spectrum of biological activities likely reflects the heterogeneity of hdl particles ; however , the hdl - protective activities can be lost in some pathological conditions and hdl can even acquire proatherogenic properties . hdl are a highly heterogeneous lipoprotein family composed by several subclasses with different density , shape , and size . the density of the hdl particles is inversely related to their size , reflecting the relative contents of low density non - polar core lipid , and high density surface protein . most part of plasma hdl has a globular shape , the central core is composed by non - polar lipids ( triglycerides and cholesteryl esters ) surrounded by a monolayer of polar lipids ( phospholipids and unesterified cholesterol ) and apolipoproteins . a minor fraction of plasma hdl has a non - spherical structure and they consist in discoidal bilayer of polar lipids , in which non - polar core is lacking ; apolipoproteins run from side to side of the disk , with polar residues facing the aqueous phase and non - polar residues facing the acyl chains of the lipid bilayer . the protein component of hdl is formed mainly by apolipoprotein a - i ( apoa - i ) , for the 70% , and apolipoprotein a - ii ( apoa - ii ) , for the 20% . two major particle subclasses have been identified on the basis of major apolipoprotein composition : particles containing only apoa - i ( lpa - i ) , and particles containing both apoa - i and apoa - ii ( lpa - i : a - ii ) . recent shotgun proteomic analysis showed that hdl contain 48 or more proteins , among these apoa - iv , apocs , apoe , lecithin : cholesterol acyltransferase ( lcat ) , cetp , phospholipid transfer protein ( pltp ) , paraoxonase ( pon ) , and platelet - activating factor acetylhydrolase ( paf - ah ) circulate in plasma bound to hdl . most of the proteins carried by hdl are not apolipoproteins , and represent very minor components of these particles . it is also possible to classify hdl on the basis of density ( hdl2 , with density of 1.063 to 1.120 g / ml , and hdl3 , 1.120 to 1.210 according to charge , hdl can be divided into - and pre--migrating particles on agarose gel , and combining charge and size these two subclass - es can be divided into 12 distinct apoa - i - containing particles , referred to as pre- ( pre-1 and pre-2 ) , ( 1 , 2 , and 3 ) and pre- ( pre-1 , pre-2 , and pre-3 ) on the basis of mobility that is slower or faster than albumin , respectively , and decreasing size . due to its highly dynamic nature , apoa - i is involved in major pathways of hdl metabolism . apoa - i and apoa - ii are synthe - sized mainly by the liver and , to a lesser extent , by the small intestine and are secreted as components of triglyceride - rich lipoproteins . the regulatory role of pltp is achieved through two main functions , phospholipid transfer activity and the capability to modulate hdl size and composition in a process called hdl conversion . hepatocytes are able to secrete apoa - i in lipid - free or lipid - poor and lipidated forms . apoa - i is secreted as pro - apoa - i and converted to a mature form by a metalloprotease in plasma . there are three potential sources of lipid - poor apoa - i in plasma : it may be released as lipid - poor protein after its synthesis in the liver and intestine , it may be released from triglyceride - rich lipoproteins that are undergoing lipolysis by lipoprotein lipase , and it may be generated in the circulation during the remodeling of mature , spherical hdl particles . the cholesterol esters generated by lcat are more hydrophobic than free cholesterol and thus migrate into the hydrophobic core of the lipoprotein , with the resulting conversion of small , discoidal pre--hdl into mature , spherical , -migrating hdl ( -hdl ) . lcat thus plays a central role in intravascular hdl metabolism and in the determination of plasma hdl level . esterification of cholesterol in plasma by lcat is also necessary for cholesterol uptake from the liver , either directly through the scavenger receptor class b member 1 ( sr - bi ) or indirectly through cetp . mature , large -hdl particles can be converted back to pre--hdl through the action of pltp and the endothelial and hepatic lipases , that hydrolyze triglycerides and phospholipids on hdl ( fig . hdl components are catabolized in different ways ; the major sites of catabolism of the protein components are liver and kidney . glomerular filtration barrier prevents access of mature hdl particles to the proximal tubules ; however , cubulin may bind filtered lipid - poor hdl . in the liver , holo - hdl particles accumulated in endosomal compartments can be transferred to lysosomes for degradation or in a small proportion , they can be resecreted into the circulation . one of the most important function of hdl is to promote the removal of cholesterol from peripheral cells , including macrophages within the arterial wall , and shuttle it to the liver for excretion through the bile and feces in a process called reverse cholesterol transport ( rct ) . this exchange can occur by several processes : via aqueous diffusion , which occurs according to the direction of cholesterol gradient , or through three main distinct and protein - mediated pathways . lipid - free / lipid - poor apolipoproteins , mainly apoa - i , represent the principal cholesterol acceptors via abca1 . the cholesterol accumulated in hdl is esterified in plasma by lcat with the resulting formation of cholesteryl esters . then , hydrophobic cholesteryl esters move to the core while unesterified cholesterol is removed from the surface of hdl , leading to the progressive enlargement of these particles . for long time , lcat has been considered necessary for efficient rct by keeping unesterified cholesterol gradient from cells to hdl , but recent data suggest that even if functional lcat is not present , macrophage cholesterol efflux and rct can occur . large amount of the cholesteryl esters formed by the lcat are exchanged with triglycerides through cetp - mediated process into apob containing lipoproteins that are finally catabolized by the liver . alternatively , hdl - cholesteryl esters are taken - up by the liver through sr - bi . atheroprotection mediated by hdl is not only through their major role in rct , but also through other relevant functions ; one of the most important and well studied is the ability of hdl to maintain endothelial cell homeostasis and integrity . hdl have potent antioxidant properties , mediated by molecules carried by hdl ( pon-1 , paf - ah , and lcat ) or by apoa - i and apoa - ii , as well as anti - inflammatory , antithrombotic , cytoprotective , vasodilatory , anti - infectious activities and the capacity to enhance insulin secretion . this wide spectrum of biological activities likely reflects the heterogeneity of hdl particles ; however , the hdl - protective activities can be lost in some pathological conditions and hdl can even acquire proatherogenic properties . lcat is principally synthetized in the liver and in little amount in other tissues , such as brain and testes , and circulates in plasma compartment at concentration of 5 mg / l mainly bound to hdl but also to ldl . lcat converts phosphatidylcholine and cholesterol into cholesteryl ester and lysophosphatidylcholine in plasma and other biological fluids . in rct pathway , lcat plays a key role and it is thought to help facilitate this process by leading formation of large and mature hdl . furthermore , it is reported that the majority of cholesteryl esters formed by lcat are removed by the liver . without lcat , hdl - c , apoa - i and apoa - ii levels in the plasma are drastically reduced for the lacking formation of mature and spherical shaped hdl and for the rapid catabolism of discoidal hdl by the kidney . on the basis of these evidences , variations in lcat activity seem to be naturally implicated in atherosclerosis prevention or development . to elucidate the role of lcat in atherosclerosis , a large number of studies have been performed in both animal models and humans ( table 1 ) . studies performed in mice in which lcat was overexpressed or downregulated suggest that activity of lcat is not associated to atheroprotection and the lack of enzyme is not associated to increased atherosclerosis , even if the hdl - c levels in plasma are very low . the increased atherosclerosis in mice with lcat overexpression is probably due to the accumulation in plasma of dysfunctional large apoe - rich hdl , which were shown to be defective in the delivery of cholesterol to the liver through sr - bi . the contradictory results obtained in the studies on animal models do not clarify the role of lcat in atherosclerosis , allowing for further consideration . the role of lcat in atherosclerosis was also explored in humans , both in general population and in subjects at high cardiovascular risk . as observed in animal studies , the epic - norfolk was the first prospective study investigating the correlation of lcat plasma levels and atherosclerosis carried out in general population in more than 2,700 subjects . one - third of enrolled subjects developed coronary artery diseases ( cads ) , but no associations between plasma lcat levels and risk to develop future cad was observed . when individuals were divided according to gender , increased lcat levels correlated with lower risk of cad only in men , while in women was the opposite . reduction of lcat concentration / activity associated with absence of cad was described in the copenhagen city heart study , that enrolled more than 10,000 participants , and in the copenhagen general population study , in which more than 50,000 subjects are involved . the variants s208 t found in the coding region of lcat gene was associated with reduction in hdl - c and apoa - i levels , but not with increased risk of myocardial infarction , ischemic heart disease , and ischemic cerebrovascular disease . in agreement with the results obtained in the general population , an observational study carried out in 540 subjects at high cardiovascular risk showed that low plasma lcat levels are not associated with higher carotid intima - media thickness ( imt ) , a marker of preclinical atherosclerosis . consistent with these results , in various studies it was demonstrated that an increased lcat concentration is associated to cad . increased levels of lcat activity was associated with increased imt in 74 subjects with metabolic syndrome , as well as in the control subjects of the study . increased lcat activity was associated with formation of small ldl particles that are more atherogenic than large particles , but no parameters of subclinical atherosclerosis were analyzed . on other side , some studies affirm the opposite : decreased lcat activity is associated with cad . few years later , in 100 subjects divided according to the degree of atherosclerotic disease , lcat activity was found positively correlated with the severity of coronary atherosclerosis . lower levels of lcat activity were also observed in patients with ischemic heart disease , and in a study on patients with acute myocardial infarction . while epidemiological studies have repeatedly shown a strong and inverse correlation between plasma hdl - c concentrations and the incidence of chd , the significance of such association for chd development has been recently questioned , and clinical trials with various drugs able to increase hdl - c levels did not show the expected benefits . hdl metabolism is regulated by a large number of factors that modify plasma levels of circulating hdl , and plasma hdl - c levels are remarkably susceptible to variations in these factors which also affect hdl shape , size , density , and lipid and apolipoprotein composition , and as a consequence hdl function . investigations of factors involved in hdl metabolism thus represent a good way to understand the relationship between hdl and chd , and will likely translate in the development of innovative therapeutic approaches to chd prevention and treatment specifically affecting hdl function independent of plasma hdl - c levels .

enteral nutrition has been used to maintain or return nutritional health in ill patients or chronic illnesses for many years . malnutrition is a common problem which many specialists may encounter in hospitalized patients especially in those with high metabolic needs . nowadays , timely provision of enteral nutrition is a therapeutic method to attenuate disease severity , adjust immune response , reduce complications and would have a favorable effect on therapeutic results of ill patients . tube feeding is used for patients who have an efficient digestive system , but are not able to receive food orally , or receive less food than they need for maintaining vital body functions . in patients who use this feeding method , two factors of nutrient contents and microbiological safety are very important and have been investigated in many studies . all types of enteral feeding formulations , whether handmade at hospital kitchen , or made by dilution of ready - to - use formulas , contain various amounts of proteins , carbohydrates and lipids in different combinations . the nature of these foods ( in terms of ph , nutrient contents , water activity , etc . ) is so that if they become contaminated , they would immediately grow microorganisms inside and put the patient at the risk of infection . in addition , microbial contamination of these foods slows the trend of patient 's recovery and can even cause dangerous conditions like pneumonia , nosocomial infections and sepsis . moreover , microbial infection can impact nutritional value of food by microbial spoilage . regarding the amount of acceptable bacterial contamination in different food products , american food and drug administration ( fda ) established standards for different aspects of these foods in 2006 , especially for health and nutritional quality . health quality means observing microbiological indexes in food . on the basis of this guideline , in addition to the elimination of pathogen microorganisms , these foods must meet certain standards , which are determined by evaluating indicator microorganisms . since the presence of certain pathogen microorganisms in food may cause severe illnesses , foods should be studied regarding the presence of such microorganisms . fda guideline has mandated to assess the foods in terms of total colony count of aerobic microorganisms , count of coliforms , escherichia coli , bacillus cereus , detecting the salmonella spp . and listeria monocytogenes and determining staphylococcal enterotoxins . according to this guideline , food products that have one of the following conditions are considered below the standards of microbial safety and inappropriate for consumption : contamination with aerobic microorganisms > 10 cfu / g in one sample , contamination of over 10 cfu / g in 3 or more samples , coliform count over 3 organisms / g , or positive for l. monocytogenes or salmonella spp . similar standards have been devised in several other countries . according to regulations in spain , unfortunately , no microbial standards have been devised for such foods in islamic republic of iran . one way is to mix the kitchen food at the hospital and the other way is to prepare the ready - to - use formula ( usually in powder form ) by diluting it with water . ready - to - use formulas have been used for over 20 years , but most hospitals prefer to make feeding solutions by mixing nutrients in the kitchen , due to economic reasons or cultural considerations . several studies have examined and compared ready - to - use formulas with handmade foods in terms of bacterial contamination . the results of all such studies confirm the fact that commercial ready - to - use formulas are considerably less contaminated than handmade formulas . in many studies , commercial formulas were completely sterile . in 2006 , a study was conducted on handmade tube feeding formulas in two educational hospitals in isfahan to assess them in terms of bacterial contamination . the results showed that bacterial contamination of these foods was much higher than fda standards . they only examined handmade formulas and finally recommended using commercial ready - to - use formulas in order to reduce the probability of contamination . in recent years , there have been a lot of studies on microbial contamination of tube feeding formulas and its contributive factors in different countries including saudi arabia , and the philippines . however , despite the grave need , such studies are rare in iran . with regard to the increasing trend of using commercial ready - to - use formulas for patients and that handmade solutions are still used in some hospitals , this study aimed to investigate the bacterial contamination of handmade and commercial ready - to - use formulas used for patients in intensive care units ( icu ) . the amount and kind of bacterial contamination was measured and then handmade and ready - to - use formulas were compared with this regard . in this study , qualitative studies were conducted to determine l. monocytogenes and salmonella spp . which were emphasized by fda standards . in this experimental study , which was done from september 2010 to january 2011 , seventy random samples of enteral feeding solutions made in one the university hospitals in isfahan , iran were studied in terms of the amount and kind of defined microorganisms . in this hospital , feeding formulas were made in two ways : handmade ( made from available nutrients in the kitchen of the hospital ) , or ready - to - use ( made by diluting commercial powders ) . commercial formulas were prepared several times , each 30 min before patient 's feeding time in the kitchen of the hospital under the supervision of the nutrition department head . the necessary amount of powder needed for 200 ml of feeding formula was added to the cool boiled water , poured in disposable glasses , capped and taken to the wards . the ingredients of handmade foods were prepared once a day ( in the morning ) by mixing different food items like dry milk , green beans , carrot , orange juice , chicken , etc . , and keeping in refrigerator . so , all the needed food volume for 1 day was made in the morning and used until the next day . for each feeding course , a part of the prepared solution was warmed to about 45c , poured in disposable dishes , capped and taken to the wards . it is noteworthy that all feeding solutions were taken to the wards at certain times and sometimes patients were not ready to take their food . the containers of the feeding solutions were kept out of the refrigerator and at room temperature at all this time elapse . samples were collected from two icus in this hospital from two types of feeds ; handmade and ready - to - use formulas . the second sampling was done 18 h after the first time for handmade solutions . at this time , sample was taken from the same solution that was prepared in the kitchen in the morning and kept in the refrigerator . for ready - to - use formulas , sampling was done immediately after the powder was mixed with water and ready for consumption . in each sampling time , 60 ml of the feeding solution was taken to sterile disposable dishes under aseptic conditions . then samples were placed in ice containers and taken to the microbiology lab in 30 min . of the 70 samples , 42 ( 21 samples in two sampling times ) were made manually in the kitchen and 28 were made from diluting commercial formulas ( mostly ensure ) . all stages of this study were conducted to assess the actual condition of the hospital without any changes in the usual settings . immediately after samples were taken to the microbiology laboratory , defined tests were conducted on them . these tests were divided into two general categories for each sample : quantitative tests , which include determining the counts of indicator microorganisms . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . quantitative tests , which include determining the counts of indicator microorganisms . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . in order to perform quantitative tests , different dilutions were prepared using serial dilution method in sterile normal saline 0.9% . then appropriate dilutions were cultured and the microorganisms colonies total colony count of viable aerobic microorganisms was measured based on standard number 356 of iranian standard and industrial research organization ( isiro ) using nutrient agar culture medium . cfu of coliforms was counted based on standard number 437 of isiro using culture medium violet red bile agar . colony count of s. aureus was performed based on standard number 1194 of isiro using baird - parker agar . qualitative tests include detection of specific microorganisms in samples such as l. monocytogenes and salmonella spp . qualitative tests include detection of specific microorganisms in samples such as l. monocytogenes and salmonella spp . detection of salmonella spp . was performed based on standard number 1810 of isiro using buffered peptone water enriching media and slenite cystine broth culture medium to boost its growth two levels of enrichment in culture media of listeria enrichment broth ( uvm - i ) and fraser broth ( uvm - ii ) were used , which yields suitable growth of listeria . then , oxford agar culture medium , which is selective for l. monocytogenes , was used . all stages of the tests were performed immediately after samples reached the laboratory , under aseptic conditions and under laminar airflow hood . standard microbial species were also used to verify the accuracy of microbiological tests . for quantitative tests , any microbial growth was considered contamination . because of limitations of serial dilution technique , it is not possible to show cfu less than 1 log cfu / g . therefore , if there was fewer than 10 colonies on each plate , the result was reported as < 10 . finally , statistical studies were conducted using spss for windows ( spss , chicago , il , usa ) version 16.0 , to compare the amount of contamination of the first and second handmade samples after calculating the log ( cfu / g ) of each sample using wilcoxon signed ranks test . this comparison was done to find the difference of contamination between the two samples regarding passage of time and quality of storing feeding solutions . furthermore , mann whitney test was applied to compare the contamination results between first - sampling handmade samples and commercial ready - to - use samples . in this hospital , feeding formulas were made in two ways : handmade ( made from available nutrients in the kitchen of the hospital ) , or ready - to - use ( made by diluting commercial powders ) . commercial formulas were prepared several times , each 30 min before patient 's feeding time in the kitchen of the hospital under the supervision of the nutrition department head . the necessary amount of powder needed for 200 ml of feeding formula was added to the cool boiled water , poured in disposable glasses , capped and taken to the wards . the ingredients of handmade foods were prepared once a day ( in the morning ) by mixing different food items like dry milk , green beans , carrot , orange juice , chicken , etc . , and keeping in refrigerator . so , all the needed food volume for 1 day was made in the morning and used until the next day . for each feeding course , a part of the prepared solution was warmed to about 45c , poured in disposable dishes , capped and taken to the wards . it is noteworthy that all feeding solutions were taken to the wards at certain times and sometimes patients were not ready to take their food . the containers of the feeding solutions were kept out of the refrigerator and at room temperature at all this time elapse . samples were collected from two icus in this hospital from two types of feeds ; handmade and ready - to - use formulas . the second sampling was done 18 h after the first time for handmade solutions . at this time , sample was taken from the same solution that was prepared in the kitchen in the morning and kept in the refrigerator . for ready - to - use formulas , sampling was done immediately after the powder was mixed with water and ready for consumption . in each sampling time , 60 ml of the feeding solution was taken to sterile disposable dishes under aseptic conditions . then samples were placed in ice containers and taken to the microbiology lab in 30 min . of the 70 samples , 42 ( 21 samples in two sampling times ) were made manually in the kitchen and 28 were made from diluting commercial formulas ( mostly ensure ) . all stages of this study were conducted to assess the actual condition of the hospital without any changes in the usual settings . immediately after samples were taken to the microbiology laboratory , defined tests were conducted on them . these tests were divided into two general categories for each sample : quantitative tests , which include determining the counts of indicator microorganisms . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . quantitative tests , which include determining the counts of indicator microorganisms . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . in order to perform quantitative tests , different dilutions were prepared using serial dilution method in sterile normal saline 0.9% . then appropriate dilutions were cultured and the microorganisms colonies were counted using pour plate method . total colony count of viable aerobic microorganisms was measured based on standard number 356 of iranian standard and industrial research organization ( isiro ) using nutrient agar culture medium . cfu of coliforms was counted based on standard number 437 of isiro using culture medium violet red bile agar . colony count of s. aureus was performed based on standard number 1194 of isiro using baird - parker agar . qualitative tests include detection of specific microorganisms in samples such as l. monocytogenes and salmonella spp . qualitative tests include detection of specific microorganisms in samples such as l. monocytogenes and salmonella spp . detection of salmonella spp . was performed based on standard number 1810 of isiro using buffered peptone water enriching media and slenite cystine broth culture medium to boost its growth two levels of enrichment in culture media of listeria enrichment broth ( uvm - i ) and fraser broth ( uvm - ii ) were used , which yields suitable growth of listeria . then , oxford agar culture medium , which is selective for l. monocytogenes , was used . all stages of the tests were performed immediately after samples reached the laboratory , under aseptic conditions and under laminar airflow hood . for quantitative tests , colony counting was done manually and the result was reported as cfu / g . any microbial growth was considered contamination . because of limitations of serial dilution technique , it is not possible to show cfu less than 1 log cfu / g . therefore , if there was fewer than 10 colonies on each plate , the result was reported as < 10 . finally , statistical studies were conducted using spss for windows ( spss , chicago , il , usa ) version 16.0 , to compare the amount of contamination of the first and second handmade samples after calculating the log ( cfu / g ) of each sample using wilcoxon signed ranks test . this comparison was done to find the difference of contamination between the two samples regarding passage of time and quality of storing feeding solutions . furthermore , mann whitney test was applied to compare the contamination results between first - sampling handmade samples and commercial ready - to - use samples . the results of microbiological tests showing the amount and kind of bacterial contamination of feeding solutions are demonstrated in table 1 . the comparison of bacterial contamination of first - time handmade samples and ready - to - use samples are shown in table 2 . considering the extensive and non - normal distribution of data , parametric statistical tests could not be used to compare the contamination of samples . therefore , non - parametric tests of mann - whitney and wilcoxon signed ranks were used . contamination of handmade samples in two sampling times comparison of the contamination of the first handmade samples with ready - to - use samples regarding that any positive bacterial growth was considered contamination , in the first - time sampling , of 21 samples , 11 ( 52% ) had coliform contamination , 5 ( 24% ) had s. aureus contamination and 16 ( 76% ) had total viable counts greater than 10 cfu / g ; whereas in the second - time sampling , of 21 samples , 16 ( 76% ) had coliform contamination , 13 ( 62% ) had s. aureus contamination and 17 ( 81% ) had total viable counts greater than 10 cfu / g . wilcoxon test was used to compare the contamination of the first- and the second - time sampling after calculating log ( cfu / g ) . as can be seen in table 1 , the difference between total viable contamination of these two groups of samples was significant ( p = 0.004 ) . for these two sampling times , maximum coliform contamination was 1.7 10 and 5.0 10 cfu / g , respectively . the results for s. aureus were 2.3 10 and 4.0 10 cfu / g , respectively . therefore , 18 h of keeping the feeds had increased coliform contamination about 1.5 logs and s. aureus contamination about 2 logs . the increased contamination after this time was not significant for coliforms ( p = 0.085 ) , but was significant for s. aureus ( p = 0.008 ) . the range of contamination of ready - to - use samples for total viable count , coliforms and s. aureus are shown in table 2 . out of 28 commercial formulas , 27 samples ( 96% ) had total viable counts greater than 10 cfu / g . also , 24 samples ( 86% ) and 27 samples ( 96% ) were contaminated with s. aureus and coliforms , respectively . in order to compare ready - to - use and handmade samples , mann - whitney test was used . as can be seen in table 2 , contamination of ready - to - use formulas in all three microbiological samples regarding that any positive bacterial growth was considered contamination , in the first - time sampling , of 21 samples , 11 ( 52% ) had coliform contamination , 5 ( 24% ) had s. aureus contamination and 16 ( 76% ) had total viable counts greater than 10 cfu / g ; whereas in the second - time sampling , of 21 samples , 16 ( 76% ) had coliform contamination , 13 ( 62% ) had s. aureus contamination and 17 ( 81% ) had total viable counts greater than 10 cfu / g . wilcoxon test was used to compare the contamination of the first- and the second - time sampling after calculating log ( cfu / g ) . as can be seen in table 1 , the difference between total viable contamination of these two groups of samples was significant ( p = 0.004 ) . for these two sampling times , maximum coliform contamination was 1.7 10 and 5.0 10 cfu / g , respectively . the results for s. aureus were 2.3 10 and 4.0 10 cfu / g , respectively . therefore , 18 h of keeping the feeds had increased coliform contamination about 1.5 logs and s. aureus contamination about 2 logs . the increased contamination after this time was not significant for coliforms ( p = 0.085 ) , but was significant for s. aureus ( p = 0.008 ) . the range of contamination of ready - to - use samples for total viable count , coliforms and s. aureus are shown in table 2 . out of 28 commercial formulas , 27 samples ( 96% ) had total viable counts greater than 10 cfu / g . also , 24 samples ( 86% ) and 27 samples ( 96% ) were contaminated with s. aureus and coliforms , respectively . in order to compare ready - to - use and handmade samples , mann - whitney test was used . as can be seen in table 2 , contamination of ready - to - use formulas in all three microbiological samples in the present study , 96.4% of the ready - to - use formulas and 78.6% of handmade formulas had total aerobic microorganism count of over 10 cfu / g . furthermore , coliforms were over 3 organisms / g in 57.1% and 17.8% of handmade and ready - to - use samples , respectively , which means they cross the standard limit ( the details are not mentioned in the result section ) . this high rate of contamination shows lack of observing health standards in different stages of preparation and transportation of both types of feeding solutions . bastow et al . compared handmade and ready - to - use foods in terms of bacterial contamination . the contamination of all handmade samples immediately after preparation was on average 10 -10 organisms / ml while none of the commercial samples were contaminated . in two other studies by muytjens et al . and simmons et al . , relevant researches have shown that contamination of ready - to - use formulas are directly related to the stages of preparation . bacterial contamination of handmade formulations could be attributed to several sources including original food items , food - making devices , blenders , environmental contamination of the kitchen regarding hygiene of the floor and air conditioner , process of food preparation , not observing the hygienic principles by kitchen staff and nurses and process of food carriage to the wards . comparison of the results of the handmade samples of both sampling times shows the significant increase of contamination in the second time . this increase indicates the sub - standard conditions of keeping feeding solutions during this period . the conditions have provided suitable medium for growth and proliferation of bacteria due to the temperature , long time of storage and environmental contamination . with regard to powder samples , there was not any data indicating their sterility neither on the packaging nor on the package inserts of these products ; contacting the manufacturing companies of these ready - to - use formulas , we realized that these products are not produced under the sterile condition , so their microorganism count could not be zero ; however , high contamination shows lack of hygiene in manufacturing companies . international organizations have established standards to control the contamination of nutritional solutions , however , inadequate observation of personal hygiene and the process of food preparation is one of the main reasons for contamination , which indicates the break between theoretical and practical standards . although ready - to - use formulas are prepared 30 min before each feeding time , total viable count of microorganisms , coliforms and s. aureus count were all higher than those in handmade formulas . it can be attributed to lack of hygiene during preparation time as well as presence the same source of contamination for both types of formulas . moreover , contamination of ready - to - use formulas can be caused by lack of hygiene in the production line of the manufacturer to some extent . in a similar study by jalali et al . they found contamination of most samples to be over the standard limits for total viable count , coliforms and s. aureus . they recommended using ready - to - use formulas instead of handmade formulas to reduce contamination . however , the present study found contamination in both handmade and ready - to - use formulas . these results call for designing comprehensive and documented guidelines for preparation , storage and transportation of these products in iran . such regulations will clarify the conditions for the involved personnel ( cooking staff , nurses and workers carrying food ) and also the basic rules of periodical monitoring of places that such foods are prepared or stored . moreover , considering the fact that ready - to - use samples used in the present study were contaminated at the time of preparation and transportation , it seems that closed system ready - to - use formulas that do not need further process for preparation and can be used at patient 's bed can reduce bacterial contamination . several studies including the present one have shown that even the commercial formulations can be contaminated at the time of opening the can , diluting the powder formulation or pouring the formula into the patient 's dish . furthermore , any steps of the processing of handmade formulas could be considered as the cause of contamination . this introduces the subject of future studies in order to determine the sources of these formulas microbial overload . moreover , the nature of these foods is so that they grow other somewhat pathogen microorganisms inside including anaerobes , molds , or yeasts . this also warrants more comprehensive studies to evaluate the probability of these kinds of contamination . the results of present study indicate that the microbial safety of the majority of enteral feeding solutions in this hospital is lower than standard values published in relevant guidelines . the presented data demonstrate that the development of protocols for clean techniques in the preparation , handling and storage of both commercial and handmade enteral feeds is necessary .

background : this study aimed to investigate and compare the bacterial safety of handmade and commercial ready - to - use enteral feeding formulas used in an iranian teaching hospital.methods:in this experimental study , a total number of 70 samples ( 21 handmade formulas sampled at two sampling times , i.e. the time of preparation and 18 h after preparation , and 28 commercial ready - to - use formulas ) were studied . total count of viable microorganisms , coliform count and staphylococcus aureus count for all samples were conducted.results:out of 42 handmade samples , 16 samples ( 76% ) had total viable counts greater than 103 cfu / g in the first sampling time and 17 samples ( 81% ) had total viable counts greater than 103 cfu / g in the second sampling time . also , 11 ( 52% ) had coliform contamination in the first sampling time which reached 76% ( 16 samples ) in the second sampling time . regarding contamination with s. aureus , 5 samples ( 24% ) were contaminated in the first- and 13 samples ( 62% ) were contaminated in the second - sampling time . out of 28 commercial formulas , 27 samples ( 96% ) had total viable counts greater than 103 cfu / g . also , 24 samples ( 86% ) were contaminated with s. aureus and 27 samples ( 96% ) were contaminated with coliforms . in order to compare these two formulas , the results of mann - whitney test showed that contamination of ready - to - use formulas in all three microbiological samples was significantly more than that for handmade samples.conclusions:the results of the present study indicate that the microbial safety of enteral feeding solutions in this hospital is much lower than standard values , demonstrating that the development of protocols for clean techniques in the preparation , handling and storage of both commercial and handmade enteral feeds is necessary .

INTRODUCTION METHODS Preparation, storage and administration of feeding solutions Data collection Laboratory examinations of the samples Data analysis RESULTS Handmade samples Commercial ready-to-use samples DISCUSSION CONCLUSIONS

nowadays , timely provision of enteral nutrition is a therapeutic method to attenuate disease severity , adjust immune response , reduce complications and would have a favorable effect on therapeutic results of ill patients . all types of enteral feeding formulations , whether handmade at hospital kitchen , or made by dilution of ready - to - use formulas , contain various amounts of proteins , carbohydrates and lipids in different combinations . in addition , microbial contamination of these foods slows the trend of patient 's recovery and can even cause dangerous conditions like pneumonia , nosocomial infections and sepsis . regarding the amount of acceptable bacterial contamination in different food products , american food and drug administration ( fda ) established standards for different aspects of these foods in 2006 , especially for health and nutritional quality . on the basis of this guideline , in addition to the elimination of pathogen microorganisms , these foods must meet certain standards , which are determined by evaluating indicator microorganisms . fda guideline has mandated to assess the foods in terms of total colony count of aerobic microorganisms , count of coliforms , escherichia coli , bacillus cereus , detecting the salmonella spp . according to this guideline , food products that have one of the following conditions are considered below the standards of microbial safety and inappropriate for consumption : contamination with aerobic microorganisms > 10 cfu / g in one sample , contamination of over 10 cfu / g in 3 or more samples , coliform count over 3 organisms / g , or positive for l. monocytogenes or salmonella spp . one way is to mix the kitchen food at the hospital and the other way is to prepare the ready - to - use formula ( usually in powder form ) by diluting it with water . ready - to - use formulas have been used for over 20 years , but most hospitals prefer to make feeding solutions by mixing nutrients in the kitchen , due to economic reasons or cultural considerations . several studies have examined and compared ready - to - use formulas with handmade foods in terms of bacterial contamination . the results of all such studies confirm the fact that commercial ready - to - use formulas are considerably less contaminated than handmade formulas . in many studies , commercial formulas were completely sterile . in 2006 , a study was conducted on handmade tube feeding formulas in two educational hospitals in isfahan to assess them in terms of bacterial contamination . the results showed that bacterial contamination of these foods was much higher than fda standards . they only examined handmade formulas and finally recommended using commercial ready - to - use formulas in order to reduce the probability of contamination . in recent years , there have been a lot of studies on microbial contamination of tube feeding formulas and its contributive factors in different countries including saudi arabia , and the philippines . with regard to the increasing trend of using commercial ready - to - use formulas for patients and that handmade solutions are still used in some hospitals , this study aimed to investigate the bacterial contamination of handmade and commercial ready - to - use formulas used for patients in intensive care units ( icu ) . the amount and kind of bacterial contamination was measured and then handmade and ready - to - use formulas were compared with this regard . in this study , qualitative studies were conducted to determine l. monocytogenes and salmonella spp . in this experimental study , which was done from september 2010 to january 2011 , seventy random samples of enteral feeding solutions made in one the university hospitals in isfahan , iran were studied in terms of the amount and kind of defined microorganisms . in this hospital , feeding formulas were made in two ways : handmade ( made from available nutrients in the kitchen of the hospital ) , or ready - to - use ( made by diluting commercial powders ) . commercial formulas were prepared several times , each 30 min before patient 's feeding time in the kitchen of the hospital under the supervision of the nutrition department head . the ingredients of handmade foods were prepared once a day ( in the morning ) by mixing different food items like dry milk , green beans , carrot , orange juice , chicken , etc . , and keeping in refrigerator . so , all the needed food volume for 1 day was made in the morning and used until the next day . for each feeding course , a part of the prepared solution was warmed to about 45c , poured in disposable dishes , capped and taken to the wards . it is noteworthy that all feeding solutions were taken to the wards at certain times and sometimes patients were not ready to take their food . the containers of the feeding solutions were kept out of the refrigerator and at room temperature at all this time elapse . samples were collected from two icus in this hospital from two types of feeds ; handmade and ready - to - use formulas . the second sampling was done 18 h after the first time for handmade solutions . at this time , sample was taken from the same solution that was prepared in the kitchen in the morning and kept in the refrigerator . for ready - to - use formulas , sampling was done immediately after the powder was mixed with water and ready for consumption . in each sampling time , 60 ml of the feeding solution was taken to sterile disposable dishes under aseptic conditions . then samples were placed in ice containers and taken to the microbiology lab in 30 min . of the 70 samples , 42 ( 21 samples in two sampling times ) were made manually in the kitchen and 28 were made from diluting commercial formulas ( mostly ensure ) . all stages of this study were conducted to assess the actual condition of the hospital without any changes in the usual settings . immediately after samples were taken to the microbiology laboratory , defined tests were conducted on them . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . in order to perform quantitative tests , different dilutions were prepared using serial dilution method in sterile normal saline 0.9% . then appropriate dilutions were cultured and the microorganisms colonies total colony count of viable aerobic microorganisms was measured based on standard number 356 of iranian standard and industrial research organization ( isiro ) using nutrient agar culture medium . colony count of s. aureus was performed based on standard number 1194 of isiro using baird - parker agar . all stages of the tests were performed immediately after samples reached the laboratory , under aseptic conditions and under laminar airflow hood . because of limitations of serial dilution technique , it is not possible to show cfu less than 1 log cfu / g . therefore , if there was fewer than 10 colonies on each plate , the result was reported as < 10 . finally , statistical studies were conducted using spss for windows ( spss , chicago , il , usa ) version 16.0 , to compare the amount of contamination of the first and second handmade samples after calculating the log ( cfu / g ) of each sample using wilcoxon signed ranks test . this comparison was done to find the difference of contamination between the two samples regarding passage of time and quality of storing feeding solutions . furthermore , mann whitney test was applied to compare the contamination results between first - sampling handmade samples and commercial ready - to - use samples . in this hospital , feeding formulas were made in two ways : handmade ( made from available nutrients in the kitchen of the hospital ) , or ready - to - use ( made by diluting commercial powders ) . commercial formulas were prepared several times , each 30 min before patient 's feeding time in the kitchen of the hospital under the supervision of the nutrition department head . the ingredients of handmade foods were prepared once a day ( in the morning ) by mixing different food items like dry milk , green beans , carrot , orange juice , chicken , etc . , and keeping in refrigerator . so , all the needed food volume for 1 day was made in the morning and used until the next day . for each feeding course , a part of the prepared solution was warmed to about 45c , poured in disposable dishes , capped and taken to the wards . it is noteworthy that all feeding solutions were taken to the wards at certain times and sometimes patients were not ready to take their food . the containers of the feeding solutions were kept out of the refrigerator and at room temperature at all this time elapse . samples were collected from two icus in this hospital from two types of feeds ; handmade and ready - to - use formulas . the second sampling was done 18 h after the first time for handmade solutions . at this time , sample was taken from the same solution that was prepared in the kitchen in the morning and kept in the refrigerator . for ready - to - use formulas , sampling was done immediately after the powder was mixed with water and ready for consumption . in each sampling time , 60 ml of the feeding solution was taken to sterile disposable dishes under aseptic conditions . then samples were placed in ice containers and taken to the microbiology lab in 30 min . of the 70 samples , 42 ( 21 samples in two sampling times ) were made manually in the kitchen and 28 were made from diluting commercial formulas ( mostly ensure ) . all stages of this study were conducted to assess the actual condition of the hospital without any changes in the usual settings . immediately after samples were taken to the microbiology laboratory , defined tests were conducted on them . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . to do so , total colony count of viable aerobic microorganisms , count of coagulase positive s. aureus and coliforms were performed . in order to perform quantitative tests , different dilutions were prepared using serial dilution method in sterile normal saline 0.9% . total colony count of viable aerobic microorganisms was measured based on standard number 356 of iranian standard and industrial research organization ( isiro ) using nutrient agar culture medium . colony count of s. aureus was performed based on standard number 1194 of isiro using baird - parker agar . was performed based on standard number 1810 of isiro using buffered peptone water enriching media and slenite cystine broth culture medium to boost its growth two levels of enrichment in culture media of listeria enrichment broth ( uvm - i ) and fraser broth ( uvm - ii ) were used , which yields suitable growth of listeria . for quantitative tests , colony counting was done manually and the result was reported as cfu / g . because of limitations of serial dilution technique , it is not possible to show cfu less than 1 log cfu / g . finally , statistical studies were conducted using spss for windows ( spss , chicago , il , usa ) version 16.0 , to compare the amount of contamination of the first and second handmade samples after calculating the log ( cfu / g ) of each sample using wilcoxon signed ranks test . this comparison was done to find the difference of contamination between the two samples regarding passage of time and quality of storing feeding solutions . furthermore , mann whitney test was applied to compare the contamination results between first - sampling handmade samples and commercial ready - to - use samples . the results of microbiological tests showing the amount and kind of bacterial contamination of feeding solutions are demonstrated in table 1 . the comparison of bacterial contamination of first - time handmade samples and ready - to - use samples are shown in table 2 . considering the extensive and non - normal distribution of data , parametric statistical tests could not be used to compare the contamination of samples . therefore , non - parametric tests of mann - whitney and wilcoxon signed ranks were used . contamination of handmade samples in two sampling times comparison of the contamination of the first handmade samples with ready - to - use samples regarding that any positive bacterial growth was considered contamination , in the first - time sampling , of 21 samples , 11 ( 52% ) had coliform contamination , 5 ( 24% ) had s. aureus contamination and 16 ( 76% ) had total viable counts greater than 10 cfu / g ; whereas in the second - time sampling , of 21 samples , 16 ( 76% ) had coliform contamination , 13 ( 62% ) had s. aureus contamination and 17 ( 81% ) had total viable counts greater than 10 cfu / g . wilcoxon test was used to compare the contamination of the first- and the second - time sampling after calculating log ( cfu / g ) . as can be seen in table 1 , the difference between total viable contamination of these two groups of samples was significant ( p = 0.004 ) . for these two sampling times , maximum coliform contamination was 1.7 10 and 5.0 10 cfu / g , respectively . the results for s. aureus were 2.3 10 and 4.0 10 cfu / g , respectively . therefore , 18 h of keeping the feeds had increased coliform contamination about 1.5 logs and s. aureus contamination about 2 logs . the increased contamination after this time was not significant for coliforms ( p = 0.085 ) , but was significant for s. aureus ( p = 0.008 ) . the range of contamination of ready - to - use samples for total viable count , coliforms and s. aureus are shown in table 2 . out of 28 commercial formulas , 27 samples ( 96% ) had total viable counts greater than 10 cfu / g . also , 24 samples ( 86% ) and 27 samples ( 96% ) were contaminated with s. aureus and coliforms , respectively . in order to compare ready - to - use and handmade samples , mann - whitney test was used . as can be seen in table 2 , contamination of ready - to - use formulas in all three microbiological samples regarding that any positive bacterial growth was considered contamination , in the first - time sampling , of 21 samples , 11 ( 52% ) had coliform contamination , 5 ( 24% ) had s. aureus contamination and 16 ( 76% ) had total viable counts greater than 10 cfu / g ; whereas in the second - time sampling , of 21 samples , 16 ( 76% ) had coliform contamination , 13 ( 62% ) had s. aureus contamination and 17 ( 81% ) had total viable counts greater than 10 cfu / g . wilcoxon test was used to compare the contamination of the first- and the second - time sampling after calculating log ( cfu / g ) . as can be seen in table 1 , the difference between total viable contamination of these two groups of samples was significant ( p = 0.004 ) . for these two sampling times , maximum coliform contamination was 1.7 10 and 5.0 10 cfu / g , respectively . the results for s. aureus were 2.3 10 and 4.0 10 cfu / g , respectively . therefore , 18 h of keeping the feeds had increased coliform contamination about 1.5 logs and s. aureus contamination about 2 logs . the increased contamination after this time was not significant for coliforms ( p = 0.085 ) , but was significant for s. aureus ( p = 0.008 ) . the range of contamination of ready - to - use samples for total viable count , coliforms and s. aureus are shown in table 2 . out of 28 commercial formulas , 27 samples ( 96% ) had total viable counts greater than 10 cfu / g . also , 24 samples ( 86% ) and 27 samples ( 96% ) were contaminated with s. aureus and coliforms , respectively . in order to compare ready - to - use and handmade samples , mann - whitney test was used . as can be seen in table 2 , contamination of ready - to - use formulas in all three microbiological samples in the present study , 96.4% of the ready - to - use formulas and 78.6% of handmade formulas had total aerobic microorganism count of over 10 cfu / g . furthermore , coliforms were over 3 organisms / g in 57.1% and 17.8% of handmade and ready - to - use samples , respectively , which means they cross the standard limit ( the details are not mentioned in the result section ) . this high rate of contamination shows lack of observing health standards in different stages of preparation and transportation of both types of feeding solutions . compared handmade and ready - to - use foods in terms of bacterial contamination . the contamination of all handmade samples immediately after preparation was on average 10 -10 organisms / ml while none of the commercial samples were contaminated . , relevant researches have shown that contamination of ready - to - use formulas are directly related to the stages of preparation . bacterial contamination of handmade formulations could be attributed to several sources including original food items , food - making devices , blenders , environmental contamination of the kitchen regarding hygiene of the floor and air conditioner , process of food preparation , not observing the hygienic principles by kitchen staff and nurses and process of food carriage to the wards . comparison of the results of the handmade samples of both sampling times shows the significant increase of contamination in the second time . this increase indicates the sub - standard conditions of keeping feeding solutions during this period . the conditions have provided suitable medium for growth and proliferation of bacteria due to the temperature , long time of storage and environmental contamination . with regard to powder samples , there was not any data indicating their sterility neither on the packaging nor on the package inserts of these products ; contacting the manufacturing companies of these ready - to - use formulas , we realized that these products are not produced under the sterile condition , so their microorganism count could not be zero ; however , high contamination shows lack of hygiene in manufacturing companies . international organizations have established standards to control the contamination of nutritional solutions , however , inadequate observation of personal hygiene and the process of food preparation is one of the main reasons for contamination , which indicates the break between theoretical and practical standards . although ready - to - use formulas are prepared 30 min before each feeding time , total viable count of microorganisms , coliforms and s. aureus count were all higher than those in handmade formulas . moreover , contamination of ready - to - use formulas can be caused by lack of hygiene in the production line of the manufacturer to some extent . they found contamination of most samples to be over the standard limits for total viable count , coliforms and s. aureus . they recommended using ready - to - use formulas instead of handmade formulas to reduce contamination . however , the present study found contamination in both handmade and ready - to - use formulas . these results call for designing comprehensive and documented guidelines for preparation , storage and transportation of these products in iran . moreover , considering the fact that ready - to - use samples used in the present study were contaminated at the time of preparation and transportation , it seems that closed system ready - to - use formulas that do not need further process for preparation and can be used at patient 's bed can reduce bacterial contamination . several studies including the present one have shown that even the commercial formulations can be contaminated at the time of opening the can , diluting the powder formulation or pouring the formula into the patient 's dish . furthermore , any steps of the processing of handmade formulas could be considered as the cause of contamination . this introduces the subject of future studies in order to determine the sources of these formulas microbial overload . the results of present study indicate that the microbial safety of the majority of enteral feeding solutions in this hospital is lower than standard values published in relevant guidelines . the presented data demonstrate that the development of protocols for clean techniques in the preparation , handling and storage of both commercial and handmade enteral feeds is necessary .

the biotechnology revolution has made it possible to produce highly purified polypeptides in large quantities . development of polypeptide therapeutics , however , may bring challenges , due to the susceptibility to proteolysis , their short shelf or halflife , low solubility , rapid elimination and their potential to cause specific immune responses 1 , 2 . one strategy to minimize such properties is conjugation with an inert and hydrophilic macromolecule that does not alter the safety or efficacy profile of the parent molecule . clinical experience over the last decades has shown that polyethylene glycol ( peg ) is a suitable polymer for this purpose ; chemical conjugation with peg to improve the pharmacokinetic ( pk ) profile of a biopharmaceutical is generally known as pegylation 2 , 3 . pegylation has been shown to improve the properties of native proteins by reducing renal elimination , providing steric shielding against immunological recognition , and decreasing receptormediated clearance or enzymatic attack . today , pegylation is the dominant technique to improve properties of a biopharmaceutical by polymer conjugation 4 , 5 . numerous pegylated biopharmaceuticals for parenteral administration are currently approved for use in humans or are in clinical development . the first approved pegylated biotherapeutic was adagen ( enzon , 1990 ) ; the most recently approved pegylated biopharmaceutical is plegridy ( biogen idec , 2014 ) 2 , 6 . pegylated biopharmaceuticals , either commercially available or in clinical development , are modified with peg polymers of 560 kda in size . many pegylated drug candidates are currently in various phases of development , including several indicated for the treatment of haemophilia a. bayer and novo nordisk have developed pegylated variants of recombinant bdomain deleted rfviii ( bddrfviii ) , which are currently under evaluation in clinical phase iii trials . bayers bay 949027 is a bddrfviii that carries an engineered amino acid sequence to allow cysteinedirected pegylation with a 60 kda branched peg 7 . glycopegylated fviii ( n8gp ) developed by novo nordisk is a bddrfviii with a truncated bdomain that is modified with a branched 40 kda peg on olinked glycans using enzymatic glycoconjugation technology 8 . in contrast , baxaltas bax 855 is a fulllength rfviii conjugated with 20 kda branched peg molecules that consist of two chains , each 10 kda in size . bax 855 is synthesized using a proprietary pegylation methodology developed by nektar therapeutics ( huntsville , al , usa ) that targets the epsilonamino groups of lysines 9 . bax 855 contains the same original , native fulllength rfviii protein used in advate ( rfviii , recombinant antihaemophilic factor , plasma / albuminfree method ) . the aim of pegylated rfviii variants was to prolong fviii activity ( i.e. increase the halflife ) , which would allow for less frequent infusion ( or higher trough levels ) while maintaining efficacy of the parent rfviii product . the benefits of longer halflife , however , must not be compromised by introducing an increased safety risk attributable to combination with a chemical polymer . the safety of peg and pegylated products has been demonstrated in numerous studies 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 . in developing bax 855 , the applied pegylation technology was optimized to retain functionality of the fviii molecule , improve its pk properties and provide additional therapeutic options in the personalization of haemophilic care . in addition , nonclinical studies conducted by baxalta , have demonstrated that the excellent safety profile established for advate remains unchanged for the pegylated rfviii product , bax 855 . the safety evaluation of bax 855 was based on data from animal model studies with bax 855 , and published data on pegylated biopharmaceuticals extracted to determine whether chemical modification with peg impaired the safety profile of parent drug molecules . data from a repeated dose toxicity study with an unbound pegacid ( peg2ru20kcooh ) most relevant to bax 855 as it would be liberated from the conjugate after 100% catabolism of the protein were also evaluated . the hypothetical elimination pathways of bax 855 and peg are shown in fig . 1 . hypothetical degradation and elimination pathways of pegrfviii conjugate ( bax 855 ) ; pegacid is the final degradation product , which is primarily eliminated via kidney and liver . the toxicity study with peg2ru20kcooh performed in the context of preclinical development of bax 855 contributed to the publically available safety reports on peg . published information on distribution , metabolism and excretion , which are highly relevant to the safety of peg and pegylated biopharmaceuticals , are also described . as a whole , these comprehensive data aim to demonstrate the safety of bax 855 . distribution , metabolism and excretion from the organism are important determinants for the safety of a compound . peg is the acronym for a chemical family of uncharged polyethers consisting of repeated units of ethylene oxide and having amphiphilic properties and very low chemical reactivity ; only the terminal hydroxyl groups of the molecule may be accessible for covalent bonding . as with the peg used for conjugation to rfviii in bax 855 , pegs are frequently modified at the chain terminus , rendering them less reactive ( methoxypeg , mpeg ; endcapped pegs ) . the pharmacokinetics , metabolism and distribution of peg after parenteral application were recently discussed in detail by baumann et al . 20 . briefly , peg was shown to be readily distributed and excreted in several animal species . some reports referring to enzymatic metabolism of peg molecules via alcohol ( adh ) and aldehyde dehydrogenase might be misinterpreted 22 , 23 . because peg represents a family of compounds with large variations in terminal groups and size , it is important to distinguish literature covering pegs with free hydroxy groups from references discussing endcapped pegs used to modify pharmaceuticals . still , peg may be metabolized at its terminal ends , but the ratio is rendered negligible due to modification of the polyethylene glycol chain ends with , e.g. methoxycapping 20 . ether linkages connecting ethylene glycol subunits in the peg chain are highly stable in vivo due to the absence of etherases in eukaryotes . nevertheless , the formation of peg peroxides in vitro is known to occur when peg solutions are stored in the presence of oxygen 24 . peg chains may be cleaved by free radicals in vivo after pino or phagocytosis of peg or pegcontaining particles when reactive oxygen species are present in the ( phago)lysosome , but at a minor rate , with no detectable toxicological relevance 24 . for nonglycosylated globular proteins , peg molecules differ from globular proteins as peg chains are highly hydrated , which amplifies the hydrodynamic radius of peg in aqueous solutions . furthermore , the flexible and linear peg molecules are able to migrate through glomerular pores despite their large polymer size , a process referred to as reptation ( lat . therefore , no clear threshold for glomerular filtration can be determined for peg . however , renal clearance is significantly slower for nonconjugated pegs larger than 30 kda 21 . a peg size > 30 kda triggers increased halflife and favours other than renal clearance pathways , such as hepatic clearance or pinocytosis / phagocytosis by cells of the mononuclear phagocyte system ( mps ) , also known as the reticuloendothelial system ( res ) or lymphoreticular system . mps cells are able to engulf particles ( phagocytosis ) to form an intracellular phagosome , which fuses with the lysosome , the compartment involved in the breakdown of cellular components , thereby generating the phagolysosome . inside the phagolysosome , contents are subsequently degraded and released intra or extracellularly for further processing or elimination . peg molecules with increasing mw tend to have higher rates of cellular clearance ; similarly , macrophage uptake of the inert polymer polyvinyl alcohol tends to increase with increasing mw 25 . it has been established that the physicochemical properties of a particle 's surface , such as surface charge , size , functional groups and hydrophobicity , affect the uptake of particles by phagocytic cells . in vitro experiments with nanoparticles coated with peg of varying mw showed that the lower negative charge of the surface after pegylation reduced the uptake by macrophages 25 . peg inside of phagocytic cells may be released into the circulation by decomposition of cell membranes after apoptotic decay of the phagocytic cell or by exocytosis 20 ; renal or biliar excretion follows . the vasculature of the liver is composed of discontinuous capillary walls that allow small and large polymeric molecules such as peg to diffuse from the blood to the extravascular region . elimination might also occur via paracellular pathways , such as transepithelial movement across intercellular junctional complexes 26 . liver clearance via parenchymal cells leading to excretion via bile and faeces decreases with increasing mw . nonclinical studies for approved pegylated proteins have demonstrated that peg / pegylated proteins distribute to various compartments 20 . variability in the distribution pattern of pegylated biopharmaceuticals , their metabolism , and excretion complicate our ability to clearly understand the specific effects of pegylation . however , where proteolysis and kidney clearance are the primary degradation routes for the parent protein , increasing overall size of the pegylated conjugate will limit renal clearance . when proteins themselves are too large for renal clearance , the protein will dominate the clearance mechanism . there appear to be no explicit rules with respect to peg size in the case of clearance by receptormediated processes . thus , the variation in nonclinical data from available distribution studies in fda and ema databases1 , 2 is considered to be attributable to the different biological components of the conjugates ( protein or dna of varying size , clearance mechanism and pharmacological target ) and to the size and quantity / dose of the peg molecules used for chemical modification . an adme study with radiolabelled bax 855 investigated the distribution and excretion of bax 855 after a single high dose given intravenously to male and female rats . radiolabelling of a pegprotein conjugate with a tritium ( t ; h ) label directly on the peg was a pioneering chemical synthesis ( to be published separately ) , as previous studies with radiolabelled pegylated biotherapeutics were synthesized with the radiolabel on the protein moiety or the linker between peg and the biologically active constituent . this was an important step , as peg is almost inert to chemical modification except at its terminal hydroxyl group ( which is blocked in pegprotein conjugates ) 27 or at linker structures that are highly reactive and have many different potential labelling sites . the disadvantage of using radiolabelled linkers is that the biological distribution of peg can only be investigated as long as the linker is attached to the peg 28 . the resulting test item for the adme study in rats is shown in fig . 2 . ( t ; h ) labelled pegylated rfviii , test item of the singledose adme study in rats . starting material for synthesis of tritium ( t ; h ) labelled pegylated rfviii was peg2ru20kcooh ( pegacid ) , which was also investigated in a repeated dose toxicity study in rats , with 10 000fold exposure compared with peg administered during prophylaxis with bax 855 . coupling of ( t)peg2ru20knhs reagent to fulllength rfviii was performed in a manner similar to the modification step used to generate bax 855 . the nhs ester of the tritiumlabelled reagent reacts with the epsilonamino groups of lysines in rfviii to form stable amide bonds . after the reaction , the conjugate is purified by ionexchange chromatography and subsequently concentrated by ultrafiltration / diafiltration ( uf / df ) . tritium exchange as a radiolabelling technique has the risk that tritium may be lost through exchange with water . therefore integrity and stability of the labelled pegrfviii conjugate was tested before , during and 1 year after the adme study . the presence of tritium on the peg portion of the pegrfviii conjugate was measured by radioimmunoblot and western blot by staining with a monoclonal antipeg antibody . in addition , the agent tested in the adme study was characterized for its fviiispecific activity and pharmacokinetic studies were performed in fviiideficient mice in comparison to nonpeg tritiated bax 855 . these studies confirmed that ( i ) radiolabelled pegrfviii was stable throughout the study , ( ii ) radiolabel was present on peg , ( iii ) fviii activity was maintained , despite introduction of the label and ( iv ) prolongation of the halflife of fviii was not impeded by radiolabelling ( data not shown ) . single doses of 1 and 2 mg fviii protein kg body weight ( bw ) corresponded to fviii activities of 2088 and 4176 iu kg bw respectively . these activity doses were accompanied by peg doses of approximately 0.12 and 0.24 mg peg kg bw respectively . urine , faeces and selected tissues were collected up to 1008 h ( 6 weeks ) postdose . the distribution of drugderived radioactivity was extensive , with the highest concentrations of radioactivity observed in plasma , blood , mesenteric lymph nodes , spleen , liver , adrenal glands and kidneys . however , after high doses of radiolabelled bax 855 ( 2550 x the maximum clinical dose of 80 iu kg ) , only marginal radioactivity levels were measured in the brain and spinal cord over 6 weeks . the average brain : plasma concentration ratio was clearly < 1 ( 0.0130.333 ) for all time points measured . these data demonstrate that even at very high doses of bax 855 , peg did not reach the brain or related tissues at clinically relevant levels . furthermore , radioactivity was completely excreted within 6 weeks , indicating that peg that had been distributed into tissues was subsequently redistributed to the circulation and excreted ( see fig . 1 ) 29 . based on this stability , certain peg and pegcontaining biopharmaceuticals may cause vacuolation of certain cell types after repeated treatment with high parenteral doses in animals . based on the absence of tissue damage or any other signs of toxicity , vacuolated cells observed in some animal studies are considered to be a nonadverse consequence of an adaptive peg removal process . tumour necrosis factor binding protein ( tnfbp ) pegylated with a 20 kda peg caused renal tubular vacuolation in rats at daily i.v . doses of 10 , 20 or 40 mg kg bw for 3 months . vacuolation resulting from the high doses ( 20 and 40 mg kg day ) was only partly reversible within the recovery period of 2 months . tubular vacuolation did not lead to necrosis , but did cause distortion of tubular profiles and compression of nuclei 31 . the mode of action is considered to be related to specific binding of the tnfbp to tubular epithelial cells and subsequent receptormediated uptake and pinocytosis 31 . vacuole formation has also been reported for haemoglobin ( hb ) and bovine serum albumin ( bsa ) , both conjugated to a 5 kda peg 32 . dose of ~500 mg kg bw of both peghb and pegbsa were examined for vacuole formation in the heart , lung , liver , spleen and kidneys up to 30 days post infusion . seven days after treatment , renal tubular cell vacuoles and splenic vacuolated macrophages were recorded in all animals treated with peghb . after 30 days , only 20% of animals had vacuoles in tubular cells , and no vacuolated macrophages were present in the spleen . rats receiving pegbsa were free of renal tubular cell vacuoles at both time points , but all animals had splenic vacuolated macrophages , with decreasing severity at the later time point . formation of vacuoles in the proximal tubules is also a normal process whereby proteins such as haemoglobin are recycled and supplied to metabolic pathways . within this process , saturation of the tubular lumen with haemoglobin can result in bulbular enlargements that pinch off from cell membranes to form absorption droplets . vacuolated cells have also been observed in nonclinical studies conducted for several approved pegylated biopharmaceuticals ( somavert , macugen , cimzia , krystexxa and omontys ) 33 , 34 , 35 , 36 , 37 . the distribution and toxicity of three peg molecules ( 100 mg kg of linear 10 , 20 and 40 kda peg ) was investigated after repeated i.v . injection in rats for 3 months ; 10 kda peg was injected daily , 20 kda peg every other day and 40 kda once weekly . it was demonstrated that increasing peg mw strongly reduced peg immunehistoreactivity in renal tubules of rats . the peg immunehistoreactivity increased , however , in other cell populations with increasing mw of the peg , especially splenic macrophages and choroid plexus epithelial cells . pegrelated histological changes were generally limited to rats dosed with 40 kda peg , where peg immunehistoreactivity was associated with macrophage and choroid plexus epithelial cell vacuolation , as well as epithelial vacuolation and degeneration of renal tubules . based on these data , the effect of a peg size of 1040 kda on cellular distribution of immunereactive peg and pegrelated histological changes was clearly demonstrated . chronic dosing of biologics conjugated to peg 40 kda may have greater potential for vacuolation in macrophages , choroid plexus epithelial cells and renal tubular epithelial cells 38 . the occurrence of vacuolated cells in choroid plexus epithelial cells ( ependymal cells ) has been widely discussed in relation to the safe use of pegylated biopharmaceuticals . the ema 's chmp safety working party reviewed repeated dose toxicity studies for pegylated biologics that are approved or under development for use in children ( including study results outside the public domain ) and concluded that peg vacuolation of ependymal cells in animals was only observed if several conditions were met 39 , i.e. the study was conducted in macaques , a peg moiety size of at least 40 kda was applied , the study lasted 6 weeks and the administered dose comprised at least a cumulative monthly peg exposure of 0.4 mol kg month . in 2013 , an article discussing pegassociated vacuolation in macrophages was published 30 . vacuolation was predominantly reported for tissues comprising the res and was without apparent toxicological significance . following high exposure to pegylated biopharmaceuticals , vacuolated macrophages were observed in several tissues , including the liver , kidney , bladder and choroid plexus . these findings were considered to reflect normal physiological processing of foreign material by scavenger phagocytic cells , producing no apparent effect on cell function or cell viability . other cell types , such as hepatocytes , cells of urinary bladder , epididymis , adrenal cortex , synoviocytes , ciliary bodies of the eyes and choroid plexus of the brain previously showed vacuolation after high peg exposure . for peg therapeutic proteins , dose and durationdependent peg accumulation and cytoplasmatic vacuolation was nonspecific or frequently targetassociated , as described for renal tubular epithelial cells 31 and neurons without cellular damage or apparent effect on neuronal function ( e.g. nerve conduction velocity ) 30 . in conclusion , vacuolation is considered an effect of lysosomal processing after injection of high doses of vascular persistent but highly soluble test article such as peg . the estimated peg dose for a single dose of approved pegylated biopharmaceuticals ranges from 0.026 to 176 mg peg per person per application ( table 1 ) . for bax 855 , the dose of one fviii activity unit is associated with an exposure to 0.095 g peg . prophylactic dosing in a completed phase 2/3 pivotal study with bax 855 ( clinicaltrials.gov identifier : nct01736475 ) was up to 60 iu kg twice weekly . the maximum anticipated prophylactic dose is 80 iu kg , given twice weekly . doses of up to 100 iu kg may be used preoperatively ( over a limited number of days ) in an ongoing phase 3 surgery study . table 2 lists the peg doses associated with these clinical fviii dosing regimens . the maximum anticipated prophylactic dose of 80 iu kg bax 855 would result in a peg application of 7.6 g kg bw or 0.46 mg ( for a 60 kg individual , the standard bw used in table 1 ) . higher , shortterm exposure with bax 855 before or after surgery is not taken into account as the contribution to an overall increase in cumulative monthly or lifelong peg exposure would be minimal . this absolute dose of < 1 mg results in a lower peg exposure than that of several approved pegylated proteins such as pegaspargase ( oncaspar ) , pegfilgrastim ( neulasta ) , pegvisomant ( somavert ) , certolizumab pegol ( cimzia ) , pegloticase ( krystexxa ) and peginesatide ( omontys ) ( see table 1 ) . compared with onceamonth treatment with certolizumab ( 176 mg of 40 kda peg for 60 kg individual ) in particular , the monthly peg exposure with bax 855 dosed 10 times ( twice weekly application of 80 iu kg bw resulting in a peg dose of 76 g kg bw or 4.6 mg per person ) would be 38 times lower . peg exposure with approved pegylated biopharmaceuticals data from specified reference and/or http://www.rxlist.com ; n.a . , information not available . based on an adult with 60 kg bw and 1.6 m body surface peg dose with expected doses of bax 855 in clinical studies based on 60 kg body weight and a peg dose of 0.095 g per iu fviii . the cumulative peg dose is important in considering possible accumulation and vacuolation of ependymal cells in vivo . as described above , the chmp safety working party recently discussed the risk of ependymal cell vacuolation caused by pegs ( mainly > 40 kda ) at a monthly peg exposure of 0.4 mol kg month and recommended addressing this risk before conducting longer term clinical trials in children . in applying this recommendation to the bax 855 programme , the dose of 7.6 g peg kg per 80 iu kg bw dose can be converted to mol . the mw of the peg attached to rfviii in bax 855 is 20 kda , hence the clinical peg exposure of 7.6 g peg kg bw per day equals 7.6 g/20 000 g/mol 10 doses per month = 0.0038 mol / kg bw / month , more than 100 times below the threshold of 0.4 mol kg bw month . it can be concluded that for bax 855 , ( i ) the monthly peg exposure is substantially lower than for other approved pegylated products , ( ii ) the peg exposure is substantially lower than the threshold for ependymal cell vacuolation observed in animal studies ( 0.4 mol kg bw month ) and ( iii ) the size of the peg ( 20 kda ) allows complete elimination from the body without any risk of accumulation . thus , the peg exposure derived from bax 855 over a chronic treatment period is considered to be low and to pose no safety concern . peg is found in pharmaceuticals for topical ( ointments ) , oral ( excipients , laxatives ) , ophthalmic ( eye drops and creams ) , rectal ( suppositories ) and parenteral ( excipients ) administration , providing evidence for its clinical safety . the wide use of peg is further demonstrated by numerous entries in the fda inactive ingredient list3 . several other frequently used pharmaceutical excipients , such as tween 20/80 ( polysorbate 20/80 ) or poloxamer 188 ( pluronic f68 ; sigmaaldrich , st . louis , mo , usa ) , also contain repeated ethoxygroups and release small peg molecules when metabolized . as early as 1950 , smyth comprehensively summarized the toxicity of pegs of 62 da ( ethylene glycol ) to 10 kda 40 . however , these results are of questionable relevance as peg materials were less pure at that time and contained a broad distribution of peg moieties . more recent reviews were published in 2005 and 2007 19 , 27 . even for the most toxic ethylene glycol , the ld50 value ( for a single oral dose in guinea pigs ) was as high as 6.6 g kg bw , due to the toxic metabolite , oxalic acid . studies of chronic oral toxicity in dogs revealed no adverse effects for pegs of 200 da to 6 kda at doses of 2% of the diet for 1 year . with increasing peg size , oral toxicity ( ld50 ) decreased to > 50 g kg bw in rats for 10 kda peg . no absorption via the gastrointestinal tract was reported for pegs > 6 kda 41 . dosing with amounts matching the g kg bw in several species caused no adverse events , adverse reproductive or teratogenic effects . peg toxicity studies using intravenous application extensive safety evaluation on peg alone was conducted by scheringplough for submission of pegintron . the 12 kda mpeg was not mutagenic in the standard battery of salmonella and escherichia strains , and a chromosomal aberration test in human lymphocytes was negative . a mouse micronucleus study showed that mpeg did not cause micronucleus formation after intraperitoneal ( i.p . ) injection . a 13week study in rats , in which mpeg was administered s.c . twice weekly at doses up to 2276 g m week ( 379 g kg week ) , revealed no mpegrelated findings under macroscopic or microscopic examination . in a 13week ( 4week recovery ) twice weekly at doses up to 2276 g m week ; 190 g kg week ) , no mpegrelated findings were observed under macroscopic or microscopic examination . an embryo foetal development study in rats with mpeg administered daily at doses up to 800 g m per day ( 133 g kg ) from day 6 through 17 after mating showed no signs of toxicity , no maternal or in utero effects . no signs of toxicity , maternal or in utero effects were noted in an embryofoetal development study in rabbits with daily doses up to 800 g m ( 67 g kg ) from day 7 through 19 after mating 42 . the peg size used for bax 855 ( 20 kda ) is within the range currently used in approved pegylated biotherapeutics , i.e. 540 kda ( table 1 ) . according to yamaoka et al . administration is approximately 3 h ( 169 20 min ) , indicating that exposure is limited and peg of this size is quickly eliminated from the body 21 . while the 20 kda peg is small enough to assure rapid elimination ( primarily via the kidneys and in bile after liberation from rfviii ) , it is still able to sterically shield rfviii from clearance receptors , thereby prolonging its halflife . based on the stability of peg in vivo and stable covalent bonds , metabolism of pegrfviii is expected to liberate peg2ru20kcooh ( pegacid ) after catabolism of the protein part ( fig . 1 , for structure see fig . the nonclinical safety of pegacid was also investigated in a 28day study conducted by baxalta . doses of 0.65 , 6.5 and 65 mg kg twice weekly for a total of 8 doses ( for experimental details see data s1 ) . the dose levels selected were based on an estimation of the cumulative lifetime exposure of ~6.5 mg peg / kg bw considering a bax 855 dose of 80 iu kg bw twice weekly over 70 years . furthermore , the high dose of 65 mg peg kg bw is ~10 000 times the peg exposure of a single clinical dose of bax 855 and 10 times the estimation of the cumulative lifetime exposure . delayed onset or reversibility of toxicity was assessed during a 4 and 13week treatmentfree period . all doses were well tolerated with no negative clinical observations , effects on bw or food consumption , ophthalmoscopy or clinical or anatomical pathology . there were no macroscopic or microscopic findings ( including no signs of cell vacuolation ) due to administration of pegacid . peg2ru20kcooh ( pegacid ) , expected final degradation product of bax 855 that was also investigated in a repeated dose toxicity study in rats with 10 000fold exposure compared with peg administered during prophylaxis with bax 855 . overall , acute and chronic administration of various pegs by relevant routes of application was evaluated in several animal species . signs of toxicity only appeared at very high doses in the g kg bw range ( table 3 ) . preclinical evaluation of approved pegylated biopharmaceuticals ( adagen , oncaspar , pegintron , pegasys , neulasta , somavert , macugen , mircera , cimzia , krystexxa , omontys and plegridy ) included comprehensive toxicology studies with the pegconjugate and partly with the peg alone . several available publications also deal with adverse effects of pegprotein conjugates , however , without relation to peg 42 , 43 , 44 , 45 , 46 . if toxic effects were observed in nonclinical studies with the currently available pegylated products , they were caused by the protein portion of the conjugate and exaggerated pharmacological effects , not by the peg . the nonadverse formation of pegassociated vacuoles was observed in several animal studies , but was considered to reflect normal processing of foreign material 38 . safety pharmacology and single and repeated dose toxicity studies were conducted in mice , rats , rabbits and macaques . histopathological evaluation in rats and macaques repeatedly treated with bax 855 revealed no adverse findings in tissues . importantly , specific histopathological examination of the brain and spinal cord of these species confirmed the absence of any vacuolation in organs of all animals that received bax 855 47 . results of comparative immunogenicity studies in mice and macaques indicate that bax 855 has a similar immunogenicity profile to advate 48 . bax 855 is a human , fulllength recombinant factor viii ( rfviii , used in advate ) modified with polyethylene glycol ( peg ) to extend its circulating halflife . as the safety profile of advate is well established , assessment of bax 855 focused on possible safety issues that could arise from conjugation with peg . peg itself is a highly stable and inert molecule , generally considered as safe and has been used for decades in personal care products and as an excipient or drug component in various parenteral drug products and devices . the first pegylated biopharmaceuticals produced using pegylation technology similar to that of bax 855 were marketed in 1990 . safety data for similar peg molecules alone or in pegprotein conjugates with peg of comparable or larger size and much higher peg doses than is intended for bax 855 indicate no safety concerns caused by or related to peg at clinically relevant exposure levels . the only pegrelated finding reported in the literature and observed in some animal studies at extreme peg doses after repeated administration was vacuolation of certain cell types , including ependymal cells in the choroid plexus . however , it is important to note that no such vacuolation occurred in animal studies with bax 855 or with the unconjugated 20 kda peg . the peg size and clinical peg doses applied with bax 855 are within a range in which vacuolation is not expected and did not occur during nonclinical testing . nonclinical studies conducted with bax 855 in mice , rats , rabbits and macaques revealed no adverse effects related to pegylation of rfviii . dose of radiolabelled bax 855 in rats was completely excreted within 6 weeks . a 28day repeated dose toxicity study in rats , using peg2ru20kcooh ( the final and significant in vivo degradation product of bax 855 derived from peg in animals and humans ) , was conducted at dose levels representing multiples of a cumulative lifetime dose of peg related to bax 855 therapy . peg2ru20kcooh did not cause any adverse or nonadverse effects ( including vacuolation of macrophages or other cells ) . in conclusion , safety evaluation of peg and baxalta 's pegylated rfviii , bax 855 , based on extensive literature analysis and data from nonclinical studies identified no pegrelated safety concerns . now undergoing clinical trials , bax 855 may in the future offer an important therapeutic option for patients with haemophilia a. r. stidl , s. fuchs , j. siekmann , m. putz and p. l. turecek are fulltime employees of baxalta innovations gmbh ; m. bossar d is a fulltime employee of nektar therapeutics .

introduction bax 855 is a pegylated human fulllength recombinant factor viii ( rfviii ) based on licensed rfviii ( advate ) . the applied pegylation technology has been optimized to retain functionality of the fviii molecule , improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy.aimthe aim of this study was to confirm that the excellent safety profile of advate remains unchanged after pegylation.methodsnonclinical safety studies with bax 855 and its respective unbound polyethylene glycol ( peg ) were conducted in several species . the distribution of a single dose of radiolabelled bax 855 was further investigated in rats . publically available safety data on peg alone and pegylated biomolecules were summarized and reviewed for specific safety findings attributable to peg or pegylated biopharmaceuticals.resultssafety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues . results of a distribution study in rats administered radiolabelled bax 855 showed that radioactivity was completely excreted ; urine was the major elimination route . a 28day study in rats dosed with the unbound peg constituent ( peg2ru20kcooh ) of bax 855 showed no adverse or nonadverse effects.safety data for peg and pegprotein conjugates indicate no safety concerns associated with peg at clinically relevant dose levels . although vacuolation of certain cell types has been reported in mammals , no such vacuolation was observed with bax 855 or with the unbound peg constituent.conclusionnonclinical safety evaluation of peg and bax 855 identified no safety signals ; the compound is now in clinical development for the treatment of patients with haemophilia a.

Introduction Distribution, metabolism and excretion of unbound PEG, PEGylated biopharmaceuticals and BAX 855 Distribution, metabolism and excretion of BAX 855 Cell vacuolation after administration of unconjugated PEG and PEGprotein conjugates PEG exposure with PEGylated biopharmaceuticals and BAX 855 Safety data on unbound PEG Safety data for PEGylated proteins Summary of nonclinical safety of BAX 855 Summary and conclusion Disclosures Supporting information

clinical experience over the last decades has shown that polyethylene glycol ( peg ) is a suitable polymer for this purpose ; chemical conjugation with peg to improve the pharmacokinetic ( pk ) profile of a biopharmaceutical is generally known as pegylation 2 , 3 . numerous pegylated biopharmaceuticals for parenteral administration are currently approved for use in humans or are in clinical development . pegylated biopharmaceuticals , either commercially available or in clinical development , are modified with peg polymers of 560 kda in size . many pegylated drug candidates are currently in various phases of development , including several indicated for the treatment of haemophilia a. bayer and novo nordisk have developed pegylated variants of recombinant bdomain deleted rfviii ( bddrfviii ) , which are currently under evaluation in clinical phase iii trials . in contrast , baxaltas bax 855 is a fulllength rfviii conjugated with 20 kda branched peg molecules that consist of two chains , each 10 kda in size . bax 855 contains the same original , native fulllength rfviii protein used in advate ( rfviii , recombinant antihaemophilic factor , plasma / albuminfree method ) . the aim of pegylated rfviii variants was to prolong fviii activity ( i.e. increase the halflife ) , which would allow for less frequent infusion ( or higher trough levels ) while maintaining efficacy of the parent rfviii product . the safety of peg and pegylated products has been demonstrated in numerous studies 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 . in developing bax 855 , the applied pegylation technology was optimized to retain functionality of the fviii molecule , improve its pk properties and provide additional therapeutic options in the personalization of haemophilic care . in addition , nonclinical studies conducted by baxalta , have demonstrated that the excellent safety profile established for advate remains unchanged for the pegylated rfviii product , bax 855 . the safety evaluation of bax 855 was based on data from animal model studies with bax 855 , and published data on pegylated biopharmaceuticals extracted to determine whether chemical modification with peg impaired the safety profile of parent drug molecules . data from a repeated dose toxicity study with an unbound pegacid ( peg2ru20kcooh ) most relevant to bax 855 as it would be liberated from the conjugate after 100% catabolism of the protein were also evaluated . the hypothetical elimination pathways of bax 855 and peg are shown in fig . the toxicity study with peg2ru20kcooh performed in the context of preclinical development of bax 855 contributed to the publically available safety reports on peg . published information on distribution , metabolism and excretion , which are highly relevant to the safety of peg and pegylated biopharmaceuticals , are also described . as a whole , these comprehensive data aim to demonstrate the safety of bax 855 . distribution , metabolism and excretion from the organism are important determinants for the safety of a compound . peg is the acronym for a chemical family of uncharged polyethers consisting of repeated units of ethylene oxide and having amphiphilic properties and very low chemical reactivity ; only the terminal hydroxyl groups of the molecule may be accessible for covalent bonding . the pharmacokinetics , metabolism and distribution of peg after parenteral application were recently discussed in detail by baumann et al . still , peg may be metabolized at its terminal ends , but the ratio is rendered negligible due to modification of the polyethylene glycol chain ends with , e.g. peg chains may be cleaved by free radicals in vivo after pino or phagocytosis of peg or pegcontaining particles when reactive oxygen species are present in the ( phago)lysosome , but at a minor rate , with no detectable toxicological relevance 24 . therefore , no clear threshold for glomerular filtration can be determined for peg . it has been established that the physicochemical properties of a particle 's surface , such as surface charge , size , functional groups and hydrophobicity , affect the uptake of particles by phagocytic cells . in vitro experiments with nanoparticles coated with peg of varying mw showed that the lower negative charge of the surface after pegylation reduced the uptake by macrophages 25 . variability in the distribution pattern of pegylated biopharmaceuticals , their metabolism , and excretion complicate our ability to clearly understand the specific effects of pegylation . however , where proteolysis and kidney clearance are the primary degradation routes for the parent protein , increasing overall size of the pegylated conjugate will limit renal clearance . thus , the variation in nonclinical data from available distribution studies in fda and ema databases1 , 2 is considered to be attributable to the different biological components of the conjugates ( protein or dna of varying size , clearance mechanism and pharmacological target ) and to the size and quantity / dose of the peg molecules used for chemical modification . an adme study with radiolabelled bax 855 investigated the distribution and excretion of bax 855 after a single high dose given intravenously to male and female rats . radiolabelling of a pegprotein conjugate with a tritium ( t ; h ) label directly on the peg was a pioneering chemical synthesis ( to be published separately ) , as previous studies with radiolabelled pegylated biotherapeutics were synthesized with the radiolabel on the protein moiety or the linker between peg and the biologically active constituent . the disadvantage of using radiolabelled linkers is that the biological distribution of peg can only be investigated as long as the linker is attached to the peg 28 . the resulting test item for the adme study in rats is shown in fig . ( t ; h ) labelled pegylated rfviii , test item of the singledose adme study in rats . starting material for synthesis of tritium ( t ; h ) labelled pegylated rfviii was peg2ru20kcooh ( pegacid ) , which was also investigated in a repeated dose toxicity study in rats , with 10 000fold exposure compared with peg administered during prophylaxis with bax 855 . the nhs ester of the tritiumlabelled reagent reacts with the epsilonamino groups of lysines in rfviii to form stable amide bonds . in addition , the agent tested in the adme study was characterized for its fviiispecific activity and pharmacokinetic studies were performed in fviiideficient mice in comparison to nonpeg tritiated bax 855 . these studies confirmed that ( i ) radiolabelled pegrfviii was stable throughout the study , ( ii ) radiolabel was present on peg , ( iii ) fviii activity was maintained , despite introduction of the label and ( iv ) prolongation of the halflife of fviii was not impeded by radiolabelling ( data not shown ) . the distribution of drugderived radioactivity was extensive , with the highest concentrations of radioactivity observed in plasma , blood , mesenteric lymph nodes , spleen , liver , adrenal glands and kidneys . however , after high doses of radiolabelled bax 855 ( 2550 x the maximum clinical dose of 80 iu kg ) , only marginal radioactivity levels were measured in the brain and spinal cord over 6 weeks . these data demonstrate that even at very high doses of bax 855 , peg did not reach the brain or related tissues at clinically relevant levels . furthermore , radioactivity was completely excreted within 6 weeks , indicating that peg that had been distributed into tissues was subsequently redistributed to the circulation and excreted ( see fig . based on this stability , certain peg and pegcontaining biopharmaceuticals may cause vacuolation of certain cell types after repeated treatment with high parenteral doses in animals . the distribution and toxicity of three peg molecules ( 100 mg kg of linear 10 , 20 and 40 kda peg ) was investigated after repeated i.v . pegrelated histological changes were generally limited to rats dosed with 40 kda peg , where peg immunehistoreactivity was associated with macrophage and choroid plexus epithelial cell vacuolation , as well as epithelial vacuolation and degeneration of renal tubules . based on these data , the effect of a peg size of 1040 kda on cellular distribution of immunereactive peg and pegrelated histological changes was clearly demonstrated . the occurrence of vacuolated cells in choroid plexus epithelial cells ( ependymal cells ) has been widely discussed in relation to the safe use of pegylated biopharmaceuticals . the ema 's chmp safety working party reviewed repeated dose toxicity studies for pegylated biologics that are approved or under development for use in children ( including study results outside the public domain ) and concluded that peg vacuolation of ependymal cells in animals was only observed if several conditions were met 39 , i.e. the study was conducted in macaques , a peg moiety size of at least 40 kda was applied , the study lasted 6 weeks and the administered dose comprised at least a cumulative monthly peg exposure of 0.4 mol kg month . following high exposure to pegylated biopharmaceuticals , vacuolated macrophages were observed in several tissues , including the liver , kidney , bladder and choroid plexus . for peg therapeutic proteins , dose and durationdependent peg accumulation and cytoplasmatic vacuolation was nonspecific or frequently targetassociated , as described for renal tubular epithelial cells 31 and neurons without cellular damage or apparent effect on neuronal function ( e.g. the estimated peg dose for a single dose of approved pegylated biopharmaceuticals ranges from 0.026 to 176 mg peg per person per application ( table 1 ) . for bax 855 , the dose of one fviii activity unit is associated with an exposure to 0.095 g peg . prophylactic dosing in a completed phase 2/3 pivotal study with bax 855 ( clinicaltrials.gov identifier : nct01736475 ) was up to 60 iu kg twice weekly . the maximum anticipated prophylactic dose of 80 iu kg bax 855 would result in a peg application of 7.6 g kg bw or 0.46 mg ( for a 60 kg individual , the standard bw used in table 1 ) . higher , shortterm exposure with bax 855 before or after surgery is not taken into account as the contribution to an overall increase in cumulative monthly or lifelong peg exposure would be minimal . compared with onceamonth treatment with certolizumab ( 176 mg of 40 kda peg for 60 kg individual ) in particular , the monthly peg exposure with bax 855 dosed 10 times ( twice weekly application of 80 iu kg bw resulting in a peg dose of 76 g kg bw or 4.6 mg per person ) would be 38 times lower . based on an adult with 60 kg bw and 1.6 m body surface peg dose with expected doses of bax 855 in clinical studies based on 60 kg body weight and a peg dose of 0.095 g per iu fviii . the mw of the peg attached to rfviii in bax 855 is 20 kda , hence the clinical peg exposure of 7.6 g peg kg bw per day equals 7.6 g/20 000 g/mol 10 doses per month = 0.0038 mol / kg bw / month , more than 100 times below the threshold of 0.4 mol kg bw month . it can be concluded that for bax 855 , ( i ) the monthly peg exposure is substantially lower than for other approved pegylated products , ( ii ) the peg exposure is substantially lower than the threshold for ependymal cell vacuolation observed in animal studies ( 0.4 mol kg bw month ) and ( iii ) the size of the peg ( 20 kda ) allows complete elimination from the body without any risk of accumulation . thus , the peg exposure derived from bax 855 over a chronic treatment period is considered to be low and to pose no safety concern . however , these results are of questionable relevance as peg materials were less pure at that time and contained a broad distribution of peg moieties . even for the most toxic ethylene glycol , the ld50 value ( for a single oral dose in guinea pigs ) was as high as 6.6 g kg bw , due to the toxic metabolite , oxalic acid . studies of chronic oral toxicity in dogs revealed no adverse effects for pegs of 200 da to 6 kda at doses of 2% of the diet for 1 year . dosing with amounts matching the g kg bw in several species caused no adverse events , adverse reproductive or teratogenic effects . peg toxicity studies using intravenous application extensive safety evaluation on peg alone was conducted by scheringplough for submission of pegintron . a 13week study in rats , in which mpeg was administered s.c . an embryo foetal development study in rats with mpeg administered daily at doses up to 800 g m per day ( 133 g kg ) from day 6 through 17 after mating showed no signs of toxicity , no maternal or in utero effects . no signs of toxicity , maternal or in utero effects were noted in an embryofoetal development study in rabbits with daily doses up to 800 g m ( 67 g kg ) from day 7 through 19 after mating 42 . based on the stability of peg in vivo and stable covalent bonds , metabolism of pegrfviii is expected to liberate peg2ru20kcooh ( pegacid ) after catabolism of the protein part ( fig . the nonclinical safety of pegacid was also investigated in a 28day study conducted by baxalta . the dose levels selected were based on an estimation of the cumulative lifetime exposure of ~6.5 mg peg / kg bw considering a bax 855 dose of 80 iu kg bw twice weekly over 70 years . furthermore , the high dose of 65 mg peg kg bw is ~10 000 times the peg exposure of a single clinical dose of bax 855 and 10 times the estimation of the cumulative lifetime exposure . peg2ru20kcooh ( pegacid ) , expected final degradation product of bax 855 that was also investigated in a repeated dose toxicity study in rats with 10 000fold exposure compared with peg administered during prophylaxis with bax 855 . preclinical evaluation of approved pegylated biopharmaceuticals ( adagen , oncaspar , pegintron , pegasys , neulasta , somavert , macugen , mircera , cimzia , krystexxa , omontys and plegridy ) included comprehensive toxicology studies with the pegconjugate and partly with the peg alone . several available publications also deal with adverse effects of pegprotein conjugates , however , without relation to peg 42 , 43 , 44 , 45 , 46 . if toxic effects were observed in nonclinical studies with the currently available pegylated products , they were caused by the protein portion of the conjugate and exaggerated pharmacological effects , not by the peg . the nonadverse formation of pegassociated vacuoles was observed in several animal studies , but was considered to reflect normal processing of foreign material 38 . safety pharmacology and single and repeated dose toxicity studies were conducted in mice , rats , rabbits and macaques . histopathological evaluation in rats and macaques repeatedly treated with bax 855 revealed no adverse findings in tissues . importantly , specific histopathological examination of the brain and spinal cord of these species confirmed the absence of any vacuolation in organs of all animals that received bax 855 47 . results of comparative immunogenicity studies in mice and macaques indicate that bax 855 has a similar immunogenicity profile to advate 48 . bax 855 is a human , fulllength recombinant factor viii ( rfviii , used in advate ) modified with polyethylene glycol ( peg ) to extend its circulating halflife . as the safety profile of advate is well established , assessment of bax 855 focused on possible safety issues that could arise from conjugation with peg . peg itself is a highly stable and inert molecule , generally considered as safe and has been used for decades in personal care products and as an excipient or drug component in various parenteral drug products and devices . the first pegylated biopharmaceuticals produced using pegylation technology similar to that of bax 855 were marketed in 1990 . safety data for similar peg molecules alone or in pegprotein conjugates with peg of comparable or larger size and much higher peg doses than is intended for bax 855 indicate no safety concerns caused by or related to peg at clinically relevant exposure levels . the only pegrelated finding reported in the literature and observed in some animal studies at extreme peg doses after repeated administration was vacuolation of certain cell types , including ependymal cells in the choroid plexus . however , it is important to note that no such vacuolation occurred in animal studies with bax 855 or with the unconjugated 20 kda peg . the peg size and clinical peg doses applied with bax 855 are within a range in which vacuolation is not expected and did not occur during nonclinical testing . nonclinical studies conducted with bax 855 in mice , rats , rabbits and macaques revealed no adverse effects related to pegylation of rfviii . dose of radiolabelled bax 855 in rats was completely excreted within 6 weeks . a 28day repeated dose toxicity study in rats , using peg2ru20kcooh ( the final and significant in vivo degradation product of bax 855 derived from peg in animals and humans ) , was conducted at dose levels representing multiples of a cumulative lifetime dose of peg related to bax 855 therapy . peg2ru20kcooh did not cause any adverse or nonadverse effects ( including vacuolation of macrophages or other cells ) . in conclusion , safety evaluation of peg and baxalta 's pegylated rfviii , bax 855 , based on extensive literature analysis and data from nonclinical studies identified no pegrelated safety concerns . now undergoing clinical trials , bax 855 may in the future offer an important therapeutic option for patients with haemophilia a. r. stidl , s. fuchs , j. siekmann , m. putz and p. l. turecek are fulltime employees of baxalta innovations gmbh ; m. bossar d is a fulltime employee of nektar therapeutics .

at least 3040% of new drug candidates have poor water solubility and are difficult to be administrated . however , the trend of drug discovery is toward more hydrophobicity and thus better permeability to get through the gastrointestinal tract wall and cell membranes . a variety of nanoscaled particles as carriers therefore have been engineered , since they have much higher surface areas and thus dissolution rates . moreover , particles in the size range of about 50400 nm are able to accumulate in tumors during in vivo blood circulation due to the enhanced permeability and retention ( epr ) effect . compared with different nanoscaled particles , i.e. , micelles , liposomes , and nanoemulsions with a typical drug loading capacity ( cdl % ) below 20% , nanosuspensions ( usually called nanoparticles ) have a much higher drug loading and are able to show sufficient potency to kill tumors . among different techniques to produce nanoparticles , i.e. , milling , high pressure homogenization , and the supercritical fluid process , flash nanoprecipitation ( fnp ) shows advantages of fast processing , simple equipment , smaller size , and narrower size distribution . the fnp also permits combining several hydrophobic drugs and the incorporation of imaging agents . in the fnp technique ( see figure 1 ) , a highly hydrophobic drug is dissolved along with a block copolymer ( bcp ) in a water miscible organic solvent . this solution is injected into a small chamber at a high velocity along with water . the high velocity generates turbulent mixing , causing the hydrophobic drug and polymer to coprecipitate very rapidly , forming nanoscaled particles . the block copolymer is amphiphilic : typically a hydrophilic poly(ethylene glycol ) ( peg ) block covalently bonded to a hydrophobic block . the hydrophobic block precipitates with the drug , arresting particle growth , while the pendant peg blocks stabilize the particles against aggregation . schematic of impingement mixing to form block copolymer - protected nanoparticles ( 2d view of cross section ) . however , like many other techniques , the challenge of particle stability still exists . in our previous work , various polymers as the stabilizers , i.e. , water - soluble polyelectrolytes ( polylysine , polyethylene imine , and chitosan ) and nonionic amphiphilic diblock copolymers [ polystyrene - block - poly(ethylene glycol ) ( ps - b - peg ) , polycaprolactone - block - poly(ethylene glycol ) ( pcl - b - peg ) , polylactide - block - poly(ethylene glycol ) ( pla - b - peg ) , and poly(lactic - co - glycolic acid)-block - poly(ethylene glycol ) ( plga - b - peg ) ] , have been explored to investigate their effects on the particle formation and stability . up to now , little work has been reported to investigate the effects of drug compounds on the particle stability , which will be discussed in this study . as demonstrated in our previous study , biodegradable plga - b - peg ( scheme 1 ) is a suitable steric stabilizer for the fnp to inhibit the particle aggregation , since the plga block is noncrystallizable as well as has relatively high glass transition temperature and a right solubility parameter ( ) , ensuring that no unexpected particle destabilization introduced by this additive . -carotene , hydrocortisone , hydrocortisone ethoxysilicate , betulin , paclitaxel , and paclitaxel 2,7-bis(triethoxysilicate ) will be used as the drugs . ( scheme 1 ) these compounds or their analogues are listed in the us national cancer institute ( nci ) drug dictionary . the aims of this study are to give a guideline to choose the suitable drug with good particle stability for flash nanoprecipitation especially at a high drug loading ( e.g. , > 40 wt % ) , to create an approach to predict the particle stability for a randomly selected drug structure , and to give a possible approach to improve the particle stability . for the purpose of predicting particle stability , the n - octanol / water partition coefficient ( logp ) , a hydrophobicity indication , will be introduced into the fnp technique . the properties of n - octanol has been thought to resemble to those of lipid bilayer membranes , suggesting to some extent that a drug partition in octanol / water simulates its ability to passively diffuse across biological membranes . logp as a standard measurement of drug hydrophobicity has been widely used in the pharmaceutical industry . an empirical rule correlating logp with the particle stability made via fnp will be given to help enable a fast preclinical drug prescreen . -carotene is a type of antioxidant found in yellow and orange fruits and vegetables and in dark green , leafy vegetables . hydrocortisone is a steroid hormone produced by the adrenal cortex with primary glucocorticoid and minor mineralocorticoid effects . as a glucocorticoid receptor agonist , hydrocortisone promotes protein catabolism , gluconeogenesis , capillary wall stability , and renal excretion of calcium and suppresses immune and inflammatory responses . its synthetic counterpart is used , either as an injection or topically , in the treatment of inflammation , allergy , collagen diseases , asthma , adrenocortical deficiency , shock , and some neoplastic conditions . betulin is isolated from the bark of betula alba , the common white birch . its derivative , betulin acid , is a pentacyclic lupane - type triterpene with anti - inflammatory , anti - hiv , and anti - neoplastic activities . betulinic acid induces apoptosis through induction of changes in mitochondrial membrane potential , production of reactive oxygen species , and opening of mitochondrial permeability transition pores , resulting in the release of mitochondrial apogenic factors , activation of caspases , and dna fragmentation . although originally thought to exhibit specific cytotoxicity against melanoma cells , this agent has been found to be also cytotoxic against nonmelanoma tumor cell types including neuroectodermal and brain tumor cells . paclitaxel is a compound extracted from the pacific yew tree , taxus brevifolia , with antineoplastic activity . it binds to tubulin and inhibits the disassembly of microtubules , thereby resulting in the inhibition of cell division . this agent also induces apoptosis by binding to and blocking the function of the apoptosis inhibitor protein b - cell leukemia 2 ( bcl-2 ) . -carotene ( 97% ) , betulin ( 98% ) , water ( hplc grade ) , and tetrahydrofuran ( thf , hplc grade ) were purchased from aldrich and used as received . paclitaxel 2,7-bis(triethoxysilicate ) and hydrocortisone ethoxysilicate were synthesized by wohl ( see scheme s1 in supporting information ) . paclitaxel 2,7-bis(triethoxysilicate ) were chemically very stable in water in ph 7 but would hydrolyze in an acidic condition . plga(10k)-b - peg(2k ) was synthesized by the ring - opening polymerization of ( d , l)-lactide and glycolide with mpeg(2k)-oh as the initiator and tin(ii ) 2-ethylhexanoate as the catalyst in bulk at 150 c . the obtained product was diluted in thf , dialyzed ( spectra / pro 7 rc , molecular weight cut off ( mwco ) of 1000 ) with ch3oh for two days to remove unreacted monomers , and then concentrated under vacuum . mn of plga(10k)-b - peg(2k ) was determined by nmr , and mw / mn was determined by gpc as 1.46 . the plga blocks comprised 50% of lactic acid and 50% of glycolic acid confirmed by nmr and were amorphous . the two - stream mixer and the fnp process are illustrated in figure 1 . the chamber dimensions were the same as those used by johnson et al . and liu et al . ( type 500a - y2x with dimensions described in figure 4 and table 1 in ref ( 16 ) and in figure 1 in ref ( 31 ) ) . in this study , the mixer was modified to allow unequal flow momentums from two opposite jets ( see figure s1a in supporting information or figure 3 in ref ( 24 ) ) . each mixer inlet was connected to a 10 ml of gastight glass syringe ( sge inc . ) via teflon tubing with 1.6 mm i d . both syringes were loaded on an infusion syringe pump ( harvard apparatus model 975 ) . for typical mixing , 25 mg of the drug compound and 25 mg of the bcp were dissolved in 5 ml of thf and were loaded in one syringe . two streams were impinged at a high velocity inside the mixer chamber . in most cases the flow rate was 72 ml / min which produces a mean jet velocity of 6.1 m / s through the 0.500 mm diameter nozzle . the outlet of the mixer was connected via 12.7 cm of 1.6 mm i d teflon tubing to a beaker containing 90 ml of h2o to further dilute the nanoparticles . the reynolds number ( re ) , a ratio of inertial force to viscous force , was used to quantify the mixing.1where is a fluid density , is a fluid viscosity , v is a velocity , and q is a flow rate . for jets mixing , a is a diameter of an inlet nozzle , and s is a cross sectional area of an inlet nozzle.re higher than a transition value , which mainly depends on a mixer design , indicates a sufficient mixing quality producing an asymptotic mean particle size . johnson and prudhomme reported that the transition of this t - mixer corresponded to a jet velocity of 2.1 m / s by mixing thf and water at 35 c . with a room temperature correction to and , the jet velocity of 6.1 m / s in this study was still high enough for providing sufficient mixing . in most of the previous studies by others , the two stream jets mixer required an equal momentum from two opposite streams , and re was usually reported with re1 of a single stream . however , a four stream jets mixer allowed unequal momentums , and re was usually reported by accumulating multiple streams . in this study , the density ( 960 kg / m ) and viscosity ( 1.66 mpas ) of a 50:50 thf / water mixture in the chamber were used to calculate re , which to a certain extent was able to better compare with a situation of mixing two identical streams . a mean re of 1770 for a single stream and the accumulation of 3540 for two the concentration of the final product in 95 ml of h2o and 5 ml of thf was 0.05 wt % . the residence time was about 110 ms from entering the chamber to falling into the beaker . the total injection time was about 5 s. all -carotene experiments were done with a four - stream vortex mixer . typically two of the mixer inlets were connected to two gastight plastic syringes ( 60 ml , kendall monojet ) via teflon tubing with 1.6 mm i d . each plastic syringe contained 45 ml of water and was driven by an infusion syringe pump ( harvard apparatus , model 945 ) . the other two inlets were connected to two gastight glass syringes ( 10 ml , sge ) via teflon tubing . one of the syringes contained 5 ml of a -carotene ( 50 mg ) and bcp ( 50 mg ) thf solution ; another contained 5 ml of pure thf . the two glass syringes were driven by a second infusion syringe pump ( harvard apparatus , phd 2000 programmable ) . the pumps propel the four streams at high velocity into the small mixing chamber , generating high turbulence . complete dimensions ( see figure s1b in supporting information or figure 4 in ref ( 20 ) ) and evaluation of mixing performance using competitive reactions are given by liu et al . the flow rates were 120 ml / min for the plastic syringes and 13.3 ml / min for the glass syringes . from these flow rates , an re of 3000 ( higher than the transition value of 450 ) was calculated , using the relation ( eq 2 ) reported in refs ( 19 and 20):2where i is the density of the ith component , qi is the flow rate of the ith component , ai in this study is the shorter width of the ith inlet nozzle ( 1.1 10 m ) , si is the cross sectional area of the ith inlet nozzle ( 1.65 10 m ) , and i is the viscosity of the ith component . the two water streams dominate re , and this study assumes i = 1.0 10 kgm and i = 8.9 10 pas at room temperature . the outlet of the mixer was connected via a teflon tubing to a beaker , where the nanosuspensions were collected without further dilution . the concentration of the final product in 90 ml of h2o and 10 ml of thf was 0.1 wt % . the total injection time was about 23 s. all samples were analyzed in the as - mixed liquid , water with 510% of thf , and also without and with 1 wt % of saline added to this thf / water solution . saline was used to test the electrostatic stability of the particles ; 1 wt % was chosen because it is similar to the ion concentration in body fluids . the particle size and distribution were determined by dynamic light scattering ( dls ) using a zetapals ( brookhaven instruments , diode laser bi - dpss wavelength of 659 nm , round cuvette ) . the light intensity correlation function was collected at 25 c and a scattering angle of 90. the correlation function is a combination of the diffusion coefficient , di , of each particle which is converted into particle diameter , di , with the stokes einstein equation ( eq 3),3where kb is the boltzmann constant and t is the absolute temperature . repes yields a series of discrete particle diameters to represent the particle size distribution . it has been found more accurate than the cumulant model used in most commercial instruments . the software , gendist , was used to solve the repes algorithm and provided the size in an intensity distribution . the intensity averaged particle size , di , is defined in eq 4,4where ni is the number of particles with a diameter of di . the mass averaged diameter , dm , is more practically useful than the usual intensity average for estimating drug loading and availability . it is defined in eq 5,5where mi is the mass of a particle with a diameter of di . as discussed in our previous work , the systematic errors including both reproducibility of mixing and property measurements were within 10% for dm . cryogenic transmission electron microscopy ( cryo - tem ) specimens were prepared as described also in ref ( 23 ) and were imaged at about 170 c and 120 kv acceleration voltage by a gatan us1000 cooled ccd camera . the scanning electron microscopy ( sem ) specimens were prepared as described also in ref ( 23 ) and then were sputter - coated with a 30 layer of platinum and imaged with a jeol 6500 sem . high - performance liquid chromatography ( hplc ) was used to measure the concentration of the encapsulated paclitaxel in the nanoparticles and the free paclitaxel in thf and water mixture . the paclitaxel nanoparticles were removed from 0.5 ml of the suspension using a centrifugal filter ( ym-100 , microcon ) with a membrane cutoff of 100 kda ( 8 nm pore size ) under 12 000 g. the filtrate was freeze - dried . then 0.5 ml of thf the filtered nanoparticles were freeze - dried and extracted by 5 ml of methanol / thf ( 4:1 ) with stirring overnight . the carrier solvent was acetonitrile / ammonium acetate ( 10 mmoll ) in water in ph 4 ( adjusted with glacial acetic acid ) ( mobile phase ratio 55/45 ) eluted through a c18 rp ( beckman ) hplc column at a flow rate of 1 ml / min . the paclitaxel was detected by uv vis detector ( beckman 168 ) at the wavelength of 228 nm . the peak retention time was about 7 min , and the run time was 10 min . the cdl % is defined as the ratio of the mass of the drug trapped in the nanoparticles to the total mass of the nanoparticles . 90.2% of paclitaxel was precipitated as the nanoparticles , and 9.8% was in free molecules in the filtrate . the chemical structures were drawn by acd / chemsketch ( freeware downloaded from www.acdlabs.com , product version 12.01 ) , and their logp were calculated with the acdlogp add - on . there are three sources of nanoparticle instability , i.e. , aggregation , ostwald ripening , and recrystallization . as discussed in our previous work , polyelectrolytes , such as chitosan , or amphiphilic bcps , such as plga - b - peg , were able to effectively hinder the nanoparticle aggregation . in this study , plga - b - peg was used as a model surfactant , whose hydrophobic block was noncrystallizable as well as had relatively high glass transition temperature and a right solubility parameter , ensuring no unexpected particle destabilization is introduced by this additive . it was also showed that the molecular weights of the plga block over the range from 5k to 15k and the peg block over the range from 2k to 5k had insignificant effects on the particle stability . plga(10k)-b - peg(2k ) was used in this study , and figure 2 showed plga(10k)-b - peg(2k ) nanoparticles made via fnp either without or with -carotene . the plga - b - peg/-carotene particles were relatively stable for at least 3 weeks with the size slowly increasing from 57 to 90 nm . sem images ( cryo - tem insert ) and particle size distribution by dls of plga(10k)-b - peg(2k ) ( 50 mg ) nanoparticles ( a , c ) without , and ( b , d ) with -carotene ( 50 mg ) made with 10 ml of thf and 90 ml of h2o ( all scale bars are 100 nm ) . ostwald ripening is the phenomenon by which small particles are essentially consumed by large particles during the growth process , since the small particles have a higher solubility than the large particles as described by the kelvin equation . ostwald ripening was first described by lifshitz , slyozov , and wagner ( lsw ) . for a diffusion - controlled process , the average radius of the particles , r , changes with time according to67where k is a rate constant , is an interfacial energy , d is a diffusion coefficient of the solute molecule , is a volume of a solute molecule , t is time , ceq is a solute solubility , c is a solute concentration , and s is a supersaturation ratio , c / ceq . all values are in terms of the solution , such as the mixture of 10 vol % of thf and 90 vol % of h2o ( noted as 1thf/9h2o ) used in this study . the particle size is thus proportional to t. with a specific drug compound , only ceq and c alter the coarsening rate , dr/dt . with a constant c , increasing the ratio of the organic solvent over water increases ceq and thus dr/dt . to inhibit the ostwald ripening for a given hydrophobic drug , therefore , a minimum usage of the organic solvent is desired . for most hydrophobic drugs , the range of , d , and ceq dominates the ostwald ripening , and a lower ceq is desired for the stability . in experiments , the solubility of paclitaxel was 25 gml in 1thf/19h2o . the plga - b - peg / paclitaxel nanoparticles grew from about 100 nm to tens of micrometers within 90 min ( see figure 3a and b ) . the -carotene particles without adding any stabilizer spent about four hours growing from 89 nm to about 180 nm , and all of the particles sedimented on the bottom with the colorless supernatant within one day . with plga - b - peg , the particle size slowly increased from 57 to 90 nm in 3 weeks and was much more stable than plga - b - peg / paclitaxel nanoparticles . paclitaxel 2,7-bis(triethoxysilicate ) even had a lower solubility , was out of the detection limit of hplc , and was not able to be obtained in this study ( < 1 ngml ) . the particles spent 8 days growing from 55 to 153 nm ( see figure 4c and d ) . with plga - b - peg , the particle size slowly increased from 86 to 102 nm in 6 days ( see figure 5b ) . these comparisons showed that by changing the drug solute , the lower the drug solubility in the aqueous medium , the more stable the particles made via fnp . this observation was consistent with the prediction by the lsw theory above that ceq dominated the ostwald ripening , and a lower ceq was desired for the stability . on ostwald ripening of -carotene nanoparticles where the -carotene nanoparticles were made via fnp but changing the ratio of thf over water . they made the same conclusion that with fnp the lower the solute solubility in the mixture , the slower the growth rate of the particles . sem images of plga(10k)-b - peg(2k ) ( 25 mg)/paclitaxel ( 25 mg ) nanoparticles made with 5 ml of thf and 95 ml of h2o ( a ) sprayed within 1 min on a silicon wafer ( the scale bar is 100 nm ) and ( b ) sprayed within 90 min , showing grown paclitaxel needles ( the scale bar is 10 m ) . particle size and distribution ( c ) in 30 min and ( d ) in 120 min . as described by the kelvin equation ( eq 8) , small particles have a higher solubility than large particles.8where ceq(r ) is a solubility surrounding a particle of a radius r , and ceq( ) is a bulk solubility . the capillary length , l , is a characteristic length below which curvature - induced solubility is significant and defined as l 2/kbt . as predicted by the lsw theory , a higher ceq of small particles is able to accelerate ostwald ripening . therefore , the initial particle size distribution is expected to have an effect in some degree on the particle stability and is discussed here . with bcps , the particles of a hydrophobic drug made via fnp were believed to be surrounded by amphiphilic bcps via a kinetic process during drug precipitation , and the interfacial energy of the particles was reported by johnson et al . as = 1.9 10 jm . liu et al . used this value and well predicted ostwald ripening of pegylated bcp protected -carotene nanoparticles in thf / water ( 6/12030/120 ) mixtures . this value is thus taken herein . with a molecular weight of 854 gmol and an assumption of the drug density of 1.0 10 kgm , of 1.42 10 mmolecule and l of 1.33 nm at room temperature can be estimated . since l / r 1 , eq 8 can be linearized into eq 9.9if the particle size distribution at 30 min given by dls in figure 3c is considered as the initial distribution of the plga - b - peg / paclitaxel nanoparticles , the solubility ratio of the lower radius limit ( rmin ) over the upper ( rmax ) , ceq(16 nm)/ceq(314 nm ) , drug = 7.28 10 6.85 10 = 4.3 10 jm ( surface tension of drug drug = 6.85 10 jm calculated with acd / i - lab ) . l of 3.02 nm and ceq(16 nm)/ceq(314 nm ) of 1.18 were also calculated . compared with solubility changes by using other drugs , this small solubility difference ( ceq(rmin)/ceq(rmax ) 1 ) between large and small paclitaxel particles was insignificant . therefore , for plga - b - peg / paclitaxel nanoparticles the effect of the particle size distribution on ostwald ripening was negligible . drug = 3.65 10 jm ( drug = 3.63 10 jm calculated with acd / i - lab ) and molecular weight of 537 gmol . l of 16.1 nm and ceq(13 nm)/ceq(249 nm ) of 3.23 at 10 min were estimated by eq 8 . in the same way , for paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles ( see figure 4c ) with = water drug = 2.05 10 jm ( drug = 5.23 10 jm calculated with acd / i - lab ) and molecular weight of 1178 gmol , l of 19.8 nm and ceq(8 nm)/ceq(125 nm ) of 10.1 at 10 min were estimated . by comparing the initial ceq(rmin)/ceq(rmax ) , plga - b - peg / paclitaxel nanoparticles ( 1.08 ) < -carotene nanoparticles ( 3.23 ) < paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles ( 10.1 ) . as predicted by the lsw theory , the stability trend should be plga - b - peg / paclitaxel nanoparticles > -carotene nanoparticles > paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles . however , this stability trend was opposite to the observed trend . therefore , compared with the solubility changes by using different drugs , the solubility difference between the small and the large particles made via fnp was considered to have an insignificant effect on the particle stability . the particle size distributions were narrow enough in terms of ostwald ripening to study particle stability . the third instability source is recrystallization , which converts amorphous particles into crystalline ones , since amorphous drugs have a higher solubility than the crystalline counterparts . solvent molecules dissolved in the solute matrix provide free volume , facilitate the relaxation of the solute , and increase the rate of recrystallization inside the particles . since intraparticle recrystallization barely changes the nanoparticle volume and all instable systems in this study showed a significant volume increase of individual particles , intraparticle recrystallization would not be studied in this paper . the process requires solute molecules migrate from one particle to another via either ostwald ripening or particle aggregation . by adding surface steric stabilizer , such as plga - b - peg ostwald ripening again as the source of particle instability and shown above has to be considered in this study . as discussed above with the lsw theory , the solubility of ceq plays an important role on nanoparticle stability . for a given drug compound , the water solubility can be decreased by ( 1 ) decreasing the ratio of organic solvent over water , ( 2 ) choosing a relatively poor organic solvent , or ( 3 ) being chemically modified , such as bonding to a hydrophobic moiety or form a salt . however , the amount of organic solvent was limited by the solubility of a drug in it and processing conditions during feeding and can not be infinitely decreased . feeding more water to decrease the ratio of organic solvent over water will dilute the product too much , and the concentration is too low . the option of organic solvents is limited by water miscibility , solvent toxicity , and easiness of solvent removal . a possible approach is to modify the chemical structure of the drug and make it less soluble . in this study , therefore , paclitaxel was chemically bonded with ethoxysilicate ( see scheme s1a in supporting information ) , which was a hydrophobic moiety and was expected to be easily cleaved under an acidic condition in tumors . much work on hydrolysis of various paclitaxel organosilicate vs ph was studied by wohl and showed promising results for a controlled release in a mimic condition of tumor cells . while at neutral ph , it was chemically very stable . in this study , as shown in figure 4c and d , even without the steric stabilizer of plga - b - peg , dm of the paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles only increased from 55 to 153 nm in 8 days , showing significant improvement of the particle stability compared with plga - b - peg / paclitaxel particles with grown micrometer needles in 90 min ( see figure 3b ) . figure 4a and b showed that the particles remain spherical for at least 8 days . the improved stability in the morphology and the size indicated that the ostwald ripening and recrystallization were significantly inhibited . sem images of paclitaxel 2,7-bis(triethoxysilicate ) ( 35 mg , molar equivalent to 25 mg of paclitaxel ) nanoparticles made with 5 ml of thf and 95 ml of h2o ( a ) sprayed within 1 min on a silicon wafer and ( b ) sprayed in 8 days ( both scale bars are 100 nm ) . particle size and distribution w / o saline ( c ) in 10 min and ( d ) in 8 days . = + 24 mv before adding saline , and the nanoparticles grew to visually detectable size instantly after adding 1 wt % saline and had = + 2.8 mv . it was found that the paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles without the stabilizer had surface charges ( = + 24 mv ) to inhibit the aggregation . after adding 1 wt % saline , however , decreased to + 2.8 mv . the steric stabilizer , plga - b - peg , was thus used to inhibit the aggregation . as shown in figure 5b , the particles had a good stability with size increasing from 86 to 102 nm after 6 days without saline . after adding 1 wt % saline in the 6th day , no visible aggregation was observed . plga(10k)-b - peg(2k ) ( 25 mg ) protected paclitaxel 2,7-bis(triethoxysilicate ) ( 35 mg , molar equivalent to 25 mg of paclitaxel ) nanoparticles made with 5 ml of thf and 95 ml of h2o and ( a ) sprayed within 1 min on a silicon wafer ( the scale bar is 100 nm ) ; ( b ) particle stability against time for 6 days ; ( c ) particle size distribution by dls in 20 min without saline . no noticeable size increase after adding saline in the 6th day , indicating the nanoparticles were sterically stabilized by plga(10k)-b - peg(2k ) . as described with the lsw theory as well as observed in above experiments , the solubility of a hydrophobic compound has dominant effects on the stability of the formed nanoparticles in terms of ostwald ripening and interparticle recrystallization . for a random given drug to generate nanoparticles via the fnp , it would be good to measure first the solubility in the solvent / antisolvent mixture . if changing the ratio of the solvent to antisolvent is necessary to optimize the process , a phase diagram is desired as well . in practice , however , measuring the solubility and stability could be very time - consuming , a quantitative relation between solubility with stability is unclear , and generated nanoparticles are not necessarily sufficiently stable . therefore , having a theoretical indication of the solubility , the correlation of this indication with the particle stability and then a prediction of the stability are very meaningful . this approach is also very useful to give a guideline before doing any chemical modification of a drug compound . in this study , two well - known physical parameters , hildebrand solubility parameter ( ) and logp , were investigated to tentatively build the correlation between the solubility and the stability . provides a numerical estimate of the degree of interaction between materials , particularly for nonpolar materials such as many polymers . the octanol water partition coefficient is a ratio of concentrations of un - ionized compound between immiscible octanol with water . it is one of simple molecular descriptors in lipinski s rule of 5 . it serves as a quantitative indication of lipophilicity and has been widely employed in the pharmaceutical industry . hydrocortisone , hydrocortisone ethoxysilicate , and betulin were therefore added to the list for the correlation study . as reported , hydrocortisone has a water solubility of 0.3 mg / ml . after adding 10 vol % of good solvent , i.e.,thf as expected , 50 mg of hydrocortison in 10 ml of thf and 90 ml of h2o did not generate a detectable scattered intensity by dls , indicating nanoparticles were not generated since the solubility was too high . its analogue , hydrocortisone ethoxysilicate , was thus synthesized to increase the hydrophobicity and lower the solubility . the nanoparticles ( 50 mg ) of hydrocortisone ethoxysilicate had dm of 252 nm after 10 min than the formation in 10 ml of thf and 90 ml of h2o and showed fast increasing size in the next 10 min during the dls measurement . unfortunately , hydrocortisone ethoxysilicate hydrolyzed fast back into more hydrophilic hydrocortisone , and the nanoparticle size decreased as the time went during the next six days ( see figure s2 in supporting information ) . it therefore was not a good candidate for studying the nanoparticle stability herein but able to give another example of the fast size increase at least in the first 20 min . the suspension changed from water clear to cloudy with visible needles within 30 min , indicating fast ostwald ripening and recrystallization . the sem image ( see figure s3 in supporting information ) showed grown crystalline needles sprayed on a silicon wafer within 30 min after mixing . the reason could come from the limitation of , which was not suitable for polar compounds especially with hydrogen bonds ( such as water ) . on the contrary empirically , with acdlogp > 12 , nanoparticles showed good stability ; with 2 < acdlogp < 9 , nanoparticles showed fast ostwald ripening and recrystallization ; with acdlogp < 2 , the drug is too soluble and very likely difficult to generate nanoparticles . in order to fill the gap of this rule , over 2000 anticancer drugs in the nci dictionary were also screened , and very few real drugs have been found to have acdlogp of greater than 9 , since most of the super hydrophobic potential drugs had been abandoned by the pharmaceutical industry due to an extremely low dissolution rate . but one would reasonably expect that a drug with 9 < acdlogp < 12 is marginal . estimated with the hoye method as well as assumed to treat the si atom as a c atom . it was found that without adding any surface stabilizer -carotene particles made via fnp showed good short - term ( 4 h ) stability due to slightly negative surface charge as judged by zeta potential measurements . paclitaxel 2,7-bis(triethoxysilicate ) particles in this study ( see figure 4 ) showed no sediment for 8 days . compared with the time of the measurements and possible postprocessing , this stability was long enough . by using -carotene therein , the effects of the hydrophobic drug on particle instability were able to be removed so as the effects of mixer designs , mixing processes , and surface stabilizers can be individually studied . it was also found that even with sufficient mixing some polymeric stabilizers ( e.g. , pla - b - peg and pcl - b - peg ) destabilized the particles due to their undesired physical properties ( e.g. , relatively low glass transition , polymer crystallization , and unsuitable solubility parameters ) . however , plga - b - peg , an amphiphilic diblock copolymer , had no negative impact on -carotene particle stability . moreover , with very similar experimental conditions of this study , the molecular weight of plga block over the range from 5k to 15k showed an insignificant effect on controlling the particle stability . plga - b - peg as a model polymeric surfactant therefore was used in this study to investigate the effects of the hydrophobic drugs . the empirical rule above was based on this surfactant , which did not have undesired physical properties to rather destabilize the particles like pcl - b - peg or pla - b - peg . it is known that the solubility of a drug in the water / solvent mixture also depends on the type of a solvent and a feed ratio with water . the drug and polymer have to be molecularly dissolved in a solvent before jets mixing with water . with a fixed amount of drug or polymer , the solvent had a minimum amount . however , adding too much solvent required much more water to obtain either a high drug recovery or a stable nanosuspension with limited ostwald ripening and recrystallization , which decreased the final concentration of the drug suspension . it has been found that in the fnp technique for -carotene , paclitaxel and its prodrugs , 50 mg of a drug ( or with extra 10 to 50 mg of a polymer ) dissolving in 10 ml of a solvent and mixed with 90 ml of water ( 0.5 mg / ml of a drug in production ) was the most suitable combination . the above empirical rule was based on this combination at room temperature . for some cases , water was doubled in purpose , but no further dilution was taken , which would trouble the particle postprocessing by freeze or spray drying . in table 2 , thf was a relative hydrophobic solvent , and a good solvent for many organic drugs as well as for many common polymers . for drugs with acdlogp > 2 , < 2 , a less hydrophobic solvent such as acetone , ethanol ( with acdlogp slightly lower than zero ) , or their mixture with thf can be considered to generate instable naoparticles , which could decrease this empirical value . but it should be noted that logp of a substance is most relevant for neutral substances and is useful as a general reference point to help compare overall hydrophobicity trends of compounds . logp does not account for modifications in the hydrophobicity of ionizable compounds at varying ph . the appropriate descriptor for these compounds is the distribution coefficient , d ( also typically used in its logarithmic form , logd ) . since the software to calculate logd is not free for the public , logp would be better to demonstrate this work to the interested readers . the algorithm model used in this study is acdlogp developed by the acd company , since this model has been used by some of the world s largest pharmaceutical companies ( e.g. , glaxosmithkline and pfizer ) and the acd company also developed logd model . there are various similar algorithm models available ( e.g. , alogp , alogps , ablogp , aclogp , cosmofraq , clogp , mlogp , milogp , prologp , xlogp , and logkow ) . each algorithm model has its own strengths and exceptions , but the comparable logd model is not developed for all logp models . depending on the water miscible organic solvent used in the fnp ( e.g. , thf , acetone , ethanol , or their mixtures ) , different algorithm models of logp possibly need to be tested for the exceptions . in this study , the effects of the hydrophobic drug molecules on particle stability were investigated . the work demonstrated that chemically bonding a drug compound ( e.g. , paclitaxel ) with a cleavable hydrophobic moiety of organosilicate ( e.g. , triethoxysilicate ) was able to significantly improve the particle stability , expectedly due to a decreased drug solubility and thus lowered interparticle molecular migration . this modification opened an approach to enhance the particle stability generated by fnp . even without any surfactant but with slight surface charges , paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles showed moderate stability ( no sediment for 8 days ) . to better stabilize the particles , plga - b - peg was used as a model surface stabilizer , whose hydrophobic block was noncrystallizable as well as had relatively high glass transition temperature and a right solubility parameter , ensuring no unexpected particle destabilization introduced by this additive . by changing the solute with various drugs mostly from the nci drug dictionary and their analogues , the study showed that the lower the solubility in the aqueous medium the greater the particle stability in terms of ostwald ripening , which was consistent with the prediction by the lsw theory . the particle size distribution made via fnp was sufficient narrow . compared with a solubility change by using a different drug solute , the particle solubility between small and large particles showed a negligible effect on ostwald ripening . the experiments showed that the initial particle size distribution made via fnp was bimodal or even trimodal rather than lognormal . since the dls apparatus typically can not differentiate size peaks within 3-fold , in some case the distribution appeared unimodal . very little has been known about the fnp kinetics which evolves in micro to miliseconds and a nanoscale . this study considers the non - lognormal and non - unimodal size distribution as evidence for cluster cluster aggregation rather than nucleation and growth . to correlate the drug hydrophobicity with particle stability , and logp were used as hydrophobicity indications for the drug compounds . empirically , with acdlogp > 12 , nanoparticles showed good stability ; with 2 < acdlogp < 9 , nanoparticles showed fast ostwald ripening and interparticle recrystallization ; with acdlogp < 2 , the drug was too soluble and very likely difficult to generate nanoparticles . with 9 this work introduced logp into the flash nanoprecipitation , created a quick way to predict particle stability for a randomly selected drug structure enabling a fast preclinical drug screen , and provided a possible approach to enhance the particle stability .

flash nanoprecipitation ( fnp ) can generate hydrophobic drug nanoparticles in 100 nm with a much higher drug loading ( e.g. , > 40 wt % ) than traditional nanocarriers ( e.g. , < 20 wt % ) . this paper studies the effects of drug molecules on nanoparticle stability made via fnp and demonstrates that chemically bonding a drug compound ( e.g. , paclitaxel ) with a cleavable hydrophobic moiety of organosilicate ( e.g. , triethoxysilicate ) is able to enhance the particle size stability . a nonionic amphiphilic diblock copolymer , poly(lactic - co - glycolic acid)-block - poly(ethylene glycol ) ( plga - b - peg ) , is used as a model surfactant to provide steric stabilization . the experiments here show that the lower the drug solubility in the aqueous medium , the more stable the particles in terms of ostwald ripening , which are consistent with the prediction by the lsw theory . the initial particle size distribution is sufficiently narrow and of insignificance to ostwald ripening . to correlate the particle stability with hydrophobicity , this study introduces the n - octanol / water partition coefficient ( logp ) , a hydrophobicity indication , into the fnp technique . a comparison of various drugs and their analogues shows that logp of a drug is a better hydrophobicity indication than the solubility parameter ( ) and correlates well with the particle stability . empirically , with acdlogp > 12 , nanoparticles have good stability ; with 2 < acdlogp < 9 , nanoparticles show fast ostwald ripening and interparticle recrystallization ; with acdlogp < 2 , the drug is very likely difficult to form nanoparticles . this rule creates a quick way to predict particle stability for a randomly selected drug structure and helps to enable a fast preclinical drug screen .

Introduction Experimental Section Results and Discussion Conclusion

, micelles , liposomes , and nanoemulsions with a typical drug loading capacity ( cdl % ) below 20% , nanosuspensions ( usually called nanoparticles ) have a much higher drug loading and are able to show sufficient potency to kill tumors . , milling , high pressure homogenization , and the supercritical fluid process , flash nanoprecipitation ( fnp ) shows advantages of fast processing , simple equipment , smaller size , and narrower size distribution . in the fnp technique ( see figure 1 ) , a highly hydrophobic drug is dissolved along with a block copolymer ( bcp ) in a water miscible organic solvent . the block copolymer is amphiphilic : typically a hydrophilic poly(ethylene glycol ) ( peg ) block covalently bonded to a hydrophobic block . , water - soluble polyelectrolytes ( polylysine , polyethylene imine , and chitosan ) and nonionic amphiphilic diblock copolymers [ polystyrene - block - poly(ethylene glycol ) ( ps - b - peg ) , polycaprolactone - block - poly(ethylene glycol ) ( pcl - b - peg ) , polylactide - block - poly(ethylene glycol ) ( pla - b - peg ) , and poly(lactic - co - glycolic acid)-block - poly(ethylene glycol ) ( plga - b - peg ) ] , have been explored to investigate their effects on the particle formation and stability . up to now , little work has been reported to investigate the effects of drug compounds on the particle stability , which will be discussed in this study . as demonstrated in our previous study , biodegradable plga - b - peg ( scheme 1 ) is a suitable steric stabilizer for the fnp to inhibit the particle aggregation , since the plga block is noncrystallizable as well as has relatively high glass transition temperature and a right solubility parameter ( ) , ensuring that no unexpected particle destabilization introduced by this additive . the aims of this study are to give a guideline to choose the suitable drug with good particle stability for flash nanoprecipitation especially at a high drug loading ( e.g. , > 40 wt % ) , to create an approach to predict the particle stability for a randomly selected drug structure , and to give a possible approach to improve the particle stability . for the purpose of predicting particle stability , the n - octanol / water partition coefficient ( logp ) , a hydrophobicity indication , will be introduced into the fnp technique . the properties of n - octanol has been thought to resemble to those of lipid bilayer membranes , suggesting to some extent that a drug partition in octanol / water simulates its ability to passively diffuse across biological membranes . an empirical rule correlating logp with the particle stability made via fnp will be given to help enable a fast preclinical drug prescreen . the reynolds number ( re ) , a ratio of inertial force to viscous force , was used to quantify the mixing.1where is a fluid density , is a fluid viscosity , v is a velocity , and q is a flow rate . for jets mixing , a is a diameter of an inlet nozzle , and s is a cross sectional area of an inlet nozzle.re higher than a transition value , which mainly depends on a mixer design , indicates a sufficient mixing quality producing an asymptotic mean particle size . with a room temperature correction to and , the jet velocity of 6.1 m / s in this study was still high enough for providing sufficient mixing . in this study , the density ( 960 kg / m ) and viscosity ( 1.66 mpas ) of a 50:50 thf / water mixture in the chamber were used to calculate re , which to a certain extent was able to better compare with a situation of mixing two identical streams . as discussed in our previous work , polyelectrolytes , such as chitosan , or amphiphilic bcps , such as plga - b - peg , were able to effectively hinder the nanoparticle aggregation . in this study , plga - b - peg was used as a model surfactant , whose hydrophobic block was noncrystallizable as well as had relatively high glass transition temperature and a right solubility parameter , ensuring no unexpected particle destabilization is introduced by this additive . the plga - b - peg/-carotene particles were relatively stable for at least 3 weeks with the size slowly increasing from 57 to 90 nm . sem images ( cryo - tem insert ) and particle size distribution by dls of plga(10k)-b - peg(2k ) ( 50 mg ) nanoparticles ( a , c ) without , and ( b , d ) with -carotene ( 50 mg ) made with 10 ml of thf and 90 ml of h2o ( all scale bars are 100 nm ) . for a diffusion - controlled process , the average radius of the particles , r , changes with time according to67where k is a rate constant , is an interfacial energy , d is a diffusion coefficient of the solute molecule , is a volume of a solute molecule , t is time , ceq is a solute solubility , c is a solute concentration , and s is a supersaturation ratio , c / ceq . the particle size is thus proportional to t. with a specific drug compound , only ceq and c alter the coarsening rate , dr/dt . to inhibit the ostwald ripening for a given hydrophobic drug , therefore , a minimum usage of the organic solvent is desired . the plga - b - peg / paclitaxel nanoparticles grew from about 100 nm to tens of micrometers within 90 min ( see figure 3a and b ) . with plga - b - peg , the particle size slowly increased from 57 to 90 nm in 3 weeks and was much more stable than plga - b - peg / paclitaxel nanoparticles . with plga - b - peg , the particle size slowly increased from 86 to 102 nm in 6 days ( see figure 5b ) . these comparisons showed that by changing the drug solute , the lower the drug solubility in the aqueous medium , the more stable the particles made via fnp . this observation was consistent with the prediction by the lsw theory above that ceq dominated the ostwald ripening , and a lower ceq was desired for the stability . they made the same conclusion that with fnp the lower the solute solubility in the mixture , the slower the growth rate of the particles . as described by the kelvin equation ( eq 8) , small particles have a higher solubility than large particles.8where ceq(r ) is a solubility surrounding a particle of a radius r , and ceq( ) is a bulk solubility . as predicted by the lsw theory , a higher ceq of small particles is able to accelerate ostwald ripening . therefore , the initial particle size distribution is expected to have an effect in some degree on the particle stability and is discussed here . with bcps , the particles of a hydrophobic drug made via fnp were believed to be surrounded by amphiphilic bcps via a kinetic process during drug precipitation , and the interfacial energy of the particles was reported by johnson et al . since l / r 1 , eq 8 can be linearized into eq 9.9if the particle size distribution at 30 min given by dls in figure 3c is considered as the initial distribution of the plga - b - peg / paclitaxel nanoparticles , the solubility ratio of the lower radius limit ( rmin ) over the upper ( rmax ) , ceq(16 nm)/ceq(314 nm ) , drug = 7.28 10 6.85 10 = 4.3 10 jm ( surface tension of drug drug = 6.85 10 jm calculated with acd / i - lab ) . therefore , for plga - b - peg / paclitaxel nanoparticles the effect of the particle size distribution on ostwald ripening was negligible . by comparing the initial ceq(rmin)/ceq(rmax ) , plga - b - peg / paclitaxel nanoparticles ( 1.08 ) < -carotene nanoparticles ( 3.23 ) < paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles ( 10.1 ) . as predicted by the lsw theory , the stability trend should be plga - b - peg / paclitaxel nanoparticles > -carotene nanoparticles > paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles . therefore , compared with the solubility changes by using different drugs , the solubility difference between the small and the large particles made via fnp was considered to have an insignificant effect on the particle stability . the particle size distributions were narrow enough in terms of ostwald ripening to study particle stability . by adding surface steric stabilizer , such as plga - b - peg ostwald ripening again as the source of particle instability and shown above has to be considered in this study . as discussed above with the lsw theory , the solubility of ceq plays an important role on nanoparticle stability . for a given drug compound , the water solubility can be decreased by ( 1 ) decreasing the ratio of organic solvent over water , ( 2 ) choosing a relatively poor organic solvent , or ( 3 ) being chemically modified , such as bonding to a hydrophobic moiety or form a salt . however , the amount of organic solvent was limited by the solubility of a drug in it and processing conditions during feeding and can not be infinitely decreased . in this study , therefore , paclitaxel was chemically bonded with ethoxysilicate ( see scheme s1a in supporting information ) , which was a hydrophobic moiety and was expected to be easily cleaved under an acidic condition in tumors . in this study , as shown in figure 4c and d , even without the steric stabilizer of plga - b - peg , dm of the paclitaxel 2,7-bis(triethoxysilicate ) nanoparticles only increased from 55 to 153 nm in 8 days , showing significant improvement of the particle stability compared with plga - b - peg / paclitaxel particles with grown micrometer needles in 90 min ( see figure 3b ) . the improved stability in the morphology and the size indicated that the ostwald ripening and recrystallization were significantly inhibited . the steric stabilizer , plga - b - peg , was thus used to inhibit the aggregation . as shown in figure 5b , the particles had a good stability with size increasing from 86 to 102 nm after 6 days without saline . plga(10k)-b - peg(2k ) ( 25 mg ) protected paclitaxel 2,7-bis(triethoxysilicate ) ( 35 mg , molar equivalent to 25 mg of paclitaxel ) nanoparticles made with 5 ml of thf and 95 ml of h2o and ( a ) sprayed within 1 min on a silicon wafer ( the scale bar is 100 nm ) ; ( b ) particle stability against time for 6 days ; ( c ) particle size distribution by dls in 20 min without saline . as described with the lsw theory as well as observed in above experiments , the solubility of a hydrophobic compound has dominant effects on the stability of the formed nanoparticles in terms of ostwald ripening and interparticle recrystallization . for a random given drug to generate nanoparticles via the fnp , it would be good to measure first the solubility in the solvent / antisolvent mixture . therefore , having a theoretical indication of the solubility , the correlation of this indication with the particle stability and then a prediction of the stability are very meaningful . in this study , two well - known physical parameters , hildebrand solubility parameter ( ) and logp , were investigated to tentatively build the correlation between the solubility and the stability . on the contrary empirically , with acdlogp > 12 , nanoparticles showed good stability ; with 2 < acdlogp < 9 , nanoparticles showed fast ostwald ripening and recrystallization ; with acdlogp < 2 , the drug is too soluble and very likely difficult to generate nanoparticles . in order to fill the gap of this rule , over 2000 anticancer drugs in the nci dictionary were also screened , and very few real drugs have been found to have acdlogp of greater than 9 , since most of the super hydrophobic potential drugs had been abandoned by the pharmaceutical industry due to an extremely low dissolution rate . by using -carotene therein , the effects of the hydrophobic drug on particle instability were able to be removed so as the effects of mixer designs , mixing processes , and surface stabilizers can be individually studied . , pla - b - peg and pcl - b - peg ) destabilized the particles due to their undesired physical properties ( e.g. however , plga - b - peg , an amphiphilic diblock copolymer , had no negative impact on -carotene particle stability . moreover , with very similar experimental conditions of this study , the molecular weight of plga block over the range from 5k to 15k showed an insignificant effect on controlling the particle stability . plga - b - peg as a model polymeric surfactant therefore was used in this study to investigate the effects of the hydrophobic drugs . the empirical rule above was based on this surfactant , which did not have undesired physical properties to rather destabilize the particles like pcl - b - peg or pla - b - peg . it is known that the solubility of a drug in the water / solvent mixture also depends on the type of a solvent and a feed ratio with water . however , adding too much solvent required much more water to obtain either a high drug recovery or a stable nanosuspension with limited ostwald ripening and recrystallization , which decreased the final concentration of the drug suspension . it has been found that in the fnp technique for -carotene , paclitaxel and its prodrugs , 50 mg of a drug ( or with extra 10 to 50 mg of a polymer ) dissolving in 10 ml of a solvent and mixed with 90 ml of water ( 0.5 mg / ml of a drug in production ) was the most suitable combination . for drugs with acdlogp > 2 , < 2 , a less hydrophobic solvent such as acetone , ethanol ( with acdlogp slightly lower than zero ) , or their mixture with thf can be considered to generate instable naoparticles , which could decrease this empirical value . but it should be noted that logp of a substance is most relevant for neutral substances and is useful as a general reference point to help compare overall hydrophobicity trends of compounds . the algorithm model used in this study is acdlogp developed by the acd company , since this model has been used by some of the world s largest pharmaceutical companies ( e.g. depending on the water miscible organic solvent used in the fnp ( e.g. in this study , the effects of the hydrophobic drug molecules on particle stability were investigated . the work demonstrated that chemically bonding a drug compound ( e.g. , paclitaxel ) with a cleavable hydrophobic moiety of organosilicate ( e.g. , triethoxysilicate ) was able to significantly improve the particle stability , expectedly due to a decreased drug solubility and thus lowered interparticle molecular migration . this modification opened an approach to enhance the particle stability generated by fnp . to better stabilize the particles , plga - b - peg was used as a model surface stabilizer , whose hydrophobic block was noncrystallizable as well as had relatively high glass transition temperature and a right solubility parameter , ensuring no unexpected particle destabilization introduced by this additive . by changing the solute with various drugs mostly from the nci drug dictionary and their analogues , the study showed that the lower the solubility in the aqueous medium the greater the particle stability in terms of ostwald ripening , which was consistent with the prediction by the lsw theory . the particle size distribution made via fnp was sufficient narrow . compared with a solubility change by using a different drug solute , the particle solubility between small and large particles showed a negligible effect on ostwald ripening . the experiments showed that the initial particle size distribution made via fnp was bimodal or even trimodal rather than lognormal . to correlate the drug hydrophobicity with particle stability , and logp were used as hydrophobicity indications for the drug compounds . empirically , with acdlogp > 12 , nanoparticles showed good stability ; with 2 < acdlogp < 9 , nanoparticles showed fast ostwald ripening and interparticle recrystallization ; with acdlogp < 2 , the drug was too soluble and very likely difficult to generate nanoparticles . with 9 this work introduced logp into the flash nanoprecipitation , created a quick way to predict particle stability for a randomly selected drug structure enabling a fast preclinical drug screen , and provided a possible approach to enhance the particle stability .

the sacculated forestomachs of kangaroos and wallabies ( family macropodidae ) commonly contain large numbers of nematodes belonging to the strongylid sub - family cloacininae . for example , vendl and beveridge ( 2014 ) reported means and ranges in numbers of cloacinine nematodes in the stomachs of the red - necked wallaby , macropus rufogriseus , the eastern grey kangaroo , macropus giganteus and the swamp wallaby , wallabia bicolor , as 60,800 ( 2000210,000 ) , 20,500 ( 700079,000 ) and 20,000 ( 300058 , 000 ) , respectively . these data support earlier published figures for high intensities of infection in the western grey kangaroo , macropus fuliginosus , the red kangaroo , macropus rufus , and m. giganteus ( arundel et al . although there is considerable species diversity in the stomach - inhabiting cloacinine nematodes ( spratt et al . , 1991 ) , for instance , rugopharynx australis dominates the gastric helminth community of m. rufus , with mean and maximum burdens of 47,000 and 266,000 nematodes , respectively ( arundel et al . , 1979 ) . in spite of the numerical significance of this genus in the gastric helminth communities of macropodids , continuing taxonomic studies are needed , as it is unlikely that all species have yet been described and the presence of cryptic species could potentially complicate the interpretation of ecological data published to date . the genus rugopharynx was revised by beveridge ( 1982 ) , who recognised nine species . however , beveridge ( 1982 ) noted that r. australis was potentially a complex of a number of species , differentiable only by very minor and often overlapping morphological characteristics . multilocus enzyme electrophoretic ( mee ) studies , combined with morphological evidence , indicated the existence of two new species , r. sigma and rugopharynx mawsonae , both formerly confused with rugopharynx zeta ( chilton et al . 1994 ) , while an additional mee investigation of r. australis by chilton et al . subsequently , beveridge and chilton ( 1999 ) split r. australis into 10 species and resurrected r. alpha as a valid species . the latter revision was based on extrapolating the minor morphological differences identified in samples included in the earlier electrophoretic study across the entire species complex . in spite of this progress , there has been no attempt to independently verify the validity of the species erected to date using molecular methods . a closely related genus , rugonema was erected by beveridge ( 1999 ) for specimens formerly referred to as r. australis occurring in the stomach of the black - gloved wallaby , macropus irma , from western australia , based on morphological differences in the labial collar . because of this close association with rugopharynx , rugonema labiatum was also included in the present study . given the prevalence and abundance of this nematode genus in the stomachs of kangaroos and wallabies , and the diversity of species currently recognised based on minor morphological criteria , this study was undertaken to attempt to establish species boundaries within the genus based on sequences of the first and second internal transcribed spacers ( its-1 and its-2 ) of nuclear ribosomal dna . these molecular target regions have proved to be highly informative for the specific identification of a range of strongylid nematodes , including taxa within the cloacininae ( chilton , 2004 ) , the subfamily to which rugopharynx belongs . nematodes were obtained from the stomachs of a range of kangaroos and wallabies ( fig . 1 ; table 1 ) , which had been collected as fresh road - kills or from road - kills frozen prior to examination . host nomenclature follows van dyck and strahan ( 2008 ) . in instances where a nematode species occurred across a large geographical area , an attempt was made to include samples from different geographical regions of australia , particularly any occurring on the island of tasmania ( fig . 1 ) . australian state names are abbreviated as : nsw , new south wales ; qld , queensland ; sa , south australia ; tas , tasmania ; vic , victoria ; wa , western australia . nematodes were washed in saline , frozen in liquid nitrogen and stored at 80 c until morphological and molecular studies were undertaken . nematodes were then thawed , the head and tail of each worm removed , fixed in lactophenol and mounted permanently in polyvinyl lactophenol as voucher specimens . nematodes were identified according to previous descriptions ( beveridge , 1982 , beveridge et al . , 1994 , chilton et al . , 1993 , beveridge and chilton , 1999 , appan et al . , 2004 ) . voucher specimens have been deposited in the south australian museum ( sam ) , adelaide ( table 1 ) . species within the genus rugopharynx were divided into three groups based on the morphology of the buccal capsule ( table 1 ) . nematodes with a simple , cylindrical buccal capsule were designated as type i ( fig . 2 ) , while those with a buccal capsule divided into two sections were designated as type ii . these buccal capsules were further subdivided into species with a buccal capsule divided in the mid region ( r. epsilon ) ( type iia ) and those in which the division occurred in the anterior quarter ( r. rufogrisea ) ( type iib ) . species with a buccal capsule divided into three segments were designated as type iii . in some species , such as r. spratti and r. tau , the potential division of the buccal capsule is subtle ; hence , in these cases , species were classified as type i. genomic dna was isolated from the mid - body part of each nematode using a small - scale sodium - dodecyl - sulphate / proteinase k extraction method ( gasser et al . , 1993 ) , followed by purification the region of rdna comprising the its-1 , 5.8s rrna gene , its-2 and flanking sequences (= its+ ) was amplified by the pcr using primers nc16 ( forward ; 5-agttcaatcgcaatggctt-3 ) and nc2 ( reverse ; 5-ttagtttcttttcctccgct-3 ) . pcr was performed in a 50 l volume for 30 cycles at 94 c for 30 s ( denaturation ) , 55 c for 30 s ( annealing ) and 72 c for 30 s ( extension ) , followed by one cycle at 72 c for 5 min ( final extension ) . amplicons were purified using mini - columns ( using wizard pcr - preps , promega ) , and the its+ sequenced in both directions using the same primers ( separately ) as used for pcr . the sequences generated in the present study have been deposited in the genbank database ( accession numbers ln906946-ln906995 ; table 1 ) . sequences were initially aligned using the program muscle ( edgar , 2004 ) , and alignments adjusted manually using the program mesquite v.2.75 ( maddison and maddison , 2011 ) . phylogenetic analyses of the aligned sequence data were conducted by bayesian inference ( bi ) using monte carlo markov chain analysis in the program mrbayes v.3.2.2 ( ronquist and huelsenbeck , 2003 ) . the likelihood parameters set for the bi analysis of sequence data were based on the akaike information criteria test in jmodeltest v.2.1.5 ( posada , 2008 ) . the number of substitutions was set at 6 , with a gamma - distribution . for the tree , posterior probability ( pp ) values were calculated by running 2,000,000 generations with four simultaneous tree - building chains . the standard deviation of split frequencies was < 0.01 , and the potential scale reduction factor approached one . for each analysis , a 50%-majority rule consensus tree was constructed based on the final 75% of trees produced by bi . phylogenetic analyses of the its+ sequence data were also conducted using the neighbor - joining ( nj ) and maximum parsimony ( mp ) methods in paup * 4.0b10 ( swofford , 1999 ) . for the mp analyses , heuristic searches were carried out with random addition of sequences ( n = 100 ) , tree - bisection - reconstruction ( tbr ) branch swapping , the multrees option in effect , maxtrees set at 2000 and saving all equally parsimonious trees . the tree length ( l ) , consistency index , excluding uninformative characters ( ci ) and the retention index ( ri ) were recorded . bootstrap analyses ( 1000 replicates ) were conducted to determine the relative support for clades in the consensus trees ; nodal support was expressed as a percentage . the its+ sequences of labiostrongylus australis ( genbank accession numbers aj308403-aj308411 ) were used as the outgroup in the phylogenetic analyses , because this nematode species belongs to a related tribe within the same subfamily ( i.e. cloacininae ) ( see lichtenfels , 1980 ) to which rugopharynx also belongs ( beveridge , 1982 , chilton et al . , 1997 ) and because it has been relatively well studied genetically ( chilton et al . , 2009b ) . a consensus tree depicting the phylogenetic relationships of the nematodes was compared with a composite tree of the hosts . the host phylogenetic tree was based primarily on the molecular analyses of meredith et al . ( 2008 ) ; however , as some species ( e.g. , thylogale billardierii , macropus dorsalis , petrogale herberti , petrogale assimilis and petrogale inornata ) were not included in that study , the tree was modified to incorporate these hosts based on the studies of campeau - ploquin et al . , 2001 , cardillo et al . , 2004 and eldridge et al . m. ( osphranter ) bernardus and m. ( o. ) antilopinus , from which no species of rugopharynx have been reported . not all species of petrogale and thylogale are included in the phylogenetic tree , only those included in the present study . the length of the its+ region , excluding flanking regions was 744777 bp for all taxa within the genus rugopharynx and rugonema labiatum . the length of the its-1 sequences ranged from 373 bp ( rugopharynx mawsonae , r. sigma from thylogale thetis and rugonema labiatum ) to 387 bp ( r. mu , r. rufogrisea and some individuals of r. australis ) , whereas the its-2 sequences were much shorter , ranging from 216 bp ( r. longibursaris , r. omega and r. zeta ) to 313 bp ( r. alpha and r. mawsonae ) ( supplementary table 1 ) . the length ( 153 bp ) and nucleotide sequence of the 5.8s rrna gene was the same for all morphospecies within the genus rugopharynx and rugonema labiatum . there were five fixed differences ( i.e. where there are no shared nucleotides at an alignment position ) between these two taxa ; two in the its-1 and three in the its-2 . the magnitude of fixed differences in its+ sequence among morphospecies within the genus rugopharynx ranged from eight ( i.e. between r. omega and r. longibursaris ) to 84 ( i.e. between r. alpha and r. rufogrisea ) . only one fixed difference in its-1 sequence was detected between r. setonicis and rugopharynx rho compared with 10 fixed differences between these two morphospecies for the its-2 , while the lowest number of fixed differences in its-2 sequence among morphospecies ( i.e. five ) was between r. omega and r. longibursaris . for some morphospecies , there was variation among individuals in the dna sequences of the its-1 and/or its-2 ( see supplementary tables 28 ) . three variable positions ( two in the its-1 and one in the its-2 ) were detected in the aligned sequences of r. mu individuals from hosts ( w. bicolor ) collected in new south wales and victoria ; however , none of these mutations represented a fixed difference ( supplementary table 2 ) . similarly , there were no fixed differences in its+ sequence among three specimens of r. mawsonae from the same host ( m. dorsalis ) , or among two specimens of r. pi from two host species ( m. rufogriseus and m. parryi ) , even though variations were detected at nine ( four in the its-1 and five in the its-2 ) and three ( two in its-1 and one in the its-2 ) alignment positions , respectively . a similar pattern of intraspecific variation was found for three specimens of r. macropodis from two host species ( m. giganteus and m. fuliginosus ) , except for a single nucleotide insertion in the its-1 sequence for one of two individuals ( i.e. f736 ) collected from m. giganteus . specimens of r. longibursaris from m. rufogriseus collected from launceston ( tasmania ) and emu flat ( new south wales ) differed in its+ sequence at eight alignment positions ( five in the its-1 and three in the its-2 ) , whereas no genetic variation occurred in the dna sequence of r. spratti , specimens of which were collected from the same host species and localities as r. longiburaris . similarly , there were no fixed differences in its+ sequence of five r. epsilon individuals collected on the mainland ( i.e. from m. rufogriseus and w. bicolor ) and in tasmania ( from m. rufogriseus ) , except for a single insertion in the its-1 sequence of specimen f77 collected in new south wales ( supplementary table 3 ) . in contrast , fixed differences in both the its-1 and its-2 sequences were detected among individuals of four morphospecies , r. rufogrisea , r. australis , r. zeta and r. rho , collected from different host species and/or geographical regions ( see supplementary tables 48 , respectively ) . in the case of r. rufogrisea , although there were no fixed nucleotide differences in its+ sequences between specimens from m. rufogriseus collected in new south wales ( f84 ) and tasmania ( f720 ) , there were seven ( of 12 ) variable nucleotide positions ( two in the its-1 and five in the its-2 ) when compared to a specimen from m. parryi collected in queensland . similarly , there were 52 variable nucleotide positions in its+ between r. sigma specimens from thylogale stigmatica and those from t. thetis , most ( 25 in its-1 and 24 in its-2 ) representing putative fixed differences between nematodes from the two host species ; 20 of these differences ( five in its-1 and 15 in its-2 ) represented 1 - 10 bp nucleotides . in addition , nucleotide variation was detected at 20 positions in its+ sequences among individuals of r. zeta collected from different species of petrogale in queensland . there was only one fixed nucleotide difference in its-1 sequence between specimens of r. zeta and those from p. herberti and p. inornata , whereas these nematodes differed unequivocally at 13 ( 11 in its-1 and two in its-2 ) of 20 variable positions when compared with the its+ sequence from a specimen of r. zeta from p. assimilis . genetic variation was also detected at 52 nucleotide positions in the its+ sequences among seven individuals of r. australis . this morphospecies could be separated into two groups ( clades ) based on their its+ sequences . nematodes in clade 1 had fixed differences at 38 ( 14 in its-1 and 24 in its-2 ) alignment positions when compared to those in clade 2 . one eastern grey kangaroo ( m. giganteus ) collected from new south wales contained taxa from both clades 1 and 2 ( i.e. specimens f751 and f754 ) . likewise , in the case of r. rho , individuals from macropus eugenii from south australia ( f724 ) and western australia ( f905 ) had almost identical its+ sequences , which differed at 16 positions ( eight in its-1 and eight in its-2 ) from specimens from m. fuliginosus from south australia ( f780 ) and western australia ( f910 ) , and from m. irma ( g114 ) from western australia . eighteen ( five in its-1 and 13 in its-2 ) of these differences represented 14 nucleotides . phylogenetic analyses were conducted to determine whether individual morphospecies from different host species and/or geographical regions represented monophyletic assemblages . the its+ sequences were aligned over 824 positions ( 404 for its-1 , 153 for 5.8s rdna and 267 for its-2 ) , 175 of which were informative for the mp analysis . the topology of the strict consensus tree of the mp analysis ( not shown ) , based on 1217 equally most parsimonious trees ( l = 579 , ci = 0.58 , and ri = 0.83 ) , was very similar to that produced from the bi ( fig . 3 ) and the nj analyses ( fig . 4 ) . in all phylogenetic analyses , there was no support for all specimens of r. australis representing a monophyletic assemblage . however , there was absolute support ( pp = 1.0 in the bi analysis and bs = 100% in both the nj and mp trees ) for the separation of r. australis into two clades . similarly , there was no support for r. sigma from the two host species ( t. stigmatica and t. thetis ) forming a monophyletic assemblage . there was strong support ( pp = 0.999 and 1 , and bs = 95100% ) for the separation of r. rho into two clades , one containing individuals from m. eugenii and the other containing individuals from m. fuliginosus and m. irma . however , there was support in the bi analysis ( pp = 0.994 ) for a sister taxa relationship for the two clades of r. rho , and absolute support ( pp = 1 ) for a sister relationship of the two clades with r. setonicis . in contrast , in the mp and nj analyses , there was no support for a sister taxon relationship between the two clades of r. rho . however , there was strong support ( bs = 90% ) in the nj analysis for an assemblage comprising r. setonicis and the two clades of r. rho . there was also strong support ( pp = 1.0 in the bi analysis and bs = 9697% in the mp and nj analyses ) for r. zeta from the three host species , with support ( bs = 7095% , pp = 0.831 ) for r. zeta from p. inornata and p. herberti forming a clade to the exclusion of r. zeta from p. assimilis . similarly , there was absolute support ( pp = 1.0 ; bs = 100% ) for r. rufogrisea representing a monophyletic clade , and support ( bs = 85100% ; pp = 0.882 ) for r. rufogrisea collected from m. rufogriseus in tasmania and new south wales forming a clade to the exclusion of r. rufogrisea collected from m. parryi in queensland . in the phylogenetic analysis of its+ sequence data for the morphospecies , there was support in the bi and mp analyses ( i.e. pp = 0.973 ; bs = 86% ) for a clade consisting of r. macropodis , r. rosemariae , r. rufogrisea and r. theta forming a clade with respect to the other species . there was also some support ( i.e. pp = 0.995 ; bs = 7175% ) for a clade containing r. longibursaris , r. omega , r. tau and r. spratii ( fig . comparison of the phylogeny of the nematodes , derived from the analysis of its+ sequence data , with that currently available for the host species ( fig . 5 ) this comparison revealed that a simple buccal capsule ( i ) appears to be the plesiomorphic state ( as for r. alpha ) . type i buccal capsules occurred in all clades , being the exclusive buccal capsule type in one clade , but was mixed with type ii capsules in a second clade and mixed with type iii capsules in the third clade . to date , species of the genus rugopharynx found in the stomachs of macropodid marsupials have been identified solely on the basis of morphological criteria ( beveridge , 1982 ) or the combination of morphological and mee data ( chilton et al . , 1993 , 1994 ) . in describing new species within the r. australis complex , beveridge and chilton ( 1999 ) relied on limited mee data ( chilton et al . , 1996 ) and extrapolated from this base in describing nine new species based on small but potentially significant morphological characters . the present study represents the first detailed examination of the genus using dna sequence data and therefore represents the first test of the validity of the range of species currently recognised either exclusively on morphological grounds or on the basis of morphological and mee data . the current molecular analyses included most of the currently known species , apart from r. longispicularis and r. petrogale . both of these species occur in hosts , such as the parma wallaby , macropus parma , and the brush - tailed rock wallaby , petrogale penicillata , which are currently considered to be rare or vulnerable species ( maynes , 2008 , eldridge and close , 2008 ) ; therefore , it is challenging to obtain parasite material from these host species for molecular studies . no species of rugopharynx are known to occur in m. ( n. ) agilis , m.(o . ) antilopinus and m. ( o. ) bernardus ( spratt et al . , 1991 ) . in the current study , all of the species of rugopharynx presently recognised based on morphological differences ( i.e. morphospecies ) , in some cases with supporting mee data , had a unique set of its+ sequences , thereby providing additional evidence in support of their validity . fixed differences in the its-1 and its-2 sequences between or among morphospecies were limited ( e.g. , 0.3% between r. setonicis and r. rho , and 2.3% between r. omega and r. longibursaris , for the its-1 and its-2 , respectively ) , and data from a single pair of ribosomal dna spacers may not always provide unequivocal support for specific status ( nadler and prez - ponce de lon , 2011 ) . however , given the reliability of its-1 and its-2 sequences for identifying and distinguishing nematode species from macropodids to date ( e.g. , chilton et al . , 2009a , chilton et al . , 2012 ) , the evidence presented here is relatively strong . in some instances , the morphological and genetic differentiation is supported by the occurrence of formerly cryptic species in the same host individual . in the case of r. australis and r. macropodis ( i.e. previously included within r. australis ) , the former occurring in kangaroos in arid environments and the latter in areas of higher rainfall ( beveridge and chilton , 1999 ) , both species were found at one intermediate location in victoria ( pine plains station ) ( beveridge and chilton , 1999 ) , indicating genetic isolation and providing further support for the validity of the two species . the data presented here suggest that additional cryptic ( i.e. genetically distinct but morphologically similar ) species of rugopharynx remain to be described . specimens of r. australis occurred in two quite distinct clades , although some specimens from the two clades were collected from the same individual host ( m. giganteus ) . the magnitude of fixed differences in its-1 and its-2 sequences between members of the two r. australis clades ( 3.6% and 10.4% , respectively ) was greater than that between related morphospecies ( e.g. , 0.8% and 2.3% , respectively , between r. omega and r. longibursaris ) . furthermore , there was no support for the two r. australis clades forming a monophyletic assemblage . examination of the voucher specimens involved in this study suggested that the two specimens were differentiable based on spicule lengths , with those from m. giganteus from trangie , nsw ( f751 ) being 2.0 mm , and those of the additional specimen from the same individual host ( f754 ) being only 1.28 mm long ; the spicules of a similar specimen from m. fuliginosus from hattah lakes , victoria ( f784 ) , were 1.36 mm long . beveridge and chilton ( 1999 ) gave the spicule lengths of r. australis as 1.441.95 mm , which virtually encompasses the range of the specimens used in this study . the spicule lengths of the most ( morphologically ) similar species to r. australis , ( i.e. r. macropodis ) are 1.141.23 mm ( beveridge and chilton , 1999 ) . in addition , beveridge and chilton ( 1999 ) noted significant differences in bursal morphology within this species . therefore , it appears that r. australis , as currently defined , is a composite of at least two species . in the case of r. sigma , there was limited variation ( i.e. two fixed differences in its-2 but none in its-1 ) in the its+ sequences of two specimens from t. stigmatica , collected 1200 km from one another in queensland , whereas they had 49 fixed differences ( 25 in its-1 and 24 in its-2 ) when compared with r. sigma from t. thetis . this magnitude of sequence difference ( 6.5% and 10.3% for its-1 and its-2 , respectively ) exceeded that among many morphospecies within the genus . furthermore , specimens f794 from t. thetis and g384 from t. stigmatica were collected at the same locality ( lamington national park , qld ) , thus being in sympatry . the phylogenetic analyses also showed that r. sigma from the two host species did not form a monophyletic clade , providing additional support that they represent cryptic species . examination of the female voucher specimen of r. sigma from t. thetis ( f794 ) indicates a tail length of 0.21 mm compared with 0.390.45 mm for specimens from t. stigmatica and the distance of the vulva from the posterior end as 0.30 mm compared with 0.600.70 mm in specimens from t. stigmatica ( see chilton et al . , 1993 ) . hence , there appear to be morphological features supporting the molecular differences for these specimens . ( 2000 ) concluded that the helminth communities of these two host species in southern queensland , where they are sympatric , were essentially similar . specimens of r. zeta from p. inornata and p. herberti formed a strongly supported clade to the exclusion of specimens from p. assimilis . these three closely related species of rock wallabies have parapatric distributions along the eastern coast of queensland ( potter et al . , 2012 ) . ( 2009a ) examined three species of cloacinine nematodes , cloacina caenis , c. pearsoni and c. robertsi , which occur in these related rock wallaby species and demonstrated genetic differences between them , suggesting the existence of cryptic species . there was one fixed difference in its+ between specimens of r. zeta from p. inornata and p. herberti ( but none in the its-2 ) , whereas they had 13 fixed differences ( 11 in its-1 and 2 in its-1 ) when compared to the r. zeta from p. assimilis . the magnitude of sequence difference in its-1 ( 2.8% ) exceeds that detected between r. omega and r. longibursaris , whereas the 0.9% sequence differences in the its-2 is less than between these two morphospecies . therefore , additional molecular investigations are required to test the hypothesis that r. zeta represents a species complex . the current data suggest the existence of one species in p. assimilis and a second species in both p. inornata and p. herberti . rugopharynx zeta also occurs in a number of related species of rock wallabies ( petrogale mareeba , petrogale sharmani and p. penicillata ) ( spratt et al . , 1991 ) and specimens from these additional hosts would need to be included in future studies . rugopharynx rho from m. fuliginosus from both south australia and western australia , together with m. irma from western australia , formed a clade distinct from the same morphospecies obtained from m. eugenii in south australia and western australia . there was also no support in the mp and nj analyses for these two clades forming a monophyletic assemblage . as m. fuliginosus and m. eugenii are sympatric at both localities ( van dyck and strahan , 2008 ) , the data suggest that cryptic species may also exist within this taxon . in western australia , m. irma and m. fuliginosus are sympatric ( van dyck and strahan , 2008 ) and the occurrence of this species in m. irma may have resulted from host switching . consequently the data presented here suggest that additional cryptic species may exist within r. australis , r. rho , r. sigma and r. zeta . there were no fixed differences in its+ sequence between specimens of r. rufogrisea from m. rufogriseus collected on the mainland of australia and the island state of tasmania , which is consistent with the findings for other morphospecies ( e.g. , r. epsilon and r. spratti ) that parasitise m. rufogriseus . however , the magnitude of the fixed sequence differences between r. rufogrisea from m. rufogriseus and m. parryi ( 0.5% and 2.1% in its-1 and its-2 , respectively ) is very similar to that between r. omega and r. longibursaris , two other species that parasitise m. rufogriseus ( beveridge , 1982 ) . the current analysis also provides some insight into host specificity within the genus , although many of the species examined in this study appear to be moderately host specific , occurring in one or two host species ( i.e. r. alpha , r. chi , r. delta , r. longibursaris , r. mawsonae , r. mu , r. omega , r. pi , r. spratti , r. tau and r. theta ) , other species included in this study appear to have a wide host range . thus , r. australis was identified in m. rufus ( the type host for the species ) , as well as in m. fuliginosus , m. giganteus , macropus robustus and m. dorsalis . specimens from the first four host species were collected in arid or semiarid regions of australia ( table 1 ) where these host species are sympatric and r. australis is a common parasite in each of them ( arundel et al . , 1979 , beveridge et al . , 1998 ) . however , r. australis is an uncommon parasite of m. dorsalis ( see beveridge et al . , 1998 ) and the specimen collected here was in an area in which m. dorsalis is sympatric with kangaroo species commonly parasitized by this nematode . analyses of its+ sequence data of r. epsilon specimens from different host species ( i.e. m. rufogriseus and w. bicolor ) suggest that it represents a single species with a broad host range , given that r. epsilon parasitizes a variety of macropodid hosts ( spratt et al . , 1991 ) . therefore , examination of additional specimens of r. epsilon from other host species needs to be studied to test this proposal . the genus rugonema was erected ( beveridge , 1999 ) for a single species of nematode , ru . labiatum , from the stomach of m. irma which resembled rugopharynx but in which the labial collar , instead of forming an annulus , was prominently four - lobed , similar to the genus wallabinema . however , wallabinema lacks a striated buccal capsule and differs in the morphology of the oesophagus . the sole distinguishing morphological feature of rugonema , that is the four lip - like lobes of the labial collar , is an autapomorphy within the tribe pharyngostrongylinea . based on the molecular data and a reconsideration of its morphological differentiation , rugonema is here made a synonym of rugopharynx with its sole species becoming rugopharynx labiatum ( beveridge , 1999 ) n. comb . this species was most genetically similar to r. pi and belonged to a clade that also included r. mu . beveridge ( 1982 ) divided rugopharynx into three groups based on the morphology of the buccal capsule , with either a simple cylindrical buccal capsule , a bilobed or a trilobed buccal capsule . the group with bilobed buccal capsules consisted of r. epsilon and r. rufogrisea , with the indentation occurring in the mid region in r. epsilon and in the anterior quarter in r. rufogrisea . however , among the new species described by beveridge and chilton ( 1999 ) , some were difficult to allocate to a particular group ( i.e. , r. tau and r. petrogale ) because the division of the buccal capsule was subtle ( r. tau ) or variable ( r. petrogale ) . the mapping of the morphological data on to the consensus nematode phylogenetic tree ( fig . 5 ) suggest that the simple buccal capsule is the plesiomorphic state within the genus and that bilobed buccal capsules have evolved independently . the findings of the present study also suggest that the trilobed buccal capsules are a derived character . the lack of resolution in the cladogram prevents more detailed conclusions from being drawn on the evolution of buccal capsule shapes within the genus . there appears to be no co - evolutionary relationship between nematodes and hosts . based on molecular evidence , setonix diverged within the macropodine lineage about 10 million years ago , while thylogale and petrogale are sister taxa to the clade that contains macropus and wallabia , their estimated time of divergence being about eight million years ( meredith et al . , 2008 ) . the largest clade of the molecular phylogenetic tree of the nematodes contains species from each of these macropodine genera apart from setonix . beveridge and chilton ( 2001 ) undertook a morphological phylogenetic analysis of the r. australis complex , which produced a completely unresolved tree , and consequently these authors concluded that there was no obvious co - evolutionary association with hosts . comparable studies of other cloacinine genera also supported the hypothesis that evolution within this group of nematodes was primarily by host switching ( beveridge and chilton , 2001 ) , a hypothesis concordant with the data presented above . ( 2016 ) have provided molecular evidence for host switching in the related cloacinine genus cyclostrongylus . in summary , the molecular data presented here support the earlier morphological studies of the genus rugopharynx and provide additional evidence that the species of rugopharynx currently established , many of them based on minor morphological differences and mee data , are indeed valid . the study has also revealed the existence of additional cryptic species within the genus that need to be characterised morphologically . comparisons of buccal capsule morphology with the phylogenetic tree provided some insights into the evolution of more complex buccal capsules , but there were no obvious co - evolutionary associations with hosts , the data instead suggesting a pattern of host switching in the evolution of the genus .

sequences of the internal transcribed spacers of nuclear ribosomal dna ( its-1 and its-2 ) were determined for species of the genus rugopharynx and rugonema labiatum , nematodes from the stomachs of macropodid marsupials . phylogenetic analyses of the aligned sequence data were conducted . the relationships provided molecular support for all species currently recognised , some of which are based on minor morphological differences and on multilocus enzyme electrophoretic data , but also indicated that additional , cryptic species exist within the genus . in addition , the genus rugonema is placed as a synonym of rugopharynx , its sole species becoming rugopharynx labiatum n. comb . the molecular data provided some insights into the evolution of complex buccal capsule morphologies within the genus , but there was no evidence of co - evolution between the macropodid hosts and their parasites .

Introduction Materials and methods Results Discussion Conflicts of interest

for example , vendl and beveridge ( 2014 ) reported means and ranges in numbers of cloacinine nematodes in the stomachs of the red - necked wallaby , macropus rufogriseus , the eastern grey kangaroo , macropus giganteus and the swamp wallaby , wallabia bicolor , as 60,800 ( 2000210,000 ) , 20,500 ( 700079,000 ) and 20,000 ( 300058 , 000 ) , respectively . in spite of the numerical significance of this genus in the gastric helminth communities of macropodids , continuing taxonomic studies are needed , as it is unlikely that all species have yet been described and the presence of cryptic species could potentially complicate the interpretation of ecological data published to date . the genus rugopharynx was revised by beveridge ( 1982 ) , who recognised nine species . multilocus enzyme electrophoretic ( mee ) studies , combined with morphological evidence , indicated the existence of two new species , r. sigma and rugopharynx mawsonae , both formerly confused with rugopharynx zeta ( chilton et al . the latter revision was based on extrapolating the minor morphological differences identified in samples included in the earlier electrophoretic study across the entire species complex . a closely related genus , rugonema was erected by beveridge ( 1999 ) for specimens formerly referred to as r. australis occurring in the stomach of the black - gloved wallaby , macropus irma , from western australia , based on morphological differences in the labial collar . because of this close association with rugopharynx , rugonema labiatum was also included in the present study . given the prevalence and abundance of this nematode genus in the stomachs of kangaroos and wallabies , and the diversity of species currently recognised based on minor morphological criteria , this study was undertaken to attempt to establish species boundaries within the genus based on sequences of the first and second internal transcribed spacers ( its-1 and its-2 ) of nuclear ribosomal dna . these molecular target regions have proved to be highly informative for the specific identification of a range of strongylid nematodes , including taxa within the cloacininae ( chilton , 2004 ) , the subfamily to which rugopharynx belongs . nematodes were obtained from the stomachs of a range of kangaroos and wallabies ( fig . species within the genus rugopharynx were divided into three groups based on the morphology of the buccal capsule ( table 1 ) . in some species , such as r. spratti and r. tau , the potential division of the buccal capsule is subtle ; hence , in these cases , species were classified as type i. genomic dna was isolated from the mid - body part of each nematode using a small - scale sodium - dodecyl - sulphate / proteinase k extraction method ( gasser et al . phylogenetic analyses of the aligned sequence data were conducted by bayesian inference ( bi ) using monte carlo markov chain analysis in the program mrbayes v.3.2.2 ( ronquist and huelsenbeck , 2003 ) . the likelihood parameters set for the bi analysis of sequence data were based on the akaike information criteria test in jmodeltest v.2.1.5 ( posada , 2008 ) . phylogenetic analyses of the its+ sequence data were also conducted using the neighbor - joining ( nj ) and maximum parsimony ( mp ) methods in paup * 4.0b10 ( swofford , 1999 ) . bootstrap analyses ( 1000 replicates ) were conducted to determine the relative support for clades in the consensus trees ; nodal support was expressed as a percentage . the its+ sequences of labiostrongylus australis ( genbank accession numbers aj308403-aj308411 ) were used as the outgroup in the phylogenetic analyses , because this nematode species belongs to a related tribe within the same subfamily ( i.e. the host phylogenetic tree was based primarily on the molecular analyses of meredith et al . , thylogale billardierii , macropus dorsalis , petrogale herberti , petrogale assimilis and petrogale inornata ) were not included in that study , the tree was modified to incorporate these hosts based on the studies of campeau - ploquin et al . antilopinus , from which no species of rugopharynx have been reported . the length of the its+ region , excluding flanking regions was 744777 bp for all taxa within the genus rugopharynx and rugonema labiatum . the length of the its-1 sequences ranged from 373 bp ( rugopharynx mawsonae , r. sigma from thylogale thetis and rugonema labiatum ) to 387 bp ( r. mu , r. rufogrisea and some individuals of r. australis ) , whereas the its-2 sequences were much shorter , ranging from 216 bp ( r. longibursaris , r. omega and r. zeta ) to 313 bp ( r. alpha and r. mawsonae ) ( supplementary table 1 ) . the length ( 153 bp ) and nucleotide sequence of the 5.8s rrna gene was the same for all morphospecies within the genus rugopharynx and rugonema labiatum . the magnitude of fixed differences in its+ sequence among morphospecies within the genus rugopharynx ranged from eight ( i.e. for some morphospecies , there was variation among individuals in the dna sequences of the its-1 and/or its-2 ( see supplementary tables 28 ) . three variable positions ( two in the its-1 and one in the its-2 ) were detected in the aligned sequences of r. mu individuals from hosts ( w. bicolor ) collected in new south wales and victoria ; however , none of these mutations represented a fixed difference ( supplementary table 2 ) . similarly , there were no fixed differences in its+ sequence among three specimens of r. mawsonae from the same host ( m. dorsalis ) , or among two specimens of r. pi from two host species ( m. rufogriseus and m. parryi ) , even though variations were detected at nine ( four in the its-1 and five in the its-2 ) and three ( two in its-1 and one in the its-2 ) alignment positions , respectively . specimens of r. longibursaris from m. rufogriseus collected from launceston ( tasmania ) and emu flat ( new south wales ) differed in its+ sequence at eight alignment positions ( five in the its-1 and three in the its-2 ) , whereas no genetic variation occurred in the dna sequence of r. spratti , specimens of which were collected from the same host species and localities as r. longiburaris . in contrast , fixed differences in both the its-1 and its-2 sequences were detected among individuals of four morphospecies , r. rufogrisea , r. australis , r. zeta and r. rho , collected from different host species and/or geographical regions ( see supplementary tables 48 , respectively ) . similarly , there were 52 variable nucleotide positions in its+ between r. sigma specimens from thylogale stigmatica and those from t. thetis , most ( 25 in its-1 and 24 in its-2 ) representing putative fixed differences between nematodes from the two host species ; 20 of these differences ( five in its-1 and 15 in its-2 ) represented 1 - 10 bp nucleotides . in addition , nucleotide variation was detected at 20 positions in its+ sequences among individuals of r. zeta collected from different species of petrogale in queensland . there was only one fixed nucleotide difference in its-1 sequence between specimens of r. zeta and those from p. herberti and p. inornata , whereas these nematodes differed unequivocally at 13 ( 11 in its-1 and two in its-2 ) of 20 variable positions when compared with the its+ sequence from a specimen of r. zeta from p. assimilis . likewise , in the case of r. rho , individuals from macropus eugenii from south australia ( f724 ) and western australia ( f905 ) had almost identical its+ sequences , which differed at 16 positions ( eight in its-1 and eight in its-2 ) from specimens from m. fuliginosus from south australia ( f780 ) and western australia ( f910 ) , and from m. irma ( g114 ) from western australia . eighteen ( five in its-1 and 13 in its-2 ) of these differences represented 14 nucleotides . phylogenetic analyses were conducted to determine whether individual morphospecies from different host species and/or geographical regions represented monophyletic assemblages . the its+ sequences were aligned over 824 positions ( 404 for its-1 , 153 for 5.8s rdna and 267 for its-2 ) , 175 of which were informative for the mp analysis . the topology of the strict consensus tree of the mp analysis ( not shown ) , based on 1217 equally most parsimonious trees ( l = 579 , ci = 0.58 , and ri = 0.83 ) , was very similar to that produced from the bi ( fig . in all phylogenetic analyses , there was no support for all specimens of r. australis representing a monophyletic assemblage . similarly , there was no support for r. sigma from the two host species ( t. stigmatica and t. thetis ) forming a monophyletic assemblage . in contrast , in the mp and nj analyses , there was no support for a sister taxon relationship between the two clades of r. rho . there was also strong support ( pp = 1.0 in the bi analysis and bs = 9697% in the mp and nj analyses ) for r. zeta from the three host species , with support ( bs = 7095% , pp = 0.831 ) for r. zeta from p. inornata and p. herberti forming a clade to the exclusion of r. zeta from p. assimilis . comparison of the phylogeny of the nematodes , derived from the analysis of its+ sequence data , with that currently available for the host species ( fig . type i buccal capsules occurred in all clades , being the exclusive buccal capsule type in one clade , but was mixed with type ii capsules in a second clade and mixed with type iii capsules in the third clade . to date , species of the genus rugopharynx found in the stomachs of macropodid marsupials have been identified solely on the basis of morphological criteria ( beveridge , 1982 ) or the combination of morphological and mee data ( chilton et al . the present study represents the first detailed examination of the genus using dna sequence data and therefore represents the first test of the validity of the range of species currently recognised either exclusively on morphological grounds or on the basis of morphological and mee data . in the current study , all of the species of rugopharynx presently recognised based on morphological differences ( i.e. , 0.3% between r. setonicis and r. rho , and 2.3% between r. omega and r. longibursaris , for the its-1 and its-2 , respectively ) , and data from a single pair of ribosomal dna spacers may not always provide unequivocal support for specific status ( nadler and prez - ponce de lon , 2011 ) . however , given the reliability of its-1 and its-2 sequences for identifying and distinguishing nematode species from macropodids to date ( e.g. in some instances , the morphological and genetic differentiation is supported by the occurrence of formerly cryptic species in the same host individual . previously included within r. australis ) , the former occurring in kangaroos in arid environments and the latter in areas of higher rainfall ( beveridge and chilton , 1999 ) , both species were found at one intermediate location in victoria ( pine plains station ) ( beveridge and chilton , 1999 ) , indicating genetic isolation and providing further support for the validity of the two species . genetically distinct but morphologically similar ) species of rugopharynx remain to be described . the magnitude of fixed differences in its-1 and its-2 sequences between members of the two r. australis clades ( 3.6% and 10.4% , respectively ) was greater than that between related morphospecies ( e.g. furthermore , there was no support for the two r. australis clades forming a monophyletic assemblage . examination of the voucher specimens involved in this study suggested that the two specimens were differentiable based on spicule lengths , with those from m. giganteus from trangie , nsw ( f751 ) being 2.0 mm , and those of the additional specimen from the same individual host ( f754 ) being only 1.28 mm long ; the spicules of a similar specimen from m. fuliginosus from hattah lakes , victoria ( f784 ) , were 1.36 mm long . two fixed differences in its-2 but none in its-1 ) in the its+ sequences of two specimens from t. stigmatica , collected 1200 km from one another in queensland , whereas they had 49 fixed differences ( 25 in its-1 and 24 in its-2 ) when compared with r. sigma from t. thetis . this magnitude of sequence difference ( 6.5% and 10.3% for its-1 and its-2 , respectively ) exceeded that among many morphospecies within the genus . the phylogenetic analyses also showed that r. sigma from the two host species did not form a monophyletic clade , providing additional support that they represent cryptic species . ( 2009a ) examined three species of cloacinine nematodes , cloacina caenis , c. pearsoni and c. robertsi , which occur in these related rock wallaby species and demonstrated genetic differences between them , suggesting the existence of cryptic species . there was one fixed difference in its+ between specimens of r. zeta from p. inornata and p. herberti ( but none in the its-2 ) , whereas they had 13 fixed differences ( 11 in its-1 and 2 in its-1 ) when compared to the r. zeta from p. assimilis . as m. fuliginosus and m. eugenii are sympatric at both localities ( van dyck and strahan , 2008 ) , the data suggest that cryptic species may also exist within this taxon . consequently the data presented here suggest that additional cryptic species may exist within r. australis , r. rho , r. sigma and r. zeta . however , the magnitude of the fixed sequence differences between r. rufogrisea from m. rufogriseus and m. parryi ( 0.5% and 2.1% in its-1 and its-2 , respectively ) is very similar to that between r. omega and r. longibursaris , two other species that parasitise m. rufogriseus ( beveridge , 1982 ) . the current analysis also provides some insight into host specificity within the genus , although many of the species examined in this study appear to be moderately host specific , occurring in one or two host species ( i.e. analyses of its+ sequence data of r. epsilon specimens from different host species ( i.e. the genus rugonema was erected ( beveridge , 1999 ) for a single species of nematode , ru . labiatum , from the stomach of m. irma which resembled rugopharynx but in which the labial collar , instead of forming an annulus , was prominently four - lobed , similar to the genus wallabinema . however , wallabinema lacks a striated buccal capsule and differs in the morphology of the oesophagus . the sole distinguishing morphological feature of rugonema , that is the four lip - like lobes of the labial collar , is an autapomorphy within the tribe pharyngostrongylinea . based on the molecular data and a reconsideration of its morphological differentiation , rugonema is here made a synonym of rugopharynx with its sole species becoming rugopharynx labiatum ( beveridge , 1999 ) n. comb . beveridge ( 1982 ) divided rugopharynx into three groups based on the morphology of the buccal capsule , with either a simple cylindrical buccal capsule , a bilobed or a trilobed buccal capsule . 5 ) suggest that the simple buccal capsule is the plesiomorphic state within the genus and that bilobed buccal capsules have evolved independently . the lack of resolution in the cladogram prevents more detailed conclusions from being drawn on the evolution of buccal capsule shapes within the genus . based on molecular evidence , setonix diverged within the macropodine lineage about 10 million years ago , while thylogale and petrogale are sister taxa to the clade that contains macropus and wallabia , their estimated time of divergence being about eight million years ( meredith et al . the largest clade of the molecular phylogenetic tree of the nematodes contains species from each of these macropodine genera apart from setonix . beveridge and chilton ( 2001 ) undertook a morphological phylogenetic analysis of the r. australis complex , which produced a completely unresolved tree , and consequently these authors concluded that there was no obvious co - evolutionary association with hosts . in summary , the molecular data presented here support the earlier morphological studies of the genus rugopharynx and provide additional evidence that the species of rugopharynx currently established , many of them based on minor morphological differences and mee data , are indeed valid . the study has also revealed the existence of additional cryptic species within the genus that need to be characterised morphologically . comparisons of buccal capsule morphology with the phylogenetic tree provided some insights into the evolution of more complex buccal capsules , but there were no obvious co - evolutionary associations with hosts , the data instead suggesting a pattern of host switching in the evolution of the genus .

transdermal glyceryl trinitrate is safe in patients with acute stroke.glyceryl trinitrate may improve outcome if administered within 6 h of stroke onset.the implications for clinical practice are substantial if efficacy is confirmed . an inexpensive and effective treatment for hyperacute stroke could be adopted globally in low- , middle- and high - income countries . no has vasodilatory , pro - endothelial , anti - proliferative ( vascular smooth muscle cell ) , antiplatelet [ 2 , 3 ] , anti - leucocyte , anti - inflammatory , neuroprotective , neurotransmitter and neuromodulator [ 5 , 6 ] properties . brain barrier integrity , cerebral blood flow ( cbf ) , auto- and chemo - regulation [ 79 ] , and inhibition of apoptosis . no is an endogenous inorganic soluble gas synthesised from l - arginine by three forms of no synthase ( nos ) : endothelial ( enos ) ; inducible ( inos ) ; and neuronal ( nnos ) [ 11 , 12 ] . the second messenger cyclic guanosine monophosphate , which is broken down by phosphodiesterase , is the main mediator of downstream signalling of no . no is broken down via oxidation to nitrite and ultimately nitrate , and it is now apparent that no may be made by reduction of nitrite and nitrate . up - regulation of the l - arginine / nitrite - no - cyclic guanosine monophosphate pathway can be achieved by a number of means : increased substrate , administration of no gas , induction of no synthase activity ; administration of no donors ; or inhibition of phosphodiesterase . pre - clinical experimental studies in models of ischaemia have demonstrated the role of no in a time - dependent manner . models of focal ischaemia have shown that no production is increased , through nnos activation , for up to half an hour after middle cerebral artery occlusion [ 14 , 15 ] . during the first minutes following arterial occlusion , enos and nnos activity increases up regulation of inos occurs from 12 h after the onset of ischaemia and persists for up to 7 days , whilst no within brain tissue is undetectable during this period . l - arginine administered intravenously following middle cerebral artery occlusion in a rat model improved ischaemic penumbral blood flow and reduced infarct size and volume . this effect was not seen in enos - deficient mice who developed smaller penumbral regions , larger infarcts and absent angiogenesis leading to further post - ischaemic injury [ 1921 ] . therefore , enos and enos - derived no are neuroprotective in focal ischaemia , whilst nnos- and inos - derived no have deleterious effects on tissue survival with resultant poor neurological outcomes [ 15 , 22 ] . although neurotoxic in acute stroke , inos and nnos are involved in neurogenesis following stroke [ 23 , 24 ] . in therapeutic studies , no donors reduced infarct size in both permanent and transient models of ischaemia , and increased cerebral blood flow in permanent models , but only if administered soon after stroke induction . blood pressure ( bp ) is high in 75% of people with acute stroke and is associated independently with poor functional outcome and increased death ( regardless of stroke type ) [ 27 , 28 ] , stroke recurrence in ischaemic stroke ( is ) and haematoma expansion in intracerebral haemorrhage ( ich ) . high admission bp has been associated with lower rates of recanalisation in is patients treated with thrombolysis , and with increased infarct volume and poor functional outcome in patients with large vessel occlusion . modulation of bp in acute stroke has long been debated ; treatment of raised bp in ich is recommended , and is safe in is [ 34 , 35 ] . owing to the myriad effects of no described above and the low levels of endogenous no seen in both is and ich [ 36 , 37 ] , supplementation through administration of no donors might be beneficial . hence , lowering no can lead to worse outcomes in acute stroke , whilst increasing no may be beneficial . here , we discuss the evidence to date , potential mechanisms of action and future possibilities , including unanswered questions , for the therapeutic potential of the no donor glyceryl trinitrate ( gtn ) in acute stroke . no donors can be broadly categorised into organic ( e.g. gtn ) and inorganic ( e.g. sodium nitroprusside ) nitrates , although there are many subtypes . transdermal gtn has been administered as a transdermal patch to patients with acute and subacute stroke in three phase ii trials ; gtn lowered bp ( peripheral and central ) , 24-h bp , peak systolic bp ( sbp ) , pulse pressure and pulse pressure index ; increased heart rate ; improved vascular compliance ; and did not alter cerebral blood flow and velocity , or induce cerebral steal or increase intracranial pressure ( table 1 ) [ 4145 ] . while intravenous sodium nitroprusside has antiplatelet properties , gtn had no such impact on platelet function and can therefore be administered in patients with ich . none of these earlier studies were powered for efficacy , and this was assessed in the large efficacy of nitric oxide in stroke ( enos ) trial .table 1effects of gtn in acute / subacute strokegtn-1 2001 gtn-2 2003 gtn-3 2006 right 2013 enos 2015 gtn 1 - 2 gtn 1 - 3 systolic bp ( mmhg) 13 ( 7.8%) 23 ( 14%) 21 7 9.4 9.8diastolic bp ( mmhg) 5.2 ( 5.4%) 4 ( 3%) 6 3.5 4.8 4.4heart rate ( bpm)no changeno changeno changeno change 1.7 4.1 3.9map ( mmhg) 6.2% 5.0 6.4pp ( mmhg) 3.9 16 6.1ppi 0.03rpp ( mmhg.bpm)no change 323augmentation indeximprovedimprovedcerebral blood flow velocityno changeno changecerebral blood flowno changezero flow pressureno changeplatelet functionno changegtn supplier ( 5 mg)schwarz pharmaschwarz pharma ( 5 and 10 mg)novartis ( transiderm - nitro)msd schering - plough ( nitrodur)ucb pharma ( deponit-5 ) in 38% of sites ( 29%)novartis ( transiderm - nitro ) in 28% of sites ( 34%)msd / schering - plough ( nitrodur ) in 25% of sites ( 19%)meda/3 m healthcare ( minitran ) in 10% of sites ( 3%)wuhan jianmin in 1% of sites ( 2% ) bp blood pressure , bpm beats per minute , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , rpp rate pressure product , indicates a decrease , indicates an increase percentage of sites for enos - early ( within 6 h ) effects of gtn in acute / subacute stroke bp blood pressure , bpm beats per minute , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , rpp rate pressure product , indicates a decrease , indicates an increase percentage of sites for enos - early ( within 6 h ) enos enrolled 4011 participants with acute stroke ( within 48 h of onset ) and raised systolic bp ( 140220 mmhg ) and randomised these to transdermal gtn patch ( 5 mg ) or no patch . overall , there was no significant shift in functional outcome measured using the modified rankin scale at day 90 ( primary outcome , adjusted common odds ratio 1.01 , 95% confidence intervals [ ci ] 0.911.13 ) or of any secondary outcomes ; further , gtn was safe with no increased reporting of serious adverse events . gtn lowered bp by 7.0/3.5 mmhg as compared with control at day 1 . in a pre - defined subgroup by time to randomisation ( enos - early ) , those who received gtn within 6 h of stroke onset had a favourable shift in the modified rankin scale ( adjusted common odds ratio 0.51 , 95% ci 0.320.80 ) , less death , less disability ( barthel index ) , less mood disturbance ( zung depression scale ) , and improved cognition ( telephone mini - mental state examination ) and quality of life ( euro - quality of life visual analogue scale and health utility scale ) . the small ambulance - based rapid intervention with glyceryl trinitrate in hypertensive stroke trial ( right ) also found that transdermal gtn 5 mg , given in the pre - hospital setting by paramedics within 4 h of ictus , improved the modified rankin scale at day 90 . an individual patient data meta - analysis using data from the five completed gtn trials ( gtn-1/2/3 , enos , right , n = 4197 ) supported the findings that treatment with gtn within 6 h of onset ( n = 312 ) , but not later , improved functional outcome and secondary outcomes across a range of domains : cognition ; death ; disability ; mood ; and quality of life ( table 2 ) . the time - dependent effect on functional outcome was seen in both is and ich ; a finding supported by a subgroup analysis of participants with ich from the enos trial . those with ich who received gtn within 6 h of onset had significant improvements in functional outcome , cognition , disability , mood and quality of life at 90 days compared with those who did not receive gtn . in the aforementioned meta - analysis , those with is who received thrombolysis , given either before or after randomisation , had a significant shift to less death or dependency in the presence of gtn . a tendency to improved outcome was also seen in those with is who did not receive intravenous alteplase .table 2clinical outcomes with gtn in acute stroke all gtnrandomisation 6 hpatients ( % ) 4197312 ( 7.4)end of treatment death1.15 ( 0.79 , 1.68)0.94 ( 0.23 , 3.81 ) neurological deterioration1.29 ( 1.00 , 1.66)0.58 ( 0.28 , 1.23 ) stroke recurrence1.39 ( 0.88 , 2.18)0.46 ( 0.14 , 1.54 ) sss ( /58)0.33 ( 0.26 , 0.91)2.33 ( 0.42 , 5.09 ) nihss 0.28 ( 0.70 , 0.14)2.07 ( 3.81 , 0.34 ) day 7 non - oral feeding 0.97 ( 0.82 , 1.15)0.59 ( 0.32 , 1.08)hospital length of stay ( days)0.07 ( 1.30 , 1.44)0.02 ( 4.59 , 4.63 ) physiotherapy 0.94 ( 0.79 , 1.12)0.90 ( 0.40 , 2.05 ) occupational therapy 1.01 ( 0.72 , 1.41)1.15 ( 0.36 , 3.68 ) speech therapy 0.95 ( 0.84 , 1.08)1.01 ( 0.44 , 2.29)day 90 modified rankin scale0.99 ( 0.89 , 1.10 ) 0.52 ( 0.34 , 0.78 ) death0.87 ( 0.71 , 1.07 ) 0.32 ( 0.14 , 0.78 ) barthel index1.73 ( 0.08 , 3.55 ) 9.64 ( 3.19 , 16.09 ) quality of life ( hus)0 ( 0.02 , 0.02)0.05 ( 0.02 , 0.13 ) quality of life ( eq - vas)0.69 ( 1.06 , 2.43)5.97 ( 0.30 , 12.24 ) mood ( zds)0.38 ( 1.80 , 1.04)8.34 ( 13.32 , 3.36 ) cognition ( t - mmse)0.34 ( 0.16 , 0.84 ) 2.09 ( 0.65 , 3.54 ) cognition ( tics - m)0.16 ( 0.55 , 0.88 ) 3.56 ( 1.20 , 5.91 ) cognition ( animal naming)0.06 ( 0.60 , 0.47)1.63 ( 0.13 , 3.39)data are number ( % ) , mean difference , or odds ratio with 95% confidence intervals . comparisons by binary logistic regression , ordinal logistic regression or multiple linear regression , with adjustment for trial . significant ( p < 0.05 ) results are in bold bi barthel index , eq - vas euro - quality of life visual analogue scale , gtn glyceryl trinitrate , hus health utility scale ( derived from euro - quality of life 5-dimensions ( eq5d ) ) , nihss national institutes of health stroke scale , sss scandinavian stroke scale , tics - m telephone interview cognition scale , t - mmse telephone mini - mental state examination , zds zung depression scale unadjusted using the mantel haenszel random - effects model clinical outcomes with gtn in acute stroke data are number ( % ) , mean difference , or odds ratio with 95% confidence intervals . comparisons by binary logistic regression , ordinal logistic regression or multiple linear regression , with adjustment for trial . significant ( p < 0.05 ) results are in bold bi barthel index , eq - vas euro - quality of life visual analogue scale , gtn glyceryl trinitrate , hus health utility scale ( derived from euro - quality of life 5-dimensions ( eq5d ) ) , nihss national institutes of health stroke scale , sss scandinavian stroke scale , tics - m telephone interview cognition scale , t - mmse telephone mini - mental state examination , zds zung depression scale unadjusted using the mantel haenszel random - effects model further trials are needed to confirm whether gtn is efficacious when given early in patients with acute stroke ; one study , the rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 ( right-2 ) , is assessing transdermal 5-mg gtn patch vs. sham in 850 patients with presumed stroke within 4 h of onset , sbp > 120 mmhg and fast score 2 or 3 ; paramedics perform consent , recruitment and treatment in the pre - hospital setting . if transdermal gtn is found to be beneficial in the management of acute stroke , there are several potential mechanisms through which its actions may be mediated ( table 3 ) . first , bp lowering may reduce early recurrent events in is and haematoma expansion in ich . the ability of gtn to lower bp without reducing cbf or cerebral perfusion pressure , or increasing intracranial pressure , may be due to its vasodilatory effect thus increasing blood flow exiting the cranium . second , in addition to high sbp being associated with poor clinical outcomes in acute stroke , other haemodynamic variables including higher peak sbp , mean arterial pressure , pulse pressure , pulse pressure index and increased sbp variability are independently associated with worse functional outcome , death , recurrent stroke and early neurological deterioration [ 5254 ] . the published effects of gtn on haemodynamic measures in acute stroke are detailed in table 1 . new data on sbp variability from the four pilot studies ( gtn 1 - 3 and right ) are included in table 4 . between - visit systolic bp variability was calculated as standard deviation ( sd ) and coefficient of variation ( cov = sd / mean ) of sbp over days 17 across each trial individually and across all four trials as a whole . the mean difference between gtn and no gtn was calculated using analysis of covariance with adjustment for baseline sbp and trial as appropriate . gtn reduced sbp variability ( sd and cov ) over days 17 when given within 4 h in the right pre - hospital trial in both adjusted and unadjusted analyses . when pooled together , there was significant heterogeneity seen across the trials for both sd ( i = 75% ) and cov ( i = 79% ) . this heterogeneity likely represents the small sample sizes of each of the trials and the varying time from stroke onset to randomisation . therefore , adjustment was made for baseline sbp and trial , after which gtn significantly reduced sbp variability ( sd ) compared with no gtn . pre - specified secondary analysis of haemodynamic parameters in enos is awaited .table 3gtn in acute stroke : mechanisms of action and unanswered questionsissueprior observationscommenttimereperfusion therapies exhibit time dependency : thrombolysis and thrombectomy apparent time - dependent effect mirrors thrombolysis and thrombectomystroke type ischaemic strokebp lowering may reduce recurrent eventsapparent benefit : vasodilatory effects may improve blood flow in large arteries ( front door ) and pial arteries / collaterals ( back door ) ; new data awaited intracerebral haemorrhagebp lowering may reduce haematoma expansion apparent benefit ; new data awaitedstroke syndrome lacunarunclear effect ; further analyses and new data awaited total anterior circulationapparent benefit : tendency for improved functional outcome in total anterior circulation in enos ; further analyses and new data awaitedstroke severity severityapparent benefit : tendency for improved functional outcome in severe stroke in enos ; further analyses and new data awaitedsafety carotid stenosisbp lowering might reduce perfusioninitial safety seen in all levels of ipsilateral carotid stenosis in enos ; further analyses and new data awaited large vessel occlusionunknown ; further analyses and new data awaited dehydration and strokelarge drops in bp may occur in dehydrated patients given antihypertensive medicationrelevance to gtn unknown ; further analyses and new data awaited stroke mimicsno adverse effect seen in right ; new data awaitedduration of therapytachyphylaxis seen in gtn-1/2 and enos [ 41 , 42 , 47 ] . right-2 is assessing 4 days of treatment whilst planned trials will assess 1 or 2 days of treatmentroute of administrationtransdermal drugs allow easy application and removal without need for swallowing assessment or intravenous accessgtn given by transdermal patchpre - stroke bp - lowering therapyantihypertensive medication is regularly taken prior to stroke no interaction between this and gtn seen in enos haemodynamics bp derivativesincreased map , pp , ppi , peak sbp and sbp variability are associated with poor outcomesgtn reduces map , pp , ppi , peak sbp and variability [ 44 , 55 ] heart ratehigh heart rate and impaired heart rate variability are associated with worse outcomegtn increases heart rate by 24 beats per minute . tendencies to reduced rate pressure product ( rpp = sbp hr ) suggest that the effect of gtn on hr is more than offset by bp reduction ; further analyses and new data awaited . effect on heart rate variability to be ascertained bp blood pressure , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right2 rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 , sbp systolic bp table 4effect of gtn on blood pressure variability in acute / subacute stroketrialpatients ( n)otr ( h)sd sbp days 17cov sbp days 17 ( % ) unadjusted p valueadjusted p valueunadjusted p valueadjusted p valuegtn-1 37<1202.0 ( 6.4 , 2.4)0.362.7 ( 6.8 , 1.4)0.191.2 ( 3.7 , 1.3)0.321.5 ( 4.0 , 1.0)0.23gtn-2 90<720.1 ( 2.2 , 2.3)0.960.1 ( 2.0 , 2.3)0.920.2 ( 1.4 , 1.7)0.830.2 ( 1.3 , 1.7)0.81gtn-3 18<1201.2 ( 3.7 , 6.0)0.621.4 ( 3.4 , 6.3)0.542.4 ( 0.5 , 5.2)0.102.5 ( 0.5 , 5.4)0.10right 41<47.7 ( 12.1 , 3.3 ) 0.001 7.2 ( 11.5 , 2.8 ) 0.002 4.9 ( 8.0 , 1.8 ) 0.003 4.9 ( 8.1 , 1.7 ) 0.003 combined1861.6 ( 3.8 , 0.6)0.142.1 ( 3.8 , 0.4 ) 0.019 0.7 ( 2.2 , 0.7)0.311.2 ( 2.4 , 0.0)0.058data are mean difference ( 95% confidence intervals ) with adjustment for baseline sbp trial . results are in bold cov coefficient of variation , gtn glyceryl trinitrate , otr onset to randomization , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , sbp systolic blood pressure , sd standard deviation gtn in acute stroke : mechanisms of action and unanswered questions bp blood pressure , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right2 rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 , sbp systolic bp effect of gtn on blood pressure variability in acute / subacute stroke data are mean difference ( 95% confidence intervals ) with adjustment for baseline sbp trial . significant ( p < 0.05 ) results are in bold cov coefficient of variation , gtn glyceryl trinitrate , otr onset to randomization , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , sbp systolic blood pressure , sd standard deviation third , the time dependency of early treatment with gtn is akin to that seen in both thrombolysis and endovascular therapy , so - called reperfusion treatments [ 56 , 57 ] . as described , gtn is a potent vasodilator , which may have effects in different parts of the vascular tree : large cerebral arteries , increasing front door and peri - lesional perfusion without inducing cerebral steal ; and surface pial arteries , increasing collateral ( back door ) perfusion . fourth , in the context of thrombolysis , gtn may be synergistic through vasodilatation of the occluded or partially occluded artery , which may allow exogenous and endogenous thrombolytic compounds better access to clot . gtn also appears to prepare patients for thrombolysis by lowering systolic bp below the licensed threshold of 185 mmhg . a non - significant increase in both rates of thrombolysis and earlier treatment was seen in right . last , the neuroprotective effects of gtn mediated via no may prevent cell death from ischaemia [ 10 , 18 ] . although enos confirmed the overall safety of gtn in acute stroke , several distinct scenarios warrant further discussion ( table 3 ) . first , patients with carotid stenosis as the cause of their stroke often have high bp at presentation and whether to lower their bp is subject to debate . owing to dysfunctional cerebral autoregulation , higher bp leads to higher cerebral perfusion pressure , increasing the risk of cerebral oedema and haemorrhagic transformation of infarction , whilst bp reduction may compromise cbf extending infarction . across all levels of ipsilateral carotid artery stenosis within enos ( 2038 participants with carotid imaging data ) , however , data for patients with severe bilateral carotid stenosis are sparse , given its rarity , although a meta - analysis of three trials found that lower bp was associated with increased stroke recurrence . in this group , bp lowering should be avoided pending further data . a post - hoc analysis of the enos dataset is planned for further clarification of this important patient subgroup . second , and similarly , the advent of endovascular therapy for proximal anterior circulation vessel occlusions in acute is poses several challenges including how to manage bp without potentially compromising cbf ; the ongoing right-2 trial will include a subgroup of patients who have thrombectomy following gtn - sham treatment . third , dehydration is a common finding in patients with acute stroke and was associated with poor outcomes in a stroke registry study . bp lowering in the setting of dehydration may lead to precipitous drops in bp , which could be harmful ; the effect of gtn in this scenario is unclear and a subgroup analysis of enos may prove illuminating . last , the administration of an agent that may improve outcome when given as early as possible will inevitably mean that patients with conditions mimicking stroke will receive treatment . therefore , it is imperative that gtn is safe in this group ; a question for ongoing and future trials . as previously alluded to , gtn positively influenced several clinical outcomes when given early in both is and ich . whether gtn has the same effects in patients with lacunar syndromes , lacunar strokes or small vessel disease is unclear and further analysis is required . in addition to answering these and other questions , current and future trials will need to record data on other outcomes including thrombolysis , thrombectomy , neurosurgical procedures ( e.g. hemicraniectomy ) , feeding status , therapy usage and length of stay ( overall , intensive care unit ) , as these may be influenced by the efficacy of gtn . indeed , early gtn was associated with less nasogastric and more oral feeding in enos - early ( unpublished ) . importantly , all the trial data for gtn in acute stroke are from one group of authors ; others need to replicate and confirm these findings in differing countries , healthcare settings and stroke populations . there are few evidence - based treatments for the management of acute stroke . in acute is , intravenous thrombolysis , thrombectomy and decompressive hemicraniectomy each have high efficacy but low utility , whilst aspirin has high utility but low efficacy . managing patients with all stroke types in stroke units has very high utility with medium - level efficacy . in comparison , gtn has high utility , low cost ( 5/$7 per patient ) , is easily administered and should be easy to implement if efficacy is confirmed . importantly , the source of the patch does not influence efficacy ; participants within enos ( including those randomised within 6 h ) received patches from different manufacturers , whilst the other gtn trials used one manufacturer to supply each trial ( table 1 ) . in the future , gtn could be used on a global scale in developing and developed countries , rural and urban areas , before and in hospital , and in a variety of healthcare settings . the possibility of an inexpensive and effective intervention for the management of acute stroke is a welcome prospect in the current economic climate . open access costs were covered by the national institute of health research ( nihr ) health technology assessment programme ( 10/104/24 ) . jpa is funded by the british heart foundation ( bhf , cs/14/4/30972 ) and national institute of health research ( nihr ) health technology assessment programme ( 10/104/24 ) . pmb was / is chief investigator of the trials involving gtn ( gtn-1/2/3 , enos , and right-1/2 ) , is the lead applicant on the bhf grant funding the right-2 trial , is stroke association professor of stroke medicine , and is a nihr senior investigator .

the nitric oxide donor , glyceryl trinitrate ( gtn ) , is a candidate treatment for the management of acute stroke with haemodynamic and potential reperfusion and neuroprotective effects . when administered as a transdermal patch during the acute and subacute phases after stroke , gtn was safe , lowered blood pressure , maintained cerebral blood flow , and did not induce cerebral steal or alter functional outcome . however , when given within 6 h of stroke onset , gtn reduced death and dependency ( odds ratio 0.52 ; 95% confidence interval 0.340.78 ) , death , disability , cognitive impairment and mood disturbance , and improved quality of life ( data from two trials , n = 312 ) . in a pooled analysis of four studies ( n = 186 ) , gtn reduced between - visit systolic blood pressure variability over days 17 compared with no gtn ( mean difference 2.09 ; 95% confidence interval 3.83 to 0.35 ; p = 0.019 ) . the efficacy of gtn given in the ultra - acute / pre - hospital setting is currently being assessed and , if found to be beneficial , the implications for hyperacute stroke practice are significant . here , we discuss the evidence to date , potential mechanisms of action and future possibilities , including unanswered questions , for the therapeutic potential of gtn in acute stroke .

Key Points Introduction Nitric Oxide Donors and Acute Stroke Mechanisms of Action of Glyceryl Trinitrate in Acute Stroke Unanswered Questions Glyceryl Trinitrate in Acute Stroke: What Does the Future Hold? Funding Conflict of interest

transdermal glyceryl trinitrate is safe in patients with acute stroke.glyceryl trinitrate may improve outcome if administered within 6 h of stroke onset.the implications for clinical practice are substantial if efficacy is confirmed . an inexpensive and effective treatment for hyperacute stroke could be adopted globally in low- , middle- and high - income countries . brain barrier integrity , cerebral blood flow ( cbf ) , auto- and chemo - regulation [ 79 ] , and inhibition of apoptosis . in therapeutic studies , no donors reduced infarct size in both permanent and transient models of ischaemia , and increased cerebral blood flow in permanent models , but only if administered soon after stroke induction . blood pressure ( bp ) is high in 75% of people with acute stroke and is associated independently with poor functional outcome and increased death ( regardless of stroke type ) [ 27 , 28 ] , stroke recurrence in ischaemic stroke ( is ) and haematoma expansion in intracerebral haemorrhage ( ich ) . hence , lowering no can lead to worse outcomes in acute stroke , whilst increasing no may be beneficial . here , we discuss the evidence to date , potential mechanisms of action and future possibilities , including unanswered questions , for the therapeutic potential of the no donor glyceryl trinitrate ( gtn ) in acute stroke . transdermal gtn has been administered as a transdermal patch to patients with acute and subacute stroke in three phase ii trials ; gtn lowered bp ( peripheral and central ) , 24-h bp , peak systolic bp ( sbp ) , pulse pressure and pulse pressure index ; increased heart rate ; improved vascular compliance ; and did not alter cerebral blood flow and velocity , or induce cerebral steal or increase intracranial pressure ( table 1 ) [ 4145 ] . none of these earlier studies were powered for efficacy , and this was assessed in the large efficacy of nitric oxide in stroke ( enos ) trial .table 1effects of gtn in acute / subacute strokegtn-1 2001 gtn-2 2003 gtn-3 2006 right 2013 enos 2015 gtn 1 - 2 gtn 1 - 3 systolic bp ( mmhg) 13 ( 7.8%) 23 ( 14%) 21 7 9.4 9.8diastolic bp ( mmhg) 5.2 ( 5.4%) 4 ( 3%) 6 3.5 4.8 4.4heart rate ( bpm)no changeno changeno changeno change 1.7 4.1 3.9map ( mmhg) 6.2% 5.0 6.4pp ( mmhg) 3.9 16 6.1ppi 0.03rpp ( mmhg.bpm)no change 323augmentation indeximprovedimprovedcerebral blood flow velocityno changeno changecerebral blood flowno changezero flow pressureno changeplatelet functionno changegtn supplier ( 5 mg)schwarz pharmaschwarz pharma ( 5 and 10 mg)novartis ( transiderm - nitro)msd schering - plough ( nitrodur)ucb pharma ( deponit-5 ) in 38% of sites ( 29%)novartis ( transiderm - nitro ) in 28% of sites ( 34%)msd / schering - plough ( nitrodur ) in 25% of sites ( 19%)meda/3 m healthcare ( minitran ) in 10% of sites ( 3%)wuhan jianmin in 1% of sites ( 2% ) bp blood pressure , bpm beats per minute , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , rpp rate pressure product , indicates a decrease , indicates an increase percentage of sites for enos - early ( within 6 h ) effects of gtn in acute / subacute stroke bp blood pressure , bpm beats per minute , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , rpp rate pressure product , indicates a decrease , indicates an increase percentage of sites for enos - early ( within 6 h ) enos enrolled 4011 participants with acute stroke ( within 48 h of onset ) and raised systolic bp ( 140220 mmhg ) and randomised these to transdermal gtn patch ( 5 mg ) or no patch . overall , there was no significant shift in functional outcome measured using the modified rankin scale at day 90 ( primary outcome , adjusted common odds ratio 1.01 , 95% confidence intervals [ ci ] 0.911.13 ) or of any secondary outcomes ; further , gtn was safe with no increased reporting of serious adverse events . in a pre - defined subgroup by time to randomisation ( enos - early ) , those who received gtn within 6 h of stroke onset had a favourable shift in the modified rankin scale ( adjusted common odds ratio 0.51 , 95% ci 0.320.80 ) , less death , less disability ( barthel index ) , less mood disturbance ( zung depression scale ) , and improved cognition ( telephone mini - mental state examination ) and quality of life ( euro - quality of life visual analogue scale and health utility scale ) . the small ambulance - based rapid intervention with glyceryl trinitrate in hypertensive stroke trial ( right ) also found that transdermal gtn 5 mg , given in the pre - hospital setting by paramedics within 4 h of ictus , improved the modified rankin scale at day 90 . an individual patient data meta - analysis using data from the five completed gtn trials ( gtn-1/2/3 , enos , right , n = 4197 ) supported the findings that treatment with gtn within 6 h of onset ( n = 312 ) , but not later , improved functional outcome and secondary outcomes across a range of domains : cognition ; death ; disability ; mood ; and quality of life ( table 2 ) . those with ich who received gtn within 6 h of onset had significant improvements in functional outcome , cognition , disability , mood and quality of life at 90 days compared with those who did not receive gtn . a tendency to improved outcome was also seen in those with is who did not receive intravenous alteplase .table 2clinical outcomes with gtn in acute stroke all gtnrandomisation 6 hpatients ( % ) 4197312 ( 7.4)end of treatment death1.15 ( 0.79 , 1.68)0.94 ( 0.23 , 3.81 ) neurological deterioration1.29 ( 1.00 , 1.66)0.58 ( 0.28 , 1.23 ) stroke recurrence1.39 ( 0.88 , 2.18)0.46 ( 0.14 , 1.54 ) sss ( /58)0.33 ( 0.26 , 0.91)2.33 ( 0.42 , 5.09 ) nihss 0.28 ( 0.70 , 0.14)2.07 ( 3.81 , 0.34 ) day 7 non - oral feeding 0.97 ( 0.82 , 1.15)0.59 ( 0.32 , 1.08)hospital length of stay ( days)0.07 ( 1.30 , 1.44)0.02 ( 4.59 , 4.63 ) physiotherapy 0.94 ( 0.79 , 1.12)0.90 ( 0.40 , 2.05 ) occupational therapy 1.01 ( 0.72 , 1.41)1.15 ( 0.36 , 3.68 ) speech therapy 0.95 ( 0.84 , 1.08)1.01 ( 0.44 , 2.29)day 90 modified rankin scale0.99 ( 0.89 , 1.10 ) 0.52 ( 0.34 , 0.78 ) death0.87 ( 0.71 , 1.07 ) 0.32 ( 0.14 , 0.78 ) barthel index1.73 ( 0.08 , 3.55 ) 9.64 ( 3.19 , 16.09 ) quality of life ( hus)0 ( 0.02 , 0.02)0.05 ( 0.02 , 0.13 ) quality of life ( eq - vas)0.69 ( 1.06 , 2.43)5.97 ( 0.30 , 12.24 ) mood ( zds)0.38 ( 1.80 , 1.04)8.34 ( 13.32 , 3.36 ) cognition ( t - mmse)0.34 ( 0.16 , 0.84 ) 2.09 ( 0.65 , 3.54 ) cognition ( tics - m)0.16 ( 0.55 , 0.88 ) 3.56 ( 1.20 , 5.91 ) cognition ( animal naming)0.06 ( 0.60 , 0.47)1.63 ( 0.13 , 3.39)data are number ( % ) , mean difference , or odds ratio with 95% confidence intervals . significant ( p < 0.05 ) results are in bold bi barthel index , eq - vas euro - quality of life visual analogue scale , gtn glyceryl trinitrate , hus health utility scale ( derived from euro - quality of life 5-dimensions ( eq5d ) ) , nihss national institutes of health stroke scale , sss scandinavian stroke scale , tics - m telephone interview cognition scale , t - mmse telephone mini - mental state examination , zds zung depression scale unadjusted using the mantel haenszel random - effects model clinical outcomes with gtn in acute stroke data are number ( % ) , mean difference , or odds ratio with 95% confidence intervals . significant ( p < 0.05 ) results are in bold bi barthel index , eq - vas euro - quality of life visual analogue scale , gtn glyceryl trinitrate , hus health utility scale ( derived from euro - quality of life 5-dimensions ( eq5d ) ) , nihss national institutes of health stroke scale , sss scandinavian stroke scale , tics - m telephone interview cognition scale , t - mmse telephone mini - mental state examination , zds zung depression scale unadjusted using the mantel haenszel random - effects model further trials are needed to confirm whether gtn is efficacious when given early in patients with acute stroke ; one study , the rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 ( right-2 ) , is assessing transdermal 5-mg gtn patch vs. sham in 850 patients with presumed stroke within 4 h of onset , sbp > 120 mmhg and fast score 2 or 3 ; paramedics perform consent , recruitment and treatment in the pre - hospital setting . if transdermal gtn is found to be beneficial in the management of acute stroke , there are several potential mechanisms through which its actions may be mediated ( table 3 ) . second , in addition to high sbp being associated with poor clinical outcomes in acute stroke , other haemodynamic variables including higher peak sbp , mean arterial pressure , pulse pressure , pulse pressure index and increased sbp variability are independently associated with worse functional outcome , death , recurrent stroke and early neurological deterioration [ 5254 ] . the published effects of gtn on haemodynamic measures in acute stroke are detailed in table 1 . between - visit systolic bp variability was calculated as standard deviation ( sd ) and coefficient of variation ( cov = sd / mean ) of sbp over days 17 across each trial individually and across all four trials as a whole . the mean difference between gtn and no gtn was calculated using analysis of covariance with adjustment for baseline sbp and trial as appropriate . gtn reduced sbp variability ( sd and cov ) over days 17 when given within 4 h in the right pre - hospital trial in both adjusted and unadjusted analyses . pre - specified secondary analysis of haemodynamic parameters in enos is awaited .table 3gtn in acute stroke : mechanisms of action and unanswered questionsissueprior observationscommenttimereperfusion therapies exhibit time dependency : thrombolysis and thrombectomy apparent time - dependent effect mirrors thrombolysis and thrombectomystroke type ischaemic strokebp lowering may reduce recurrent eventsapparent benefit : vasodilatory effects may improve blood flow in large arteries ( front door ) and pial arteries / collaterals ( back door ) ; new data awaited intracerebral haemorrhagebp lowering may reduce haematoma expansion apparent benefit ; new data awaitedstroke syndrome lacunarunclear effect ; further analyses and new data awaited total anterior circulationapparent benefit : tendency for improved functional outcome in total anterior circulation in enos ; further analyses and new data awaitedstroke severity severityapparent benefit : tendency for improved functional outcome in severe stroke in enos ; further analyses and new data awaitedsafety carotid stenosisbp lowering might reduce perfusioninitial safety seen in all levels of ipsilateral carotid stenosis in enos ; further analyses and new data awaited large vessel occlusionunknown ; further analyses and new data awaited dehydration and strokelarge drops in bp may occur in dehydrated patients given antihypertensive medicationrelevance to gtn unknown ; further analyses and new data awaited stroke mimicsno adverse effect seen in right ; new data awaitedduration of therapytachyphylaxis seen in gtn-1/2 and enos [ 41 , 42 , 47 ] . effect on heart rate variability to be ascertained bp blood pressure , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right2 rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 , sbp systolic bp table 4effect of gtn on blood pressure variability in acute / subacute stroketrialpatients ( n)otr ( h)sd sbp days 17cov sbp days 17 ( % ) unadjusted p valueadjusted p valueunadjusted p valueadjusted p valuegtn-1 37<1202.0 ( 6.4 , 2.4)0.362.7 ( 6.8 , 1.4)0.191.2 ( 3.7 , 1.3)0.321.5 ( 4.0 , 1.0)0.23gtn-2 90<720.1 ( 2.2 , 2.3)0.960.1 ( 2.0 , 2.3)0.920.2 ( 1.4 , 1.7)0.830.2 ( 1.3 , 1.7)0.81gtn-3 18<1201.2 ( 3.7 , 6.0)0.621.4 ( 3.4 , 6.3)0.542.4 ( 0.5 , 5.2)0.102.5 ( 0.5 , 5.4)0.10right 41<47.7 ( 12.1 , 3.3 ) 0.001 7.2 ( 11.5 , 2.8 ) 0.002 4.9 ( 8.0 , 1.8 ) 0.003 4.9 ( 8.1 , 1.7 ) 0.003 combined1861.6 ( 3.8 , 0.6)0.142.1 ( 3.8 , 0.4 ) 0.019 0.7 ( 2.2 , 0.7)0.311.2 ( 2.4 , 0.0)0.058data are mean difference ( 95% confidence intervals ) with adjustment for baseline sbp trial . results are in bold cov coefficient of variation , gtn glyceryl trinitrate , otr onset to randomization , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , sbp systolic blood pressure , sd standard deviation gtn in acute stroke : mechanisms of action and unanswered questions bp blood pressure , enos efficacy of nitric oxide in stroke trial , gtn glyceryl trinitrate , map mean arterial pressure , pp pulse pressure , ppi pulse pressure index , right2 rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 , sbp systolic bp effect of gtn on blood pressure variability in acute / subacute stroke data are mean difference ( 95% confidence intervals ) with adjustment for baseline sbp trial . significant ( p < 0.05 ) results are in bold cov coefficient of variation , gtn glyceryl trinitrate , otr onset to randomization , right rapid intervention with glyceryl trinitrate in hypertensive stroke trial , sbp systolic blood pressure , sd standard deviation third , the time dependency of early treatment with gtn is akin to that seen in both thrombolysis and endovascular therapy , so - called reperfusion treatments [ 56 , 57 ] . although enos confirmed the overall safety of gtn in acute stroke , several distinct scenarios warrant further discussion ( table 3 ) . across all levels of ipsilateral carotid artery stenosis within enos ( 2038 participants with carotid imaging data ) , however , data for patients with severe bilateral carotid stenosis are sparse , given its rarity , although a meta - analysis of three trials found that lower bp was associated with increased stroke recurrence . bp lowering in the setting of dehydration may lead to precipitous drops in bp , which could be harmful ; the effect of gtn in this scenario is unclear and a subgroup analysis of enos may prove illuminating . hemicraniectomy ) , feeding status , therapy usage and length of stay ( overall , intensive care unit ) , as these may be influenced by the efficacy of gtn . there are few evidence - based treatments for the management of acute stroke . in the future , gtn could be used on a global scale in developing and developed countries , rural and urban areas , before and in hospital , and in a variety of healthcare settings . the possibility of an inexpensive and effective intervention for the management of acute stroke is a welcome prospect in the current economic climate . pmb was / is chief investigator of the trials involving gtn ( gtn-1/2/3 , enos , and right-1/2 ) , is the lead applicant on the bhf grant funding the right-2 trial , is stroke association professor of stroke medicine , and is a nihr senior investigator .

yeast species provide remarkable opportunities to study genomic evolution . in the two decades since the sequence of saccharomyces cerevisiae first , studies such as mutation accumulation experiments have provided a view of mutational processes and rates at unprecedented resolution , both in saccharomyces ( lynch et al . 2008 ; nishant et al . 2010 ; zhu et al . second , population genomics studies are providing extensive detail about genetic variation , both in s. cerevisiae and in its undomesticated relative s. paradoxus , which has led to inferences about the natural life cycle , the strength and direction of selection , and the nature of quantitative genetic variation of yeast phenotypes ( tsai et al . 2008 ; liti et al . third , the comprehensive annotation and wealth of experimental information about the functions of s. cerevisiae genes has provided a solid starting point for the exploration of the genomes of related fungi ( sunnerhagen and piskur 2006 ; dujon 2010 ; rozpedowska , piskur and wolfe 2011 ; zarin and moses 2014 ) . this third area 1 ) , which in turn is part of the subphylum saccharomycotina ( kurtzman 2011 ) . the most striking evolutionary event in the family was the occurrence of a whole - genome duplication ( wgd ) approximately 100200 million years ago ( wolfe and shields 1997 ) . this event defines a clade called the post - wgd species , whose genomes contain evidence of this shared event , whereas outgroup species that diverged from the s. cerevisiae lineage before the wgd occurred are called non - wgd species . the wgd increased this number transiently to about 10 000 genes , but most of the extra copies of genes were not retained and instead they became lostthat is , one of the two genes in each pair became deleted , usually without any other rearrangements in the local area of chromosome ( byrnes , morris and li 2006 ; scannell , butler and wolfe 2007a ) . post - wgd species now typically contain about 5500 genes , which includes 500 pairs of genes ( ohnologs ) that were formed by the wgd ; the other 4500 loci were not retained in duplicate and became single - copy again . phylogenetic relationships of species in the family saccharomycetaceae , and summary of some gene content differences . letters above branches indicate inferred points of loss of the dal ( d ) , gal ( g ) , bna ( b ) , his ( h ) , rnai ( r ; loss of all argonaute genes or all dicer genes ) , nhej ( n ) and dynein ( y ) pathways . gal genes refers to gal1/3 , gal4 , gal7 , gal10 and gal80 ( hittinger , rokas and carroll 2004 ) . the topology of the cladogram is from ( kurtzman 2003 ; hedtke , townsend and hillis 2006 ; gordon et al . note that tetrapisispora does not appear to be monophyletic ( gordon et al . the order of genes along chromosomes ( synteny ) is strongly conserved within the family saccharomycetaceae , although it is relatively poorly conserved in more distant comparisons between different families within saccharomycotina ( dujon 2010 ) . we developed a database and browser interface , the yeast gene order browser ( ygob http://ygob.ucd.ie ) , as a resource for exploring synteny relationships among saccharomycetaceae species ( byrne and wolfe 2005 ) . synteny comparisons , both within genomes and between post - wgd and non - wgd genomes , provided the main evidence for wgd ( wolfe and shields 1997 ; dietrich et al . 2004 ; dujon et al . 2004 ; kellis , birren and lander 2004 ) . synteny conservation also enabled our laboratory , in 2009 , to infer the approximate gene content and genome organization of the ancestral yeast organism that underwent wgd , and hence to study the chromosomal rearrangements , gene losses and gains that occurred in s. cerevisiae and other post - wgd species in their hundred - million - year descent from this ancestor ( gordon , byrne and wolfe 2009 ) . it is a description of the state of the genome immediately before the wgd occurred . our ancestral genome reconstruction was inferred manually , whereas other groups used computer - assisted methods and obtained highly congruent results ( sankoff 2009 ) . the ancestral genome reconstruction provides a convenient reference point for studying the evolution of gene content and chromosome organization in saccharomycetaceae . the ancestral genome consists of eight lists , each of which is the deduced order of genes along one of the eight ancestral chromosomes that later became duplicated by the wgd . its genes are named according to their chromosome and position , for example , anc_2.345 indicates the 345th gene along ancestral chromosome 2 . in any non - wgd species in the saccharomycetaceae , the gene order along any section of chromosome is usually similar to the ancestral order . discontinuities correspond to genomic rearrangements , either in the non - wgd species or on the lineage of the ancestor ( i.e. the post - wgd lineage prior to the occurrence of the wgd ) . in any post - wgd species , there are two genomic regions corresponding to each chromosomal region in the ancestor . each of these regions contains a subset of the ancestral genes , usually without rearrangement of gene order , and some ancestral genes remain in duplicate ( ohnologs ) and thus appear on both chromosomal regions of the post - wgd species . we now have a genome sequence from at least one species in almost every known genus of the family saccharomycetaceae as defined by kurtzman ( 2011 ) ( fig . 1 ) . the only genera classified in this family that have not yet been sequenced are zygotorulaspora ( a sister clade to torulaspora and zygosaccharomyces ; kurtzman 2003 ) and cyniclomyces whose phylogenetic position is uncertain and which may be basal to the family ( boundy - mills and miller 2011 ) . outside this family ( kurtzman 2003 ; kurtzman and robnett 2013 ) , we have relatively limited genomic data from the closest outgroup genera such as hanseniaspora / kloeckera ( giorello et al . 2012a , b ) and cyberlindnera ( tomita et al . 2012 ; freel et al . although genome evolution in the sequenced saccharomycetaceae species has largely been conservative , with similar genes being arranged in similar ways along the chromosomes of each species , the exceptions to this rule the differences between the species can often be of interest and can point to differences in the biology of the species that own the genomes . in this review , we focus on aspects that are unique to the genome of a particular species or genus . we focus in particular on seven genomes that we sequenced in 2011 , from three post - wgd genera ( naumovozyma , kazachstania and tetrapisispora ) and one non - wgd genus ( torulaspora ) . we have previously described the evolution of the mating - type ( mat ) loci of these species ( gordon et al . we sampled three post - wgd genera that had not previously been extensively studied : naumovozyma , kazachstania and tetrapisispora ( kurtzman 2003 ) . together with the recent sequencing of genomes in the nakaseomyces clade ( including candida glabrata and its asexual relatives ) by gabaldon et al . ( 2013 ) , these data mean that we now have a genome sequence for every known post - wgd genus , and multiple genomes for all post - wgd genera except vanderwaltozyma ( fig . the genome of naumovozyma castellii had been sequenced to draft level by cliften et al . ( 2003 , 2006 ) and it had been shown to be a post - wgd species with differential gene loss as compared to s. cerevisiae ( langkjaer et al . 2003 ; scannell , butler and wolfe 2007a ) . we completed the genome sequence of the type strain of n. castellii ( cbs 4309 ; this species was previously also called saccharomyces castellii and naumovia castellii ) , and also sequenced its congener n. dairenensis ( cbs 421 ) . until recently , these were the only two species known in the genus naumovozyma ( liu et al . the genus kazachstania is much more species rich and we sequenced two representatives , kazachstania naganishii ( cbs 8797 ) and k. africana ( cbs 2517 ) , that span the phylogenetic breadth of this genus ( vaughan - martini , lachance and kurtzman 2011 ) . kazachstania naganishii was previously known as s. exiguus strain yp74l-3 , before it was realized to be a separate species distinct from s. exiguus ( which itself is now called k. exigua ) . extensive methods for genetic manipulation of k. naganishii have been developed by hisatomi , kubota and tsuboi ( 1999a , b ) and sugihara et al . we sequenced two tetrapisispora species because this genus was known to be sister to vanderwaltozyma , representing the post - wgd lineage most distantly related to s. cerevisiae . analysis of the genome of vanderwaltozyma polyspora ( obsolete name : kluyveromyces polysporus ) showed that it had undergone extensive loss of duplicate genes independently of the post - wgd gene losses in s. cerevisiae ( scannell et al . we again chose two species spanning the phylogenetic breadth of the genus : tetrapisispora phaffii ( cbs 4417 ) and t. blattae ( cbs 6284 ) . a transformation system for t. phaffii has been developed ( oro et al . 2014 ) . tetrapisispora blattae is an outgroup to all other known species of tetrapisispora ( lachance 2011 ) . surprisingly , we found by phylogenomic analysis that the genus tetrapisispora is not monophyletic : t. phaffii and v. polyspora are more closely related to each other than either of them is to t. blattae ( gordon et al . excluding the rdna locus , the t. blattae genome is relatively large for a post - wgd species . the genome contains some unusually large regions of non - coding dna ( for example , flanking the mat genes ; gordon et al . it is also characterized by the presence of regions of amino acid repeats internal to many proteins . an extreme example is its ortholog of s. cerevisiae yel1 ( a gef required for localization of arf3 to the bud neck ) , which is more than twice the length of the orthologs in all other saccharomycetaceae ( 2001 residues compared to 687 in s. cerevisiae ) . across the whole proteome , t. blattae proteins have a longer median length than any of the other species discussed here ( table 1 ) . but at the same time , the t. blattae genome lacks orthologs of some well - known large genes : it has ira1 but not ira2 , tor1 but not tor2 , sph1 but not spa2 . in each of these cases , the lost gene is one member of an ohnolog pair in s. cerevisiae . we also sequenced torulaspora delbrueckii ( cbs 1146 ) , a stress - tolerant yeast that is associated with winemaking ( albertin et al . 2014 ) . torulaspora , zygosaccharomyces and a third genus zygotorulaspora comprise the clade of non - wgd species that is the closest outgroup to the post - wgd species ( fig . the genomes of two zygosaccharomyces species , z. rouxii and z. bailii , were sequenced by souciet et al . , there is a larger clade of better known non - wgd genera : kluyveromyces , lachancea and eremothecium ( ashbya ) . clade - specific gene amplifications are of interest because they can indicate recent directional evolutionary pressure on genomes . saccharomyces cerevisiae , for example , contains more hexose transporters than other saccharomycetaceae , and this may indicate adaptation to the fermentative lifestyle ( piskur et al . 2006 ; merico et al . 2007 ; lin and li 2011 ) . in many cases , however , the amplifications are of orphan genes that lack homologs in other species and whose functions are unknown . there is at least one such orphan family in s. cerevisiae itself : a family of five highly divergent but related genes including abm1 . these genes are of unknown function and appear as recent insertions into the saccharomyces genome when compared to the ancestral genome ( gordon , byrne and wolfe 2009 ) . yeasts other than s. cerevisiae also contain species - specific or clade - specific gene amplifications . in many of these cases , however , it is difficult to say anything about the functions of the amplified genes because they are completely unknown and the genes lack homologs or even recognizable protein domains . for example , the n. castellii genome has two large orphan gene families , as first described by cliften et al . it was recently identified as a transposase of the hat family , part of a mobile element that was named roamer ( sarilar , bleykasten - grosshans and neuveglise 2015 ) . the other orphan family , exemplified by ncas0g03840 , has 24 members and lacks any identifiable protein domains . despite being species - specific , this family is highly divergent in sequence with only 27% amino acid sequence identity between its most diverse members . similarly t. phaffii has a diverse nine - member family , again completely species - specific , exemplified by tpha0n00100 . the existence of such diverse gene families within a single species raises some interesting ( but currently unanswerable ) evolutionary questions about how they originated and how the members of the family became so different in sequence . in other cases orphan gene families are genus - specific , for example a family with 5 members in n. castellii ( e.g. ncas0g01740 ) and 15 members in n. dairenensis ( e.g. ndai0g00110 ) . in this last example , many of the n. dairenensis members of the family are telomeric , whereas the five n. castellii genes are not telomeric but are all clustered within a 40-kb region on chromosome 7 . we identified a singleton gene in t. blattae ( tbla0d02000 , 1032 amino acids ) with similarity to dna transposases of the mule ( mutator - like element ) family . mules are members of the mutator superfamily of dna transposons ( class ii transposable elements ) ( neuveglise et al . the t. blattae gene has highest similarity to the 3 gene of k. lactis , which functions in mating - type switching in that species ( barsoum , martinez and astrom 2010 ; rajaei et al . 2014 ) and is the only other mule - like transposase in the family saccharomycetaceae . complete mule elements have terminal inverted repeats ( tirs ) as well as a transposase gene , but the only described mule in subphylum saccharomycotina that is complete and active in transposition is the mutyl element of yarrowia lipolytica ( neuveglise et al . we also found truncated non - syntenic homologs of tbla0d02000 in other saccharomycetaceae : tpha0g02110 in t. phaffii ( 270 residues lacking the conserved transposase domain ) , and several possible pseudogene fragments in n. castellii and n. dairenensis . the mat1 gene of n. dairenensis contains two identical tandem copies of a 20-bp repeat sequence , which cause a frameshift by comparison to the 1 genes of other species . losses of genes from the mat locus have previously been identified in the ctg clade of candida species ( logue et al . 2005 ; butler 2010 ) , but are uncommon in the saccharomycetaceae where all other species have intact 1 , 2 and a1 genes . the a2 gene , coding for an hmg - domain activator of transcription of a - specific genes ( tsong et al . 2003 , 2006 ) , is present in all non - wgd saccharomycetaceae but was lost in the common ancestor of all post - wgd species , more or less concurrently with the wgd ( butler et al . 2004 ) . in k. naganishii , the hmr locus , containing a silenced copy of the mating - type a information , is not located near a telomere as in all other species . instead , it is located between orthologs of the s. cerevisiae genes ykr011c and tof2 , more than 300 kb from the nearest telomere . 2 ) shows that the copy of the a1 gene at hmr is truncated at the 5 end , which is unusual . the way these loci are organized in k. naganishii makes silencing of transcription at hmr unnecessary , because the copy of the a1 gene at hmr has no promoter or start codon . during mating - type switching , a copy of the 3 part of the a1 gene from hmr is inserted beside the complete 5 part of the gene at the mat locus to assemble a full - length and functional a1 gene . the lack of requirement for silencing has apparently removed the need for hmr to be located beside a telomere , allowing hmr to move to a new chromosomal site in this species . however , in s. cerevisiae chromatin modification at hmr has a dual role : as well as silencing transcription , it also prevents the ho endonuclease cleaving hmr ( haber 2012 ) . therefore , it is unclear what prevents k. naganishii ho endonuclease from cleaving its hmr , and laboratory experiments will be necessary to discover whether there are chromatin modifications at k. naganishii hmr . the organization of the mat , hml and hmr loci and the triplicated x and z repeat regions is shown schematically . the copy of the a1 gene at hmr lacks exon 1 and therefore does not need to be transcriptionally repressed by chromatin modification . hml is close to a telomere and transcription of hml1 and hml2 is probably repressed by sir proteins . this change is the result of a rearrangement that occurred between the hml locus and the ancestral telomere . the t. blattae hml genes ( hml1 = anc_1.1 = tbla0a07590 and hml2 = anc_1.2 = tbla0a07600 ) retain linkage to the end of ancestral chromosome 1 on one side ( they are beside anc_1.4 and anc_1.5 ) , but on the other side , where a telomere is found in most other species , they are instead adjacent to genes anc_7.365 ( pex13 ) and anc_7.366 ( mmr1 ) from the middle of ancestral chromosome 7 . consequently , t. blattae hml is more than 800 kb from the nearest telomere . it is still on the same chromosome as the mat locus as seen in all saccharomycetaceae ( gordon et al . tetrapisispora blattae has a low number of chromosomes for a post - wgd species ( 10 chromosomes ; fig . 1 ) , and this example of a telomere fusion is one of several that have occurred in the t. blattae genome . unlike the situation in k. naganishii , sir proteins are probably still required for silencing of the non - telomeric hml in t. blattae because the copy of the 1 gene at this locus is full length and intact ( hml2 is truncated at the 3 end ) . in k. africana this species has no ho endonuclease gene . instead of the usual arrangement of three mat - like loci ( mat , hml and hmr ) , this species has only two mat - like loci which we refer to as mata and mat ( fig . appear to be an hml or hmr - type silent cassette because they have no x or z repeat sequences that could allow dna exchange to occur between the two loci during mating - type switching . the situation in k. africana appears to have arisen by chromosome breakage and rearrangement ; its mat and mata loci share synteny with the left and right sides , respectively , of the mat locus in the related species k. naganishii which has an organization resembling s. cerevisiae . we are confident that our assembly of the k. africana genome is structurally correct across the mata and mat loci because our sequencing strategy generated pairs of sequence reads from the ends of clones in four genomic libraries with large insert sizes ( averaging 3 , 7 , 19 and 20 kb ) . the chromosome 4 and 7 rearrangements are supported by 134 and 160 unique pairs of reads , respectively , with no pairs supporting an unrearranged configuration . the type strain of k. africana ( cbs 2517 ) , which is the strain we sequenced , has been described as diploid and capable of sporulation ( vaughan - martini , lachance and kurtzman 2011 ) . kazachstania africana ( orange ) has a mata - like locus on chromosome 7 and a mat-like locus on chromosome 4 . it has no apparent hml or hmr loci , no z or x repeats , and no ho endonuclease gene . for comparison , both idiomorphs ( mat and mata ) of the mat locus in the related species k. naganishii are shown ( blue ) . one possible scenario to explain the rearrangement in k. africana is that a mat chromosome broke into two pieces during an attempt to switch mating types . one piece , consisting of the right - hand part of the mat chromosome ( as drawn in fig . 3 ) and the mata1 gene , gained a new telomere to form k. africana chromosome 7 . the other piece , consisting of the left - hand part of the mat chromosome and the mat genes , became fused end to end with another chromosome corresponding to k. naganishii chromosome 12 ( fig . after this rearrangement occurred , the species was unable to switch mating types so the hml , hmr and ho loci all became unnecessary and were lost . other scenarios , such as loss of ho before loss of hml and hmr and rearrangement of the mat locus , are also plausible . importantly , in the absence of any experimental data from k. africana it remains unclear how cell types are specified in this species , particularly whether mata and mat genes are both transcribed in haploid cells . all species of saccharomycetaceae contain homologs of the s. cerevisiae genes for mating pheromones ( -factor mf and a - factor mfa ) and their receptors ( ste2 and ste3 ) . the a - factor genes seem to be remarkably mobile : among 23 species we examined , mfa genes were found at 19 different genomic locations ( fig . 4a ; see also oheigeartaigh et al . some of this diversity is due to high levels of gene duplication ( for example n. castellii and t. blattae each have five mfa genes ) . the diversity of locations seen even in species with only one mfa gene may be due to cycles of gene duplication and loss from the original location . the s. cerevisiae mfa2 gene is at a position ( anc_2.114 ) that appears to be the ancestral mfa site for all post - wgd species and their close non - wgd relatives z. rouxii and to . other non - wgd species show mfa genes at four different sites and it is unclear which of these sites is ancestral to the non - wgd clade ( fig . notably , in many cases where an mfa gene has a location unique to a single species or genus , the location is also a site of rearrangement in that clade relative to the ancestral gene order ( indicated by two this suggests that the duplication of mfa genes may have occurred at the same time as the chromosomal rearrangement . the high mobility of mfa genes may be related to their small size , with a coding region of only 100 bp ( oheigeartaigh et al . complete a - factor proteins sequences are aligned and arranged into groups of orthologs according to their genomic location . genes with two anc numbers are located at points of rearrangement relative to the ancestral genome . the number of mature pheromone repeat units and the amino acid sequence of the most common repeat unit is shown . in contrast to the mfa genes , genes for -pheromone and the ste2/ste3 pheromone receptors show more sedate modes of evolution . the -pheromone gene(s ) code for precursor proteins containing one to eight repeats of an active peptide that is 13 amino acid residues long in most species ( fig . exceptions to this pattern are ashbya gossypii whose mf gene ( aar163c ) codes for a single copy of a 12-mer peptide that has been shown to be functional ( wendland , dunkler and walther 2011 ) , and to . delbrueckii whose mf gene ( tdel0g01600 ) appears to code for three copies of a 12-mer peptide ( fig . 4b ) . only two duplications of mf genes can be inferred , apart from the retention of two ohnologs of the gene at its ancestral location ( anc_6.185 ) in many species after wgd . one duplication produced a second copy in an ancestor of naumovozyma species , and the other produced a second copy in an ancestor of the eremothecium / lachancea clade ( wendland , dunkler and walther 2011 ) . interestingly , neofunctionalization of extra copies of pheromone genes appears to have occurred , separately in a. gossypii and n. dairenensis , after these two duplications . in each case , a gene was formed ( afl062w and ndai0f02280 ) that codes for an n - terminal secretion signal similar to a pheromone precursor protein , but the gene does not code for any pheromone - like repeats . the ste2 and ste3 pheromone receptor genes are single copy in all species , except v. polyspora which retained two ste2 ohnologs after wgd , and they have not moved from their ancestral locations in any species . the dal cluster of six genes , coding for the allantoin catabolism pathway , is the largest metabolic gene cluster in s. cerevisiae ( wong and wolfe 2005 ; naseeb and delneri 2012 ) . we previously showed that there is also a dal cluster in n. castellii , but not in any non - wgd species , and that this cluster was assembled by relocation of genes after the wgd ( wong and wolfe 2005 ) . naumovozyma dairenensis contains a dal cluster with identical gene content and order to n. castellii . in k. naganishii , the cluster has expanded even more by the incorporation of a seventh gene , the allantoin transporter dal5/knag0d03140 , into the cluster . seven - gene dal clusters including dal5 have also been found in nakaseomyces bacillisporus and c. castellii ( gabaldon et al . the phylogenetic distribution of the seven - gene cluster , and the fact that dal5 is at a different place in the clusters , suggests that dal5 was recruited into the cluster by two separate events in the kazachstania and nakaseomyces genera . the dal cluster in k. naganishii is not located at a site orthologous to the dal clusters in s. cerevisiae and n. castellii , but instead is at a site that corresponds to a point of rearrangement between an ancestral telomere ( anc_3.581 ) and an internal chromosomal site ( anc_4.55 ) . in stark contrast to the k. naganishii cluster , the dal genes are completely absent from the genomes of its congener k. africana and both of the tetrapisispora species , as well as the previously reported absences from v. polyspora ( scannell et al . 2007b ) and four species in the c. glabrata / nakaseomyces clade ( gabaldon et al . the dal genes are either tightly clustered in the genome or completely absent from the genome , in all post - wgd species , whereas they are scattered around the genome in all non - wgd species . it appears that the duplication of the gene pairs dal7/mls1 and dal4/fur4 , which occurred as part of the wgd , facilitated a major reorganization of the dal pathway , enabling both the formation of a cluster and multiple subsequent losses of that cluster . similar to the dal genes , the gal genes for galactose catabolism form a cluster in many yeast species but are completely absent in others ( hittinger , rokas and carroll 2004 ; slot and rokas 2010 ) ( fig . when present in saccharomycetaceae , this cluster is usually found in a conserved syntenic location corresponding to the position of s. cerevisiae gal1/3 , gal7 and gal10 ( anc_3.2173.219 ) . delbrueckii has no gal genes at this ancestral location but instead has a large cluster of gal genes near the telomere of chromosome 5 ( fig . 5a ) . the telomeric cluster spans 22 kb and contains two gal1 genes ( 74% amino acid sequence identity ) , two gal10 genes ( 79% identity ) and one gal7 gene . in addition , it contains one copy each of the genes gal4 ( transcription activator ) and gal2 ( galactose permease ) which do not form part of the cluster in other saccharomycetaceae . delbrueckii cluster also contains genes for additional enzymatic steps in the leloir pathway : it has homologs of s. cerevisiae mel1 ( secreted alpha - galactosidase , which converts extracellular melibiose into galactose and glucose ) , and pgm1 ( phosphoglucomutase , the glycolytic step immediately downstream of the gal7 step ) . the cluster also has a homolog of k. lactis hgt1 ( high - affinity glucose transporter ; billard et al . the 10-gene cluster therefore contains all the genes necessary for conversion of extracellular melibiose into glucose-6-phosphate . although its telomeric location might suggest that the cluster was gained by horizontal gene transfer into to . delbrueckii genes are of saccharomycetaceae origin and its gal7 and gal4 genes group with zygosaccharomyces ( which has a gal1-gal10-gal7 cluster at the ancestral location ) as expected in the absence of horizontal transfer . torulaspora delbrueckii also contains a third gal10 gene ( tdel0g04910 ) near another telomere ; all three gal10 proteins contain the fused epimerase and mutarotase domains typical of saccharomycetaceae ( slot and rokas 2010 ) . the multiple gal10 and gal1 genes appear to have originated by duplications that occurred within the genus torulaspora ( fig . bailii ; souciet et al . 2009 ; galeote et al . amino acid sequences were aligned using clustal omega , filtered with gblocks and trees were constructed using phyml , all as implemented in seaview ( gouy , guindon and gascuel 2010 ) . thin lines indicate branches with alrt ( approximate likelihood ratio test ) support values below 80% . trees were rooted using pachysolen tannophilus ( ptan ) gal genes ( liu et al . there is no rna interference pathway in s. cerevisiae , but there is one in n. castellii and v. polyspora ( drinnenberg et al . 1 ) , and it has been proposed that the presence of an rnai system is inversely correlated with the presence of double - strand rna killer viruses ( drinnenberg , fink and bartel 2011 ) . delbrueckii ) out of the 11 non - wgd saccharomycetaceae species whose genomes have been sequenced , although they are present in c. albicans and many other outgroup fungi ( alexandersson and sunnerhagen 2005 ) . among the post - wgd species , argonaute is present in all studied species of two very divergent clades the vanderwaltozyma / tetrapisispora clade and the naumovozyma / kazachstania clade . argonaute has been lost in the saccharomyces genus and in all studied species of the nakaseomyces / c . it is also of interest to note that two species retained both of the ohnolog copies of the argonaute gene after the wgd , which suggests that some functional divergence or specialization within the rnai pathway may have occurred in these species ( n. dairenensis and t. blattae ) . the phylogenetic distribution of dicer genes closely follows that of argonaute , except that there are two species that retain dicer but not argonaute ( c. castellii and s. uvarum , the sole member of the genus saccharomyces that has any rnai component ) . comparison of the genome sequences to the ancestral genome reveals some examples of losses of complete biochemical pathways or protein complexes . the most dramatic of these is the absence of the histidine biosynthesis pathway in t. blattae , which lacks six genes ( his1 , his2 , his3 , his4 , his5 and his7 ) from this seven - gene pathway . tetrapisispora blattae is known to be unable to grow on minimal media without histidine ( lachance 2011 ) . it is possible that this auxotrophy reflects the natural environment of t. blattae because the only two known strains of this species were isolated from cockroaches , but it is unclear whether t. blattae is an intestinal symbiont of cockroaches or merely cockroach associated ( lachance 2011 ) . almost all genes of the dynein / dynactin pathway ( winey and bloom 2012 ) , which in s. cerevisiae controls movement of the nucleus into daughter cells during budding , are missing in four species : v. polyspora ( first reported by scannell et al . the 11 missing genes are dyn1 ( = dhc1 ) , dyn3 , arp1 , arp10 , jnm1 , nip100 , pac1 , pac11 , ndl1 , ldb18 and kip1 . 1 ) shows that loss of 1112 genes from this pathway has occurred independently three times . some of the pathway components , particularly ndl1 and kip1 , have also been lost repeatedly in many other saccharomycetaceae species without complete collapse of the pathway ( fig . losses of bna genes in the seven - gene pathway for de novo nad synthesis have previously been reported in c. glabrata and other members of the nakaseomyces genus ( domergue et al . the bna pathway is also missing in the genera kluyveromyces , kazachstania and naumovozyma , and in t. blattae ( fig . two species ( n. dairenensis and c. castellii ) retain bna6 as well as bna3 , but have lost the other five genes . candida castellii bna6 is present at the gene 's ancestral location ( anc_3.563 ) , whereas n. dairenensis bna6 has transposed to a site of species - specific genomic rearrangement . phylogenetic analysis indicated accelerated evolution in both these bna6 genes , but no evidence of horizontal gene transfer ( data not shown ) . these gene distributions indicate that bna1,2,4,5,7 have been lost on a minimum of four independent occasions ( marked by 1 ) , and bna6 has been lost at least six times , during saccharomycetaceae evolution . as previously noted ( gordon , byrne and wolfe 2011b ) , l. kluyveri lacks four genes essential for non - homologous end joining ( nhej ) , a dna repair pathway that is normally used to repair double - strand dna breaks in situations where homologous recombination is not possible , such as in haploid cells . these genes are dnl4 ( dna ligase iv ) , pol4 ( dna polymerase iv ) , lif1 ( ligase interacting factor ) and nej1 ( a regulator of nhej ) ( deshpande and wilson 2007 ) . the absence of these genes is possibly a factor in the low level of rearrangement seen in the l. kluyveri genome as compared to other saccharomycetaceae ( gordon , byrne and wolfe 2011b ) . in contrast , the nhej pathway is intact in all 25 other species shown in fig . 1 , including two other lachancea species . in summary , the tree in fig . 1 indicates that there have been multiple independent losses , in different clades , of many groups of functionally related genes : three independent losses of the dal genes , four losses of gal genes , four losses of bna genes , one loss of his genes , five losses of the rnai pathway , one loss of nhej and three losses of the dynein / dynactin complex . almost every species in the tree has been affected by one or other of these losses , which are dramatic but evidently not catastrophic . these examples of interspecies functional variation highlight the shortcomings of s. cerevisiae , or indeed any single species , as a model for the biology of a wider taxonomic clade such as the saccharomycetaceae . the rnai pathway serves as a case in point : the functions of the argonaute and dicer genes in n. castellii would never have been discovered if s. cerevisiae was the only model organism , no matter how intensively s. cerevisiae was studied . conserved hypothetical genes because they had homologs in organisms such as caenorhabditis and schizosaccharomyces in which rnai had already been discovered . by extension , it is very probable that some other important biochemical pathways , protein complexes and even biological processes that are widely conserved across many yeast species remain undiscovered because they are both ( i ) absent in the model organism s. cerevisiae , and ( ii ) yeast specific so their functions can not be inferred from other eukaryotes . our understanding of saccharomycetaceae genome evolution is also incomplete because for most clades except saccharomyces we only have one genome sequence per species . population genomics has revealed that there is extensive intraspecies polymorphism of gene content , particularly at telomeric regions , leading to the concept of the pan - genome as the complete set of genes that exists within a species even if no individual member of the species contains them all ( song et al . saccharomyces kudriavzevii provides a spectacular example of intraspecies polymorphism , with the gal genes being intact in portuguese isolates but pseudogenized in japanese isolates , the result of a balanced polymorphism at multiple separate genomic loci ( hittinger et al . it is possible that a similar situation of intraspecies presence / absence polymorphism could pertain to other sets of genes that appear to have multiple independent losses in fig . 1 , such as the dal and bna genes , and that we simply have not yet sampled enough individuals from the lineages showing apparent losses . just as the lab strain s288c has turned out to be an imperfect representative of the species s. cerevisiae , with unrepresentative alleles at loci such as flo8 and mkt1 ( liu , styles and fink 1996 ; lewis et al . 2014 ) , the species s. cerevisiae is also an imperfect representative of the family saccharomycetaceae . that does not however mean that any other species could be a better model organism . instead , we may benefit by extending the concept of the pan - genome to higher taxonomic levels to describe the complete set of biological molecules , complexes and processes that exists within saccharomycetaceae , even if no single species contains the whole set . ideally , we would then like to ask the evolutionary reasons why particular parts of the pan - genome are missing in particular lineages . however , our ability to answer such questions is severely limited by the elephant in the room : our lack of knowledge about the natural environments in which the different yeast species have evolved and the niches ( if any ) to which they are adapted ( goddard and greig 2015 ) . research in our group is supported by the european research council ( advanced grant 268893 ) and science foundation ireland ( 13ia1910 ) .

many aspects of the genomes of yeast species in the family saccharomycetaceae have been well conserved during evolution . they have similar genome sizes , genome contents , and extensive collinearity of gene order along chromosomes . gene functions can often be inferred reliably by using information from saccharomyces cerevisiae . beyond this conservative picture however , there are many instances where a species or a clade diverges substantially from the s. cerevisiae paradigm for example , by the amplification of a gene family , or by the absence of a biochemical pathway or a protein complex . here , we review clade - specific features , focusing on genomes sequenced in our laboratory from the post - wgd genera naumovozyma , kazachstania and tetrapisispora , and from the non - wgd species torulaspora delbrueckii . examples include the loss of the pathway for histidine synthesis in the cockroach - associated species tetrapisispora blattae ; the presence of a large telomeric gal gene cluster in to . delbrueckii ; losses of the dynein and dynactin complexes in several independent yeast lineages ; fragmentation of the mat locus and loss of the ho gene in kazachstania africana ; and the patchy phylogenetic distribution of rnai pathway components .

INTRODUCTION CONCLUSION FUNDING

second , population genomics studies are providing extensive detail about genetic variation , both in s. cerevisiae and in its undomesticated relative s. paradoxus , which has led to inferences about the natural life cycle , the strength and direction of selection , and the nature of quantitative genetic variation of yeast phenotypes ( tsai et al . third , the comprehensive annotation and wealth of experimental information about the functions of s. cerevisiae genes has provided a solid starting point for the exploration of the genomes of related fungi ( sunnerhagen and piskur 2006 ; dujon 2010 ; rozpedowska , piskur and wolfe 2011 ; zarin and moses 2014 ) . the most striking evolutionary event in the family was the occurrence of a whole - genome duplication ( wgd ) approximately 100200 million years ago ( wolfe and shields 1997 ) . this event defines a clade called the post - wgd species , whose genomes contain evidence of this shared event , whereas outgroup species that diverged from the s. cerevisiae lineage before the wgd occurred are called non - wgd species . post - wgd species now typically contain about 5500 genes , which includes 500 pairs of genes ( ohnologs ) that were formed by the wgd ; the other 4500 loci were not retained in duplicate and became single - copy again . phylogenetic relationships of species in the family saccharomycetaceae , and summary of some gene content differences . letters above branches indicate inferred points of loss of the dal ( d ) , gal ( g ) , bna ( b ) , his ( h ) , rnai ( r ; loss of all argonaute genes or all dicer genes ) , nhej ( n ) and dynein ( y ) pathways . the order of genes along chromosomes ( synteny ) is strongly conserved within the family saccharomycetaceae , although it is relatively poorly conserved in more distant comparisons between different families within saccharomycotina ( dujon 2010 ) . synteny comparisons , both within genomes and between post - wgd and non - wgd genomes , provided the main evidence for wgd ( wolfe and shields 1997 ; dietrich et al . synteny conservation also enabled our laboratory , in 2009 , to infer the approximate gene content and genome organization of the ancestral yeast organism that underwent wgd , and hence to study the chromosomal rearrangements , gene losses and gains that occurred in s. cerevisiae and other post - wgd species in their hundred - million - year descent from this ancestor ( gordon , byrne and wolfe 2009 ) . in any non - wgd species in the saccharomycetaceae , the gene order along any section of chromosome is usually similar to the ancestral order . discontinuities correspond to genomic rearrangements , either in the non - wgd species or on the lineage of the ancestor ( i.e. the post - wgd lineage prior to the occurrence of the wgd ) . in any post - wgd species , there are two genomic regions corresponding to each chromosomal region in the ancestor . each of these regions contains a subset of the ancestral genes , usually without rearrangement of gene order , and some ancestral genes remain in duplicate ( ohnologs ) and thus appear on both chromosomal regions of the post - wgd species . we now have a genome sequence from at least one species in almost every known genus of the family saccharomycetaceae as defined by kurtzman ( 2011 ) ( fig . although genome evolution in the sequenced saccharomycetaceae species has largely been conservative , with similar genes being arranged in similar ways along the chromosomes of each species , the exceptions to this rule the differences between the species can often be of interest and can point to differences in the biology of the species that own the genomes . in this review , we focus on aspects that are unique to the genome of a particular species or genus . we focus in particular on seven genomes that we sequenced in 2011 , from three post - wgd genera ( naumovozyma , kazachstania and tetrapisispora ) and one non - wgd genus ( torulaspora ) . we sampled three post - wgd genera that had not previously been extensively studied : naumovozyma , kazachstania and tetrapisispora ( kurtzman 2003 ) . ( 2013 ) , these data mean that we now have a genome sequence for every known post - wgd genus , and multiple genomes for all post - wgd genera except vanderwaltozyma ( fig . ( 2003 , 2006 ) and it had been shown to be a post - wgd species with differential gene loss as compared to s. cerevisiae ( langkjaer et al . we sequenced two tetrapisispora species because this genus was known to be sister to vanderwaltozyma , representing the post - wgd lineage most distantly related to s. cerevisiae . analysis of the genome of vanderwaltozyma polyspora ( obsolete name : kluyveromyces polysporus ) showed that it had undergone extensive loss of duplicate genes independently of the post - wgd gene losses in s. cerevisiae ( scannell et al . excluding the rdna locus , the t. blattae genome is relatively large for a post - wgd species . the genome contains some unusually large regions of non - coding dna ( for example , flanking the mat genes ; gordon et al . it is also characterized by the presence of regions of amino acid repeats internal to many proteins . torulaspora , zygosaccharomyces and a third genus zygotorulaspora comprise the clade of non - wgd species that is the closest outgroup to the post - wgd species ( fig . , there is a larger clade of better known non - wgd genera : kluyveromyces , lachancea and eremothecium ( ashbya ) . clade - specific gene amplifications are of interest because they can indicate recent directional evolutionary pressure on genomes . saccharomyces cerevisiae , for example , contains more hexose transporters than other saccharomycetaceae , and this may indicate adaptation to the fermentative lifestyle ( piskur et al . yeasts other than s. cerevisiae also contain species - specific or clade - specific gene amplifications . in many of these cases , however , it is difficult to say anything about the functions of the amplified genes because they are completely unknown and the genes lack homologs or even recognizable protein domains . it was recently identified as a transposase of the hat family , part of a mobile element that was named roamer ( sarilar , bleykasten - grosshans and neuveglise 2015 ) . in other cases orphan gene families are genus - specific , for example a family with 5 members in n. castellii ( e.g. in this last example , many of the n. dairenensis members of the family are telomeric , whereas the five n. castellii genes are not telomeric but are all clustered within a 40-kb region on chromosome 7 . 2014 ) and is the only other mule - like transposase in the family saccharomycetaceae . losses of genes from the mat locus have previously been identified in the ctg clade of candida species ( logue et al . 2003 , 2006 ) , is present in all non - wgd saccharomycetaceae but was lost in the common ancestor of all post - wgd species , more or less concurrently with the wgd ( butler et al . instead , it is located between orthologs of the s. cerevisiae genes ykr011c and tof2 , more than 300 kb from the nearest telomere . during mating - type switching , a copy of the 3 part of the a1 gene from hmr is inserted beside the complete 5 part of the gene at the mat locus to assemble a full - length and functional a1 gene . however , in s. cerevisiae chromatin modification at hmr has a dual role : as well as silencing transcription , it also prevents the ho endonuclease cleaving hmr ( haber 2012 ) . the organization of the mat , hml and hmr loci and the triplicated x and z repeat regions is shown schematically . this change is the result of a rearrangement that occurred between the hml locus and the ancestral telomere . tetrapisispora blattae has a low number of chromosomes for a post - wgd species ( 10 chromosomes ; fig . 1 ) , and this example of a telomere fusion is one of several that have occurred in the t. blattae genome . unlike the situation in k. naganishii , sir proteins are probably still required for silencing of the non - telomeric hml in t. blattae because the copy of the 1 gene at this locus is full length and intact ( hml2 is truncated at the 3 end ) . the situation in k. africana appears to have arisen by chromosome breakage and rearrangement ; its mat and mata loci share synteny with the left and right sides , respectively , of the mat locus in the related species k. naganishii which has an organization resembling s. cerevisiae . for comparison , both idiomorphs ( mat and mata ) of the mat locus in the related species k. naganishii are shown ( blue ) . the other piece , consisting of the left - hand part of the mat chromosome and the mat genes , became fused end to end with another chromosome corresponding to k. naganishii chromosome 12 ( fig . other scenarios , such as loss of ho before loss of hml and hmr and rearrangement of the mat locus , are also plausible . importantly , in the absence of any experimental data from k. africana it remains unclear how cell types are specified in this species , particularly whether mata and mat genes are both transcribed in haploid cells . all species of saccharomycetaceae contain homologs of the s. cerevisiae genes for mating pheromones ( -factor mf and a - factor mfa ) and their receptors ( ste2 and ste3 ) . some of this diversity is due to high levels of gene duplication ( for example n. castellii and t. blattae each have five mfa genes ) . the diversity of locations seen even in species with only one mfa gene may be due to cycles of gene duplication and loss from the original location . the s. cerevisiae mfa2 gene is at a position ( anc_2.114 ) that appears to be the ancestral mfa site for all post - wgd species and their close non - wgd relatives z. rouxii and to . other non - wgd species show mfa genes at four different sites and it is unclear which of these sites is ancestral to the non - wgd clade ( fig . only two duplications of mf genes can be inferred , apart from the retention of two ohnologs of the gene at its ancestral location ( anc_6.185 ) in many species after wgd . one duplication produced a second copy in an ancestor of naumovozyma species , and the other produced a second copy in an ancestor of the eremothecium / lachancea clade ( wendland , dunkler and walther 2011 ) . the dal cluster of six genes , coding for the allantoin catabolism pathway , is the largest metabolic gene cluster in s. cerevisiae ( wong and wolfe 2005 ; naseeb and delneri 2012 ) . we previously showed that there is also a dal cluster in n. castellii , but not in any non - wgd species , and that this cluster was assembled by relocation of genes after the wgd ( wong and wolfe 2005 ) . the phylogenetic distribution of the seven - gene cluster , and the fact that dal5 is at a different place in the clusters , suggests that dal5 was recruited into the cluster by two separate events in the kazachstania and nakaseomyces genera . the dal cluster in k. naganishii is not located at a site orthologous to the dal clusters in s. cerevisiae and n. castellii , but instead is at a site that corresponds to a point of rearrangement between an ancestral telomere ( anc_3.581 ) and an internal chromosomal site ( anc_4.55 ) . in stark contrast to the k. naganishii cluster , the dal genes are completely absent from the genomes of its congener k. africana and both of the tetrapisispora species , as well as the previously reported absences from v. polyspora ( scannell et al . 2007b ) and four species in the c. glabrata / nakaseomyces clade ( gabaldon et al . the dal genes are either tightly clustered in the genome or completely absent from the genome , in all post - wgd species , whereas they are scattered around the genome in all non - wgd species . it appears that the duplication of the gene pairs dal7/mls1 and dal4/fur4 , which occurred as part of the wgd , facilitated a major reorganization of the dal pathway , enabling both the formation of a cluster and multiple subsequent losses of that cluster . delbrueckii cluster also contains genes for additional enzymatic steps in the leloir pathway : it has homologs of s. cerevisiae mel1 ( secreted alpha - galactosidase , which converts extracellular melibiose into galactose and glucose ) , and pgm1 ( phosphoglucomutase , the glycolytic step immediately downstream of the gal7 step ) . delbrueckii genes are of saccharomycetaceae origin and its gal7 and gal4 genes group with zygosaccharomyces ( which has a gal1-gal10-gal7 cluster at the ancestral location ) as expected in the absence of horizontal transfer . 1 ) , and it has been proposed that the presence of an rnai system is inversely correlated with the presence of double - strand rna killer viruses ( drinnenberg , fink and bartel 2011 ) . delbrueckii ) out of the 11 non - wgd saccharomycetaceae species whose genomes have been sequenced , although they are present in c. albicans and many other outgroup fungi ( alexandersson and sunnerhagen 2005 ) . among the post - wgd species , argonaute is present in all studied species of two very divergent clades the vanderwaltozyma / tetrapisispora clade and the naumovozyma / kazachstania clade . argonaute has been lost in the saccharomyces genus and in all studied species of the nakaseomyces / c . it is also of interest to note that two species retained both of the ohnolog copies of the argonaute gene after the wgd , which suggests that some functional divergence or specialization within the rnai pathway may have occurred in these species ( n. dairenensis and t. blattae ) . the phylogenetic distribution of dicer genes closely follows that of argonaute , except that there are two species that retain dicer but not argonaute ( c. castellii and s. uvarum , the sole member of the genus saccharomyces that has any rnai component ) . the most dramatic of these is the absence of the histidine biosynthesis pathway in t. blattae , which lacks six genes ( his1 , his2 , his3 , his4 , his5 and his7 ) from this seven - gene pathway . almost all genes of the dynein / dynactin pathway ( winey and bloom 2012 ) , which in s. cerevisiae controls movement of the nucleus into daughter cells during budding , are missing in four species : v. polyspora ( first reported by scannell et al . some of the pathway components , particularly ndl1 and kip1 , have also been lost repeatedly in many other saccharomycetaceae species without complete collapse of the pathway ( fig . losses of bna genes in the seven - gene pathway for de novo nad synthesis have previously been reported in c. glabrata and other members of the nakaseomyces genus ( domergue et al . the bna pathway is also missing in the genera kluyveromyces , kazachstania and naumovozyma , and in t. blattae ( fig . the absence of these genes is possibly a factor in the low level of rearrangement seen in the l. kluyveri genome as compared to other saccharomycetaceae ( gordon , byrne and wolfe 2011b ) . 1 indicates that there have been multiple independent losses , in different clades , of many groups of functionally related genes : three independent losses of the dal genes , four losses of gal genes , four losses of bna genes , one loss of his genes , five losses of the rnai pathway , one loss of nhej and three losses of the dynein / dynactin complex . these examples of interspecies functional variation highlight the shortcomings of s. cerevisiae , or indeed any single species , as a model for the biology of a wider taxonomic clade such as the saccharomycetaceae . the rnai pathway serves as a case in point : the functions of the argonaute and dicer genes in n. castellii would never have been discovered if s. cerevisiae was the only model organism , no matter how intensively s. cerevisiae was studied . by extension , it is very probable that some other important biochemical pathways , protein complexes and even biological processes that are widely conserved across many yeast species remain undiscovered because they are both ( i ) absent in the model organism s. cerevisiae , and ( ii ) yeast specific so their functions can not be inferred from other eukaryotes . population genomics has revealed that there is extensive intraspecies polymorphism of gene content , particularly at telomeric regions , leading to the concept of the pan - genome as the complete set of genes that exists within a species even if no individual member of the species contains them all ( song et al . just as the lab strain s288c has turned out to be an imperfect representative of the species s. cerevisiae , with unrepresentative alleles at loci such as flo8 and mkt1 ( liu , styles and fink 1996 ; lewis et al . 2014 ) , the species s. cerevisiae is also an imperfect representative of the family saccharomycetaceae . instead , we may benefit by extending the concept of the pan - genome to higher taxonomic levels to describe the complete set of biological molecules , complexes and processes that exists within saccharomycetaceae , even if no single species contains the whole set . however , our ability to answer such questions is severely limited by the elephant in the room : our lack of knowledge about the natural environments in which the different yeast species have evolved and the niches ( if any ) to which they are adapted ( goddard and greig 2015 ) . research in our group is supported by the european research council ( advanced grant 268893 ) and science foundation ireland ( 13ia1910 ) .

hematopoiesis is a complex process regulated by multiple growth factors supporting the renewal , differentiation , and survival of hematopoietic progenitors at different stages of maturation . pluripotent , multipotent , and committed precursors can undergo a number of renewal divisions ( 1 ) . during terminal differentiation , committed progenitors obey a fixed program that directs both the residual number of cell divisions and the specific timing of differentiation ( 2 , 3 ) . the correct balance between renewal and terminal differentiation is essential for homeostasis of the hematopoietic system , maintaining adequate numbers of precursors as well as mature cells , and is lost under pathological conditions such as leukemia and anemia . apoptosis is a finely regulated process equally essential for tissue development and homeostasis . during apoptosis , the cell is dismantled from within by cysteine / aspartic acid proteases ( caspases ) that cleave proteins involved in the maintenance of cell shape , the integrity of the nucleus , as well as of dna itself ( 4 , 5 ) . cleavage of these so - called death substrates causes dramatic alterations in the shape of the cells , some of which are reminiscent of the changes seen during the differentiation of erythroblasts to mature erythrocytes . indeed , caspase activation has recently been shown to be required for the differentiation of human erythroblasts in culture ( 6 ) . the raf-1 kinase has been implicated in the transduction of signals directing cell proliferation , activation , and survival . however , conventional ( 7 , 8) and conditional ( 9 ) ablation of raf-1 have revealed that the essential role of this kinase is to prevent apoptosis rather than promote proliferation . raf-1deficient embryos are anemic , exhibit various grades of growth retardation , and die at e12.5 . surprisingly in view of the anemia observed , apoptosis is restricted to the hepatoblast compartment and does not seem to affect the erythroid progenitors ( 7 ) . we demonstrate that raf-1 is degraded during erythroid maturation of primary mouse erythroblasts cultivated in physiologically relevant cytokines ( 10 ) and that this kinase delays erythroblast differentiation by restraining the caspase activation associated with it . thus , the anemic phenotype of raf-1deficient embryos is likely due to premature erythroblast differentiation at the expense of renewal , which depletes the fetal liver of erythroid precursors . our results indicate a novel role for raf-1 in erythropoiesis and demonstrate that its essential function as a regulator of caspase activity is not restricted to the control of apoptosis . primary fetal liver cells were isolated from e11.5 or e12.0 mouse embryos from heterozygous raf-1 intercrosses . bone marrow cells were isolated from mx - cre;c - raf-1 and c - raf-1 mice after the induction of cre expression by poly inosinic / cytidylic acid ( poly i / c)*treatment in vivo ( 400 g intraperitoneally , every other day , three injections total ) . cells were collected 1 wk after the last injection and the conversion of the flox to the allele was examined by pcr analysis as previously described ( 9 ) . fetal liver and bone marrow derived cells and immortal mouse erythroblasts ( i/11 , p53-deficient ; references 10 and 11 ) were expanded and maintained in serum - free erythroid medium ( stempro34 plus nutrient supplement ; life technologies ) supplemented with 2 units / ml human recombinant erythropoietin ( epo ; janssen - cilag ) , 100 ng / ml murine recombinant stem cell factor ( r&d systems ) , and 10 m dexamethasone ( sigma - aldrich ) . 40 ng / ml insulin - like growth factor 1 ( promega ) was present in all media described ( 10 ) . cultures were maintained at densities between 2 and 4 10 cells / ml and analyzed daily for cell numbers and cell size distribution in an electronic cell counter ( casy-1 ; schrfe - system ) to determine proliferation kinetics . cumulative cell numbers were calculated as previously described ( 10 ) . for the determination of erythroid cfus ( cfue ) 10 cells were seeded in duplicate in methocult m3234 ( stemcell technologies inc . ) in the presence of 0.3 u / ml epo . colonies were counted by independent investigators after 2 and 3 d in culture . for the experiments presented in fig . 4 , fetal liver or bone marrow cells were cultured for 6 d in proliferation medium to enrich for erythroid progenitors . immediately before the experiment , the cells were density purified by ficoll centrifugation in 1.078 g / cm lymphocyte separation medium ( eurobio ) to remove dead and differentiated cells and obtain homogenous populations of proliferating erythroid progenitors ( 12 ) . cells were washed twice in pbs and reseeded at 23 10 cells / ml in s-13 differentiation medium containing 12% fcs ( life technologies ) and supplemented with 10 units / ml epo , insulin ( 4 10 ie = 10 ng / ml , actrapid hm ; novo nordisk ) , 3 10 m of the dexamethasone antagonist zk112.993 ( 10 ) , and 1 mg / ml iron - saturated human transferrin ( sigma - aldrich ) . differentiating erythroblasts were maintained at densities between 2 and 4 10 cells / ml . fetal livers lacking raf-1 contain lower amounts of cfue and raf-1deficient erythroblasts show impaired expansion in culture . ( a ) single cell suspensions ( 10 , duplicates ) from fetal livers of c - raf-1 or wt mice were plated in methocult / epo . colonies were scored after 2 and 3 d. mean values of quadruplicates sd are shown . ( b ) density - purified fetal liver erythroblasts from c - raf-1 or wt mice were cultured in erythroid medium . ( c ) the morphology of c - raf-1 and wt cells was analyzed on day 6 . ( d ) cell size profiles of erythroblasts were obtained by casy measurement on day 6 . for b , the cells were preincubated for 1 h with the indicated concentrations of the broad range caspase inhibitors z - vad - fmk ( r&d systems ) or caspase inhibitor iii ( boc - d - fmk ; calbiochem - novabiochem ) . i/11 erythroblasts were infected with a bicistronic retroviral vector in which the expression of mutant forms of raf-1 coupled to enhanced green fluorescent protein ( egfp ) via an internal ribosome entry site sequence is driven by the murine stem cell virus long - terminal repeats ( 13 ) . 8 d after infection , i/11 cells were sorted on a facscalibur ( becton dickinson ) for egfp expression and single cells were expanded in 96-well plates . erythroblasts at various stages of differentiation were cytocentrifuged onto glass slides and stained with histological dyes and neutral benzidine for hemoglobin ( 14 ) . nucleated and enucleated erythrocytes ( brown- or orange - stained small cells ) , partially mature or immature cells ( larger cells with gray or blue cytoplasm ) , and apoptotic / dead cells ( fragmented / condensed nuclei , disintegrated cells ) were counted after visual inspection in the microscope as previously described ( 14 ) , evaluating > 500 cells per sample on multiple , randomly selected fields . bone marrow derived cells cultured for 1 wk in erythroid medium were stained with antibodies against c - kit apc , ter119-pe , b220-apc , and mac-1fitc ( all from bd biosciences ) before facs analysis . hemoglobin content was analyzed by removing 50 l aliquots from the cultures and photometric determination was performed as previously described ( 10 ) . values obtained from triplicate determinations were averaged and normalized to cell number and cell volume . erythroblasts were harvested after the determination of cell numbers , washed with ice cold pbs , and resuspended in 2 sds loading or lysis buffer ( 10 mm tris - hcl , ph 7.0 , 50 mm sodium chloride , 30 mm sodium pyrophosphate , 1% triton x-100 ) . insoluble material was removed by centrifugation ( 14,000 rpm for 5 min ) . because hemoglobin , present in different amounts in the cells at various stages , interferes with any standard method of protein determination , samples were normalized with respect to both cell number and size and subjected to page followed by electrophoretic transfer to nylon membranes . after blocking in ttbs ( 10 mm tris - hcl , ph 8.0 , 150 mm sodium chloride , 0.1% tween 20 ) with 5% milk powder or 2% bsa ( fraction v ; sigma - aldrich ) , the membranes were probed with the appropriate primary antibodies ( rabbit polyclonal antiserum against a cooh - terminal peptide of v - raf [ sp63 , ctlttsprlpvf ] , hsp90 , caspase-1 , caspase-3 , caspase-9 , and lamin b ; santa cruz biotechnology , inc . ) in 12% bsa in ttbs before incubation with peroxidase - conjugated secondary antibodies and detection by enhanced chemiluminescence ( pierce chemical co. ) . primary fetal liver cells were isolated from e11.5 or e12.0 mouse embryos from heterozygous raf-1 intercrosses . bone marrow cells were isolated from mx - cre;c - raf-1 and c - raf-1 mice after the induction of cre expression by poly inosinic / cytidylic acid ( poly i / c)*treatment in vivo ( 400 g intraperitoneally , every other day , three injections total ) . cells were collected 1 wk after the last injection and the conversion of the flox to the allele was examined by pcr analysis as previously described ( 9 ) . fetal liver and bone marrow derived cells and immortal mouse erythroblasts ( i/11 , p53-deficient ; references 10 and 11 ) were expanded and maintained in serum - free erythroid medium ( stempro34 plus nutrient supplement ; life technologies ) supplemented with 2 units / ml human recombinant erythropoietin ( epo ; janssen - cilag ) , 100 ng / ml murine recombinant stem cell factor ( r&d systems ) , and 10 m dexamethasone ( sigma - aldrich ) . 40 ng / ml insulin - like growth factor 1 ( promega ) was present in all media described ( 10 ) . cultures were maintained at densities between 2 and 4 10 cells / ml and analyzed daily for cell numbers and cell size distribution in an electronic cell counter ( casy-1 ; schrfe - system ) to determine proliferation kinetics . cumulative cell numbers were calculated as previously described ( 10 ) . for the determination of erythroid cfus ( cfue ) 10 cells were seeded in duplicate in methocult m3234 ( stemcell technologies inc . ) in the presence of 0.3 u / ml epo . colonies were counted by independent investigators after 2 and 3 d in culture . for the experiments presented in fig . 4 , fetal liver or bone marrow cells were cultured for 6 d in proliferation medium to enrich for erythroid progenitors . immediately before the experiment , the cells were density purified by ficoll centrifugation in 1.078 g / cm lymphocyte separation medium ( eurobio ) to remove dead and differentiated cells and obtain homogenous populations of proliferating erythroid progenitors ( 12 ) . cells were washed twice in pbs and reseeded at 23 10 cells / ml in s-13 differentiation medium containing 12% fcs ( life technologies ) and supplemented with 10 units / ml epo , insulin ( 4 10 ie = 10 ng / ml , actrapid hm ; novo nordisk ) , 3 10 m of the dexamethasone antagonist zk112.993 ( 10 ) , and 1 mg / ml iron - saturated human transferrin ( sigma - aldrich ) . differentiating erythroblasts were maintained at densities between 2 and 4 10 cells / ml . fetal livers lacking raf-1 contain lower amounts of cfue and raf-1deficient erythroblasts show impaired expansion in culture . ( a ) single cell suspensions ( 10 , duplicates ) from fetal livers of c - raf-1 or wt mice were plated in methocult / epo . colonies were scored after 2 and 3 d. mean values of quadruplicates sd are shown . ( b ) density - purified fetal liver erythroblasts from c - raf-1 or wt mice were cultured in erythroid medium . ( c ) the morphology of c - raf-1 and wt cells was analyzed on day 6 . ( d ) cell size profiles of erythroblasts were obtained by casy measurement on day 6 . for b , the cells were preincubated for 1 h with the indicated concentrations of the broad range caspase inhibitors z - vad - fmk ( r&d systems ) or caspase inhibitor iii ( boc - d - fmk ; calbiochem - novabiochem ) . i/11 erythroblasts were infected with a bicistronic retroviral vector in which the expression of mutant forms of raf-1 coupled to enhanced green fluorescent protein ( egfp ) via an internal ribosome entry site sequence is driven by the murine stem cell virus long - terminal repeats ( 13 ) . 8 d after infection , i/11 cells were sorted on a facscalibur ( becton dickinson ) for egfp expression and single cells were expanded in 96-well plates . erythroblasts at various stages of differentiation were cytocentrifuged onto glass slides and stained with histological dyes and neutral benzidine for hemoglobin ( 14 ) . nucleated and enucleated erythrocytes ( brown- or orange - stained small cells ) , partially mature or immature cells ( larger cells with gray or blue cytoplasm ) , and apoptotic / dead cells ( fragmented / condensed nuclei , disintegrated cells ) were counted after visual inspection in the microscope as previously described ( 14 ) , evaluating > 500 cells per sample on multiple , randomly selected fields . bone marrow derived cells cultured for 1 wk in erythroid medium were stained with antibodies against c - kit apc , ter119-pe , b220-apc , and mac-1fitc ( all from bd biosciences ) before facs analysis . hemoglobin content was analyzed by removing 50 l aliquots from the cultures and photometric determination was performed as previously described ( 10 ) . values obtained from triplicate determinations were averaged and normalized to cell number and cell volume . erythroblasts were harvested after the determination of cell numbers , washed with ice cold pbs , and resuspended in 2 sds loading or lysis buffer ( 10 mm tris - hcl , ph 7.0 , 50 mm sodium chloride , 30 mm sodium pyrophosphate , 1% triton x-100 ) . insoluble material was removed by centrifugation ( 14,000 rpm for 5 min ) . because hemoglobin , present in different amounts in the cells at various stages , interferes with any standard method of protein determination , samples were normalized with respect to both cell number and size and subjected to page followed by electrophoretic transfer to nylon membranes . after blocking in ttbs ( 10 mm tris - hcl , ph 8.0 , 150 mm sodium chloride , 0.1% tween 20 ) with 5% milk powder or 2% bsa ( fraction v ; sigma - aldrich ) , the membranes were probed with the appropriate primary antibodies ( rabbit polyclonal antiserum against a cooh - terminal peptide of v - raf [ sp63 , ctlttsprlpvf ] , hsp90 , caspase-1 , caspase-3 , caspase-9 , and lamin b ; santa cruz biotechnology , inc . ) in 12% bsa in ttbs before incubation with peroxidase - conjugated secondary antibodies and detection by enhanced chemiluminescence ( pierce chemical co. ) . we have previously demonstrated that raf-1deficient embryos are anemic and have hypocellular livers showing pronounced apoptosis of the hepatoblast compartment . hepatoblast failure and the resulting lack of a proper microenvironment could be responsible for the impaired hematopoiesis and the anemic phenotype . indeed , raf-1deficient fetal livers contained much less erythroid progenitors than wt livers as assessed in cfue assays ( fig . 1 a ) . to determine whether this was due to a defect in the fetal liver environment or to a cell - autonomous defect of the erythroid progenitors , we investigated the ability of raf-1deficient erythroblasts to proliferate and differentiate in vitro . purified erythroblasts from e11.5 livers were cultivated in erythroid medium to favor proliferation but not differentiation of erythroblasts ( 10 ) . under these conditions , wt cells proliferated exponentially ( eightfold total cell number increase from day 3 to 6 ) , whereas c - raf-1 cells barely doubled ( fig . this is in line with the previous observation that antisense raf-1 oligonucleotides reduce epo and insulin - like growth factor induced proliferation of human erythroid progenitors ( 15 ) . analysis of cytospin preparations after 6 d in culture showed that wt cultures contained mainly large , undifferentiated erythroid progenitors . in contrast , the raf-1deficient cultures were far more heterogeneous and contained immature cells , differentiating blasts , and mature hemoglobinized cells ( fig this heterogeneity and the presence of mature cells in the raf-1deficient cultures could be confirmed by cell size profile analysis . the wt cultures consisted mainly of large , blast - size cells ( 10 m ) and the c - raf-1 cultures contained a high proportion of smaller cells , including erythrocytes ( 5 m , fig . 1 d ) . thus , raf-1deficient fetal liver cells fail to accumulate in culture , apparently due to accelerated differentiation at the expense of renewal . to further investigate the impact of the lack of raf-1 on erythroid differentiation , density - purified erythroblasts from fetal liver cultures ( refer to materials and methods ) were induced to terminally differentiate by exposure to epo plus insulin . neither wt nor c - raf-1 cells underwent a significant degree of cell death during differentiation ( 27% dead cells / time point ) , and in both cultures differentiation was essentially complete after 48 h , although a larger residual proportion of partially mature cells was observed in the wt cultures ( fig . 2 a ) . at 24 h , however , c - raf-1 cultures showed a dramatic reduction of the number of erythroblasts ( from 82 to 27% ) compensated by a similarly dramatic increase in the numbers of nucleated ( 1347% ) and enucleated ( 322% ) erythrocytes , as assessed by analysis of cytospins and cell size profiles ( fig . 2 , a and b ) . by comparison , wt cultures after 24 h still contained 62% erythroblasts and only 1.4% enucleated erythrocytes . accelerated differentiation of raf-1deficient cells was also evident from quantitative analysis of other differentiation parameters , i.e. , faster maturation - associated proliferation , cell size decrease , and hemoglobin accumulation ( fig . 2 c ) . to confirm that the defect observed in the raf-1deficient erythroblasts was independent from the fetal liver environment , we used erythroblasts derived from the bone marrow of mx - cre;c - raf-1 mice treated with poly i / c to fully convert the flox allele to a null allele ( fig . cells derived from poly i / c treated mx - cre;c - raf-1 animals were used as controls ( c - raf-1 ) . after 6 d in culture in erythroid medium , both the c - raf-1 and c - raf-1 populations contained comparable amounts of erythroid cells , as determined by facs analysis ( fig . these populations were density purified to remove dead and differentiating cells before the induction of terminal erythroid differentiation . similar to the situation observed in fetal liver derived erythroblast , a remarkable increase in nucleated ( 27% ) and enucleated ( 22% ) erythrocytes was apparent in the c - raf-1 culture 24 h after differentiation induction , whereas c - raf-1 cultures contained only 18% nucleated and 12% enucleated erythrocytes . thus , the differentiation defect observed is intrinsic to the raf-1deficient erythroblasts and does not depend on organ environment . c - raf-1erythroid progenitors differentiate faster than their wt counterparts . ( a ) morphology of wt and c - raf-1 cells during differentiation . cytospins stained with neutral benzidine plus histological dyes ( left ) were examined at each of the time points indicated ( right ) . ( b ) cell volume profiles measured daily show the characteristic decrease from a diameter of 10 m to 5 m . ( c ) proliferation rate during differentiation ( left ) , size decrease ( middle ) , and hemoglobin content ( right ) of wt and c - raf-1 cells were determined daily during terminal differentiation . whole cell lysates from wt cultures ( 15 g ) were subjected to immunoblot analysis with an anti c - raf-1 bone marrow derived erythroid progenitors differentiate faster than their wt counterparts . ( a ) total conversion of the floxed c - raf-1 allele to a null allele shown by pcr analysis of bone marrow cells . ( b ) facs analysis and ( c ) morphology of c - raf-1 and c - raf-1 bone marrow derived cells after 6 d of culture in proliferation medium and ( d ) after 0 and 24 h of culture in differentiation medium . if so , differentiating wt cells should have a means of neutralizing this effect of raf-1 . indeed , immunoblot analysis showed that raf-1 expression declines rapidly during erythroid differentiation ( fig . these data clearly define raf-1 as a modulator of erythroid maturation , which ensures homeostasis by preventing the premature differentiation ( and thereby the depletion ) of actively proliferating erythroid precursors . this essential function of raf-1 likely contributes to the ability of its viral counterpart , v - raf , to efficiently transform erythroid cells in vitro ( 16 ) . by we have previously demonstrated that one essential function of this kinase is to restrain caspase activation during apoptosis ( 9 ) . recently , caspase activation has been found to be associated with and required for the differentiation of human erythroblasts ( 6 ) . therefore , we tested whether caspase activation was associated with the differentiation of murine erythroblasts in our in vitro system and whether raf-1 delayed erythroid differentiation by restraining caspase activation . caspases are synthesized as zymogens and converted into one large and one small subunit by limited proteolysis . we have monitored limited caspase proteolysis by immunoblotting , indicated by the disappearance of the zymogen ( caspase-1 , -3 , and -9 ) and/or the appearance of the small subunit ( caspase-1 and -9 ) . indeed , cleavage of the caspase-1 , -3 , and 9 zymogens was associated with erythroid differentiation ( fig . 4 a ) . active caspases can , however , be efficiently counteracted by a group of proteins called iaps , which bind to and inhibit the active caspase tetramer ( 17 ) . to determine whether the limited proteolysis of the caspase zymogens corresponded to caspase activation , we tested whether the cleavage of the death substrate lamin b was associated with erythroid differentiation . in this context lamin b is a relevant substrate because its cleavage is needed to dissolve the nuclear membrane and is therefore likely connected with enucleation ( 6 ) . ( a ) caspase activation accompanies the differentiation of erythroblasts . at the indicated times after the induction of differentiation , wt erythroblasts were lysed and the activation state of caspase-1 , -3 , and -9 was analyzed by immunoblotting . wt erythroblasts were pretreated for 1 h with 150 m broad - range caspase inhibitor z - vad - fmk or left untreated ( no inhibitor ) before being switched to differentiation medium ( with or without z - vad - fmk ) . levels of p70 lamin b and its cleavage product p40 in whole cell lysates ( 15 g ) were detected by western blot analysis . ( c ) cell size profiles and ( d ) hemoglobin content of cultures treated with z - vad - fmk as in b or with 150 m bocd - fmk were analyzed daily . the time points indicated , the protein levels of p70 lamin b and its cleavage product p40 were determined by western blot analysis ( 15 g ) . indeed , we observed cleavage of the 70-kd lamin b protein to a 40-kd diagnostic proteolytic fragment during erythroblast differentiation of wt cells ( fig . 4 b ) . treatment with the cell - permeable caspase inhibitor z - vad - fmk abolished the maturation - associated lamin b cleavage ( fig . in addition , both z - vad - fmk and a second broad - range caspase inhibitor , bocd - fmk , delayed erythroblast differentiation as measured by cell size decrease . after 36 h of differentiation , two distinct peaks were well discernible in the untreated culture : large - sized erythroblasts and small - sized mature cells . however , in inhibitor - treated cultures , the main peak at this time was represented by proliferating precursors ( fig . 4 c ) . similarly , the inhibitors significantly delayed and reduced the accumulation of hemoglobin in the differentiating cultures ( fig . 4 d ) . the inhibition of caspase activation , however , did not prevent differentiation - associated down - regulation of raf-1 ( fig . whether , in turn , the absence of raf-1 had an effect on differentiation - associated caspase activation , we determined the extent of lamin b cleavage in differentiating wt and c - raf-1 erythroblasts . however , more lamin b cleavage product ( p40 ) , indicative of elevated caspase activity , was present in c - raf-1 cultures at the start as well after 24 h of differentiation ( fig . 4 e ) . this was not associated with increased apoptosis ( fig . 2 a and unpublished data ) and correlated well with the differences observed in the amount of differentiated cells between wt and raf-1deficient erythroblast culture ( fig . as observed in raf-1deficient macrophages ( 9 ) and fibroblasts treated by apoptotic stimuli ( unpublished data ) , lack of raf-1 is associated with an increase in caspase activation in differentiating erythroblasts . if raf-1 is indeed responsible for regulating erythroblast differentiation via caspase - mediated cleavage of important proteins , constitutive activation of raf-1 should delay both cleavage of caspase substrates and erythroid differentiation itself . to confirm and extend these results , we have used the factor - dependent differentiation - competent erythroblast cell line i/11 ( 10 , 11 ) . as in primary fetal liver derived erythroblasts , raf-1 degradation accompanies the somewhat slower differentiation of these cells ( fig . 5 , a and b ) . to assess whether raf-1 overexpression would delay death substrate cleavage and differentiation and whether this required raf-1 kinase activity , we infected i/11 cells with retroviral constructs directing the expression of full - length raf-1 ( wt raf-1 ) and of a truncated , constitutively active form of raf-1 ( bxb raf-1 ) , and selected clones expressing these proteins . bulk populations of infected cells and clones expressing varying amounts of the exogenous proteins proliferated with similar kinetics in erythroid medium , indicating that neither wt nor constitutively active raf-1 confer a selective proliferation advantage under these conditions ( unpublished data ) . we consistently observed that the truncated , constitutively active ( bxb ) raf-1 was expressed at much higher levels than wt raf-1 . one might speculate that the overexpression of the wt protein , containing the regulatory domain that binds to upstream effectors ( e.g. , ras ) , might compete with and block the activation of other downstream effectors necessary for survival . this would result in the counter selection of cells expressing high amounts of wt raf-1 , but not of bxb . except slightly increasing proliferation during differentiation ( fig . 5 b , left ) , expression of wt raf-1 did not have a major impact on erythroid differentiation as measured by hemoglobin accumulation ( right ) , size decrease , and cytospin analysis ( c and d ) . expression of wt raf-1 reduced lamin b cleavage , albeit slightly , at all time points ( fig . 5 e , right ) , implying that a minimum threshold of caspase activity is necessary , above which differentiation starts and proceed normally . in contrast , constitutively active ( bxb ) raf-1 delayed differentiation , as indicated by ongoing proliferation after 7296 h , reduced hemoglobin accumulation ( fig . 5 b ) , increased cell size ( c ) , and persistence of partially mature and immature cells that could be switched back to proliferation conditions ( d and unpublished data ) . in parallel , , wt raf-1 was expressed at high levels compared with the endogenous protein but was down - regulated in the same manner during the course of differentiation . as mentioned above , bxb raf-1 was expressed at higher levels and because of this persisted throughout differentiation although its amount decreased at the same rate as that of wt raf-1 ( fig . overexpression of active raf-1 delays erythroid differentiation and differentiation - associated caspase activation in i/11 erythroblasts . levels of raf-1 were detected at the indicated time points by immunoblot analysis of 50 g whole cell lysate . the differentiation of uninfected i/11 was compared with that of cells expressing murine stem cell virus - egfp ( empty vector ) with full - length raf-1 ( wt raf-1 ) or a constitutively active raf ( bxb ) . differentiation parameters analyzed were proliferation during differentiation ( b , left ) , hemoglobin accumulation ( b , right ) , and size decrease ( c ; overlays of cell size profiles obtained after 0 and 72 h of differentiation ) . ( d ) after 96 h of differentiation , the morphology and hemoglobin content of cells expressing exogenous wt raf-1 and constitutively active raf ( bxb raf ) were compared . cell lysates were prepared from i/11 erythroblasts stably expressing raf-1 mutants at different time points after the induction of differentiation . the amount of raf-1 protein ( left ) as well as of lamin b ( right ) were analyzed by immunoblotting whole cell lysates ( 15 g ) . first , by showing that overexpression of raf-1 restrains caspase activation and delays erythroid differentiation , we confirm the role of raf-1 as a negative regulator of both processes . due to the differences in the expression of wt and bxb raf-1 , however , it is not possible to deduce whether bxb delays differentiation more efficiently than wt raf-1 because of its constitutive kinase activity or simply because of its higher expression . be that as it may , the experiment shows that a certain level of raf-1 expression is needed at later maturation stages for restraining differentiation - induced caspase activation , whereas high level expression at the onset of differentiation has little effect on the progress of maturation . second , the fact that the full - length protein expressed under the control of the retroviral promoter is down - regulated with the same kinetics and efficiency as endogenous raf-1 indicates that the regulation of raf-1 expression occurs at the posttranscriptional level , as also confirmed by northern blot analysis ( unpublished data ) . interestingly , we have observed posttranscriptional raf-1 down - regulation during pathogen - induced macrophage apoptosis ( 9 ) . in this case , raf-1 was degraded by the proteasome in a caspase - dependent manner . thus , in macrophages , the relationship between raf-1 and caspase activation was reciprocal , with the protease directing the degradation of the kinase that restrains its activation ( 9 ) . in contrast , raf-1 down - regulation in differentiating erythroblasts is either upstream of caspase activation or independently regulated . defining the signal directing raf-1 down - regulation and , if possible , interfering with it will be important in further elucidating the exact position of this kinase in the signal transduction cascade leading to erythroid differentiation . it is possible that raf-1 , or a downstream effector , directly modifies the initiator protease(s ) , similar to how protein kinase b phosphorylates human caspase-9 and inhibits its protease activity ( 18 ) . raf-1 might also modulate the expression or activity of caspase inhibitors ( 17 ) . in this context , in drosophila activated d - ras and d - raf inhibit apoptosis by antagonizing hid ( 19 , 20 ) , whose human orthologs have been shown to antagonize iaps ( 21 , 22 ) . d - raf , however , shares a greater degree of homology with b - raf than raf-1 . furthermore , at least in neurons , the regulation of iap is an essential function of b - raf and can not be substituted for by raf-1 ( 23 ) . raf-1 is activated by the erythroleukemia - inducing strains of friend spleen focus - forming virus , and its inhibition by antisense oligonucleotide partially reduces proliferation of virus - infected cells ( 24 ) . in addition , oncogenic v - raf versions cooperate with myc to transform erythroid cells ( 16 ) . p53 cells can be likewise transformed by v - raf , giving rise to cell lines that can be propagated continuously and are highly tumorigenic ( 25 ) . we now report a previously unrecognized function of raf-1 as a modulator of erythroid homeostasis , which , by restraining caspase activation , prevents the premature differentiation of erythroblasts and safeguards the maintenance of the appropriate amount of proliferating precursors . a delay in precursor differentiation , as the one caused by mutation / overexpression of raf-1 ( bxb raf-1 or v - raf ) , should tip the balance between proliferation and terminal maturation of erythroid cells toward precursor renewal and might represent the main contribution of raf to the establishment of leukemia .

the raf kinases are key signal transducers activated by mitogens or oncogenes . the best studied raf isoform , raf-1 , was identified as an inhibitor of apoptosis by conventional and conditional gene ablation in mice . c - raf-1/ embryos are growth retarded and anemic , and die at midgestation with anomalies in the placenta and fetal liver . here , we show that raf-1deficient primary erythroblasts can not be expanded in culture due to their accelerated differentiation into mature erythrocytes . in addition , raf-1 expression is down - regulated in differentiating wild - type cells , whereas overexpression of activated raf-1 delays differentiation . as recently described for human erythroid precursors , we find that caspase activation is necessary for the differentiation of murine fetal liver erythroblasts . differentiation - associated caspase activation is accelerated in erythroid progenitors lacking raf-1 and delayed by overexpression of the activated kinase . these results reveal an essential function of raf-1 in erythropoiesis and demonstrate that the ability of raf-1 to restrict caspase activation is biologically relevant in a context distinct from apoptosis .

Introduction Materials and Methods Cell Isolation, Culture, Differentiation, and Infection. Cell Morphology, Histological Staining, FACS Western Blot Analysis. Results and Discussion

the correct balance between renewal and terminal differentiation is essential for homeostasis of the hematopoietic system , maintaining adequate numbers of precursors as well as mature cells , and is lost under pathological conditions such as leukemia and anemia . during apoptosis , the cell is dismantled from within by cysteine / aspartic acid proteases ( caspases ) that cleave proteins involved in the maintenance of cell shape , the integrity of the nucleus , as well as of dna itself ( 4 , 5 ) . cleavage of these so - called death substrates causes dramatic alterations in the shape of the cells , some of which are reminiscent of the changes seen during the differentiation of erythroblasts to mature erythrocytes . indeed , caspase activation has recently been shown to be required for the differentiation of human erythroblasts in culture ( 6 ) . however , conventional ( 7 , 8) and conditional ( 9 ) ablation of raf-1 have revealed that the essential role of this kinase is to prevent apoptosis rather than promote proliferation . raf-1deficient embryos are anemic , exhibit various grades of growth retardation , and die at e12.5 . surprisingly in view of the anemia observed , apoptosis is restricted to the hepatoblast compartment and does not seem to affect the erythroid progenitors ( 7 ) . we demonstrate that raf-1 is degraded during erythroid maturation of primary mouse erythroblasts cultivated in physiologically relevant cytokines ( 10 ) and that this kinase delays erythroblast differentiation by restraining the caspase activation associated with it . thus , the anemic phenotype of raf-1deficient embryos is likely due to premature erythroblast differentiation at the expense of renewal , which depletes the fetal liver of erythroid precursors . our results indicate a novel role for raf-1 in erythropoiesis and demonstrate that its essential function as a regulator of caspase activity is not restricted to the control of apoptosis . bone marrow cells were isolated from mx - cre;c - raf-1 and c - raf-1 mice after the induction of cre expression by poly inosinic / cytidylic acid ( poly i / c)*treatment in vivo ( 400 g intraperitoneally , every other day , three injections total ) . fetal liver and bone marrow derived cells and immortal mouse erythroblasts ( i/11 , p53-deficient ; references 10 and 11 ) were expanded and maintained in serum - free erythroid medium ( stempro34 plus nutrient supplement ; life technologies ) supplemented with 2 units / ml human recombinant erythropoietin ( epo ; janssen - cilag ) , 100 ng / ml murine recombinant stem cell factor ( r&d systems ) , and 10 m dexamethasone ( sigma - aldrich ) . 4 , fetal liver or bone marrow cells were cultured for 6 d in proliferation medium to enrich for erythroid progenitors . cells were washed twice in pbs and reseeded at 23 10 cells / ml in s-13 differentiation medium containing 12% fcs ( life technologies ) and supplemented with 10 units / ml epo , insulin ( 4 10 ie = 10 ng / ml , actrapid hm ; novo nordisk ) , 3 10 m of the dexamethasone antagonist zk112.993 ( 10 ) , and 1 mg / ml iron - saturated human transferrin ( sigma - aldrich ) . fetal livers lacking raf-1 contain lower amounts of cfue and raf-1deficient erythroblasts show impaired expansion in culture . ( b ) density - purified fetal liver erythroblasts from c - raf-1 or wt mice were cultured in erythroid medium . ( c ) the morphology of c - raf-1 and wt cells was analyzed on day 6 . nucleated and enucleated erythrocytes ( brown- or orange - stained small cells ) , partially mature or immature cells ( larger cells with gray or blue cytoplasm ) , and apoptotic / dead cells ( fragmented / condensed nuclei , disintegrated cells ) were counted after visual inspection in the microscope as previously described ( 14 ) , evaluating > 500 cells per sample on multiple , randomly selected fields . bone marrow derived cells cultured for 1 wk in erythroid medium were stained with antibodies against c - kit apc , ter119-pe , b220-apc , and mac-1fitc ( all from bd biosciences ) before facs analysis . bone marrow cells were isolated from mx - cre;c - raf-1 and c - raf-1 mice after the induction of cre expression by poly inosinic / cytidylic acid ( poly i / c)*treatment in vivo ( 400 g intraperitoneally , every other day , three injections total ) . fetal liver and bone marrow derived cells and immortal mouse erythroblasts ( i/11 , p53-deficient ; references 10 and 11 ) were expanded and maintained in serum - free erythroid medium ( stempro34 plus nutrient supplement ; life technologies ) supplemented with 2 units / ml human recombinant erythropoietin ( epo ; janssen - cilag ) , 100 ng / ml murine recombinant stem cell factor ( r&d systems ) , and 10 m dexamethasone ( sigma - aldrich ) . 4 , fetal liver or bone marrow cells were cultured for 6 d in proliferation medium to enrich for erythroid progenitors . cells were washed twice in pbs and reseeded at 23 10 cells / ml in s-13 differentiation medium containing 12% fcs ( life technologies ) and supplemented with 10 units / ml epo , insulin ( 4 10 ie = 10 ng / ml , actrapid hm ; novo nordisk ) , 3 10 m of the dexamethasone antagonist zk112.993 ( 10 ) , and 1 mg / ml iron - saturated human transferrin ( sigma - aldrich ) . fetal livers lacking raf-1 contain lower amounts of cfue and raf-1deficient erythroblasts show impaired expansion in culture . ( b ) density - purified fetal liver erythroblasts from c - raf-1 or wt mice were cultured in erythroid medium . ( c ) the morphology of c - raf-1 and wt cells was analyzed on day 6 . nucleated and enucleated erythrocytes ( brown- or orange - stained small cells ) , partially mature or immature cells ( larger cells with gray or blue cytoplasm ) , and apoptotic / dead cells ( fragmented / condensed nuclei , disintegrated cells ) were counted after visual inspection in the microscope as previously described ( 14 ) , evaluating > 500 cells per sample on multiple , randomly selected fields . bone marrow derived cells cultured for 1 wk in erythroid medium were stained with antibodies against c - kit apc , ter119-pe , b220-apc , and mac-1fitc ( all from bd biosciences ) before facs analysis . we have previously demonstrated that raf-1deficient embryos are anemic and have hypocellular livers showing pronounced apoptosis of the hepatoblast compartment . to determine whether this was due to a defect in the fetal liver environment or to a cell - autonomous defect of the erythroid progenitors , we investigated the ability of raf-1deficient erythroblasts to proliferate and differentiate in vitro . purified erythroblasts from e11.5 livers were cultivated in erythroid medium to favor proliferation but not differentiation of erythroblasts ( 10 ) . under these conditions , wt cells proliferated exponentially ( eightfold total cell number increase from day 3 to 6 ) , whereas c - raf-1 cells barely doubled ( fig . this is in line with the previous observation that antisense raf-1 oligonucleotides reduce epo and insulin - like growth factor induced proliferation of human erythroid progenitors ( 15 ) . analysis of cytospin preparations after 6 d in culture showed that wt cultures contained mainly large , undifferentiated erythroid progenitors . in contrast , the raf-1deficient cultures were far more heterogeneous and contained immature cells , differentiating blasts , and mature hemoglobinized cells ( fig this heterogeneity and the presence of mature cells in the raf-1deficient cultures could be confirmed by cell size profile analysis . the wt cultures consisted mainly of large , blast - size cells ( 10 m ) and the c - raf-1 cultures contained a high proportion of smaller cells , including erythrocytes ( 5 m , fig . thus , raf-1deficient fetal liver cells fail to accumulate in culture , apparently due to accelerated differentiation at the expense of renewal . to further investigate the impact of the lack of raf-1 on erythroid differentiation , density - purified erythroblasts from fetal liver cultures ( refer to materials and methods ) were induced to terminally differentiate by exposure to epo plus insulin . neither wt nor c - raf-1 cells underwent a significant degree of cell death during differentiation ( 27% dead cells / time point ) , and in both cultures differentiation was essentially complete after 48 h , although a larger residual proportion of partially mature cells was observed in the wt cultures ( fig . at 24 h , however , c - raf-1 cultures showed a dramatic reduction of the number of erythroblasts ( from 82 to 27% ) compensated by a similarly dramatic increase in the numbers of nucleated ( 1347% ) and enucleated ( 322% ) erythrocytes , as assessed by analysis of cytospins and cell size profiles ( fig . accelerated differentiation of raf-1deficient cells was also evident from quantitative analysis of other differentiation parameters , i.e. , faster maturation - associated proliferation , cell size decrease , and hemoglobin accumulation ( fig . to confirm that the defect observed in the raf-1deficient erythroblasts was independent from the fetal liver environment , we used erythroblasts derived from the bone marrow of mx - cre;c - raf-1 mice treated with poly i / c to fully convert the flox allele to a null allele ( fig . after 6 d in culture in erythroid medium , both the c - raf-1 and c - raf-1 populations contained comparable amounts of erythroid cells , as determined by facs analysis ( fig . similar to the situation observed in fetal liver derived erythroblast , a remarkable increase in nucleated ( 27% ) and enucleated ( 22% ) erythrocytes was apparent in the c - raf-1 culture 24 h after differentiation induction , whereas c - raf-1 cultures contained only 18% nucleated and 12% enucleated erythrocytes . ( c ) proliferation rate during differentiation ( left ) , size decrease ( middle ) , and hemoglobin content ( right ) of wt and c - raf-1 cells were determined daily during terminal differentiation . whole cell lysates from wt cultures ( 15 g ) were subjected to immunoblot analysis with an anti c - raf-1 bone marrow derived erythroid progenitors differentiate faster than their wt counterparts . ( a ) total conversion of the floxed c - raf-1 allele to a null allele shown by pcr analysis of bone marrow cells . ( b ) facs analysis and ( c ) morphology of c - raf-1 and c - raf-1 bone marrow derived cells after 6 d of culture in proliferation medium and ( d ) after 0 and 24 h of culture in differentiation medium . this essential function of raf-1 likely contributes to the ability of its viral counterpart , v - raf , to efficiently transform erythroid cells in vitro ( 16 ) . by we have previously demonstrated that one essential function of this kinase is to restrain caspase activation during apoptosis ( 9 ) . recently , caspase activation has been found to be associated with and required for the differentiation of human erythroblasts ( 6 ) . therefore , we tested whether caspase activation was associated with the differentiation of murine erythroblasts in our in vitro system and whether raf-1 delayed erythroid differentiation by restraining caspase activation . we have monitored limited caspase proteolysis by immunoblotting , indicated by the disappearance of the zymogen ( caspase-1 , -3 , and -9 ) and/or the appearance of the small subunit ( caspase-1 and -9 ) . indeed , cleavage of the caspase-1 , -3 , and 9 zymogens was associated with erythroid differentiation ( fig . to determine whether the limited proteolysis of the caspase zymogens corresponded to caspase activation , we tested whether the cleavage of the death substrate lamin b was associated with erythroid differentiation . ( a ) caspase activation accompanies the differentiation of erythroblasts . indeed , we observed cleavage of the 70-kd lamin b protein to a 40-kd diagnostic proteolytic fragment during erythroblast differentiation of wt cells ( fig . in addition , both z - vad - fmk and a second broad - range caspase inhibitor , bocd - fmk , delayed erythroblast differentiation as measured by cell size decrease . the inhibition of caspase activation , however , did not prevent differentiation - associated down - regulation of raf-1 ( fig . whether , in turn , the absence of raf-1 had an effect on differentiation - associated caspase activation , we determined the extent of lamin b cleavage in differentiating wt and c - raf-1 erythroblasts . as observed in raf-1deficient macrophages ( 9 ) and fibroblasts treated by apoptotic stimuli ( unpublished data ) , lack of raf-1 is associated with an increase in caspase activation in differentiating erythroblasts . to confirm and extend these results , we have used the factor - dependent differentiation - competent erythroblast cell line i/11 ( 10 , 11 ) . as in primary fetal liver derived erythroblasts , raf-1 degradation accompanies the somewhat slower differentiation of these cells ( fig . to assess whether raf-1 overexpression would delay death substrate cleavage and differentiation and whether this required raf-1 kinase activity , we infected i/11 cells with retroviral constructs directing the expression of full - length raf-1 ( wt raf-1 ) and of a truncated , constitutively active form of raf-1 ( bxb raf-1 ) , and selected clones expressing these proteins . bulk populations of infected cells and clones expressing varying amounts of the exogenous proteins proliferated with similar kinetics in erythroid medium , indicating that neither wt nor constitutively active raf-1 confer a selective proliferation advantage under these conditions ( unpublished data ) . one might speculate that the overexpression of the wt protein , containing the regulatory domain that binds to upstream effectors ( e.g. this would result in the counter selection of cells expressing high amounts of wt raf-1 , but not of bxb . 5 b , left ) , expression of wt raf-1 did not have a major impact on erythroid differentiation as measured by hemoglobin accumulation ( right ) , size decrease , and cytospin analysis ( c and d ) . in parallel , , wt raf-1 was expressed at high levels compared with the endogenous protein but was down - regulated in the same manner during the course of differentiation . overexpression of active raf-1 delays erythroid differentiation and differentiation - associated caspase activation in i/11 erythroblasts . the differentiation of uninfected i/11 was compared with that of cells expressing murine stem cell virus - egfp ( empty vector ) with full - length raf-1 ( wt raf-1 ) or a constitutively active raf ( bxb ) . first , by showing that overexpression of raf-1 restrains caspase activation and delays erythroid differentiation , we confirm the role of raf-1 as a negative regulator of both processes . due to the differences in the expression of wt and bxb raf-1 , however , it is not possible to deduce whether bxb delays differentiation more efficiently than wt raf-1 because of its constitutive kinase activity or simply because of its higher expression . be that as it may , the experiment shows that a certain level of raf-1 expression is needed at later maturation stages for restraining differentiation - induced caspase activation , whereas high level expression at the onset of differentiation has little effect on the progress of maturation . second , the fact that the full - length protein expressed under the control of the retroviral promoter is down - regulated with the same kinetics and efficiency as endogenous raf-1 indicates that the regulation of raf-1 expression occurs at the posttranscriptional level , as also confirmed by northern blot analysis ( unpublished data ) . interestingly , we have observed posttranscriptional raf-1 down - regulation during pathogen - induced macrophage apoptosis ( 9 ) . in this case , raf-1 was degraded by the proteasome in a caspase - dependent manner . thus , in macrophages , the relationship between raf-1 and caspase activation was reciprocal , with the protease directing the degradation of the kinase that restrains its activation ( 9 ) . in contrast , raf-1 down - regulation in differentiating erythroblasts is either upstream of caspase activation or independently regulated . defining the signal directing raf-1 down - regulation and , if possible , interfering with it will be important in further elucidating the exact position of this kinase in the signal transduction cascade leading to erythroid differentiation . furthermore , at least in neurons , the regulation of iap is an essential function of b - raf and can not be substituted for by raf-1 ( 23 ) . raf-1 is activated by the erythroleukemia - inducing strains of friend spleen focus - forming virus , and its inhibition by antisense oligonucleotide partially reduces proliferation of virus - infected cells ( 24 ) . in addition , oncogenic v - raf versions cooperate with myc to transform erythroid cells ( 16 ) . we now report a previously unrecognized function of raf-1 as a modulator of erythroid homeostasis , which , by restraining caspase activation , prevents the premature differentiation of erythroblasts and safeguards the maintenance of the appropriate amount of proliferating precursors . a delay in precursor differentiation , as the one caused by mutation / overexpression of raf-1 ( bxb raf-1 or v - raf ) , should tip the balance between proliferation and terminal maturation of erythroid cells toward precursor renewal and might represent the main contribution of raf to the establishment of leukemia .

oral health has long been considered to be an important aspect of overall , general health . however , oral disease still continues to be one of the most prevalent problems affecting the overall wellbeing of the world 's population . prevention and general maintenance are primary and effective methods to ensure oral health in addition to patients practice of oral hygiene techniques . factors that influenced the effectiveness and adequacy of the patients oral hygiene maintenance included their knowledge , attitudes , and behavior regarding oral disease prevention . healthcare professionals perceptions and practice of oral health maintenance are typically developed during formal education . assessing these patterns of oral health attitudes and behavior among healthcare professional students are of particular importance because the development of their own perceptions and practices of oral health maintenance have a direct impact on their ability to influence their patients perceptions and practice of oral health maintenance . kawamura ( 1988 ) developed the hiroshima university - dental behavioral inventory ( hu - dbi ) to assess patients attitudes , behavior , and perception of oral health which was eventually utilized within dental schools . over the years retest reliability and validity , and thus , has been adopted in many countries , including united kingdom , finland , greece , china , saudi arabia , and the united arab emirates . the hu - dbi has been translated from japanese to chinese , korean , english , and finnish for cross - cultural comparisons of dental students around the world . even with the widespread utilization of the hu - dbi assessing students internationally , to date , very few studies in the literature have evaluated the attitudes and behavior of kuwaiti healthcare professional students . prior studies have found that the students of kuwait university health sciences center ( kuhsc ) possessed limited knowledge regarding the etiology of dental diseases and the correct methods of maintaining oral health . to date , there have been no reported studies evaluating oral health attitudes of behavior between dental and nondental healthcare professionals utilizing the hu - dbi questionnaire at kuhsc . this might be useful in assessing the existing differences in oral health practices and perceptions among dental and nondental students at kuhsc , as well as in assessing the students attitudes and behavior regarding oral health maintenance and oral disease prevention . thus , the aims of this study were to assess the attitudes and behavior of oral health maintenance among kuhsc students in four faculties ( medicine , dentistry , pharmacy , and allied health ) and to compare the oral health attitudes and behavior of all kuhsc students based on their academic level . this cross - sectional study was conducted in full accordance with the world medical association declaration of helsinki and was approved by the kuhsc ethical committee ( ref . : vdr / ec/1788 ) , and was conducted from september 1 2014 to february 27 2015 . students enrolled in the faculties of medicine , dentistry , pharmacy , and allied health of kuhsc were asked to participate in the study . the total number of students who were enrolled at the four hsc faculties for the 20142015 academic year was 1802 . the sample size covered the entire population of registered students at kuhsc , which was satisfactory for the aims of this project . an e - mail invitation with a link to the hu - dbi survey was sent to all 1802 students with kuhsc e - mail addresses via the qualtrics survey system to ensure anonymity and privacy . no pilot study was carried out as all the discrepancies and redundancies were eliminated in the original study by dr . the qualtrics web - based survey was used in this study to digitize the hu - dbi , manage responses , track participants , and generate initial statistical reports ( qualtrics , usa ) . a written consent form was approved by the kuhsc ethical committee and obtained from all participants . the study participants were 140 dental students and 533 medical students in years 1 through 7 , as well as 225 pharmacy students and 904 allied health students in years 1 through 5 . because all instructions at kuhsc are conducted in the english language , students were invited to participate in this survey using the well - established english version of the hu - dbi , consisting of 20 dichotomous questions in an agree - disagree format . a numerical estimation of oral health attitudes and behavior was calculated based on the total agree / disagree responses from 12 scored items out of a total of 20 items in the hu - dbi [ appendix 1 ] . of the 12 items , 6 items were given one point for each agreed response ( marked as a ) and zero points for each disagreed response , whereas for the subsequent 6 items , one point was given for each disagreed response ( marked as d ) , and zero points given for each agreed response . demographical questions were added to the hu - dbi to compare responses from each faculty , including gender , age , and academic level . the dental and medical school academic levels were classified into three groups , namely , a basic sciences group consisting of years 1 and 2 ( didactic lectures ) , a pre - clinical group consisting of years 3 and 4 ( laboratory courses , didactic lectures , and problem - based learning seminars ) , and a clinical group consisting of years 5 through 7 ( direct patient care ) . students enrolled in the faculty of pharmacy or allied health were also grouped into three academic levels , namely , a basic sciences group consisting of years 1 and 3 , a pre - clinical group consisting of years 3 and 4 , and a clinical group consisting of year 5 . the survey data was collected , de - identified , and organized into microsoft excel spreadsheets ( microsoft inc . , usa ) , and was statistically analyzed utilizing the statistical package for the social sciences version 20.0 software ( ibm inc . the data were analyzed for frequency distributions , and differences among the groups were assessed by the mann a factor analysis test was conducted to reduce redundancy and cluster survey questions into broader categories and represented by a common factor . p values less than 0.05 were considered to be statistically significant ( p < 0.05 ) . of the 1802 registered students at the four kuhsc faculties , 77% of the students completed the questionnaire , yielding a final sample size of 1387 students , with age ranging from 17 to 25 years . among the participants , 343 ( 24.7% ) were males and 1044 ( 75.3% ) were females , with a mean age of 21.3 1.2 years . of the 1387 participants , 397 ( 28.6% ) were from the faculty of medicine , 141 ( 10.2% ) were from the faculty of dentistry , 329 ( 23.7% ) were from the faculty of pharmacy , and 520 ( 37.5% ) were from the faculty of allied health . the distribution of students by gender , age , academic level , and response rate is presented in table 1 . demographic distributions of kuhsc total sample ( n=1387 ) the mean hu - dbi score for the entire sampled population was 5.14 0.94 . the mean hu - dbi score was 5.22 0.28 for male students and 5.31 0.21 for female students . the mean scores of the hu - dbi based on academic level were 5.37 0.22 , 5.64 0.13 , and 5.75 0.21 for the basic sciences , pre - clinical , and clinical groups , respectively . the mean hu - dbi score was 5.43 0.47 for the students enrolled in the faculty of medicine , 5.74 0.23 for the faculty of dentistry , 4.73 0.33 for the faculty of pharmacy , and 4.55 0.24 for the faculty of allied health ; all the scores were statistically significant at p < 0.05 . the results showed that there were statistically significant differences among the responses when students from all the four faculties were grouped by academic level ( basic , pre - clinical , or clinical ) for questionnaire items 3 , 9 , 10 , 17 , and 19 [ table 2 ] . thirty - two percent of pre - clinical students in all the four faculties were worried about the color of their teeth , which was lower than that of the students in the basic sciences and clinical years ( item 3 , p < 0.01 ) . forty - three percent of students in their clinical years responded that they were never taught professionally how to brush , which was higher than that of the students in the basic sciences and pre - clinical groups ( item 10 , p < 0.01 ) . questionnaire items and percentages of agree responses by academic level , faculty , and gender eighteen percent of the students in the basic sciences group reported that they brushed each of their teeth carefully , which was lower compared to the students in the pre - clinical and clinical groups ( item 9 , p < 0.05 ) . thirty - three percent of basic sciences students were more likely to use a toothbrush with hard bristles , a higher rate when compared to the pre - clinical and clinical students ( item 17 , p < 0.05 ) . thirty - one percent of students in the clinical years reported spending too much time brushing their teeth , which was a higher rate than that of the students in the basic sciences and pre - clinical years ( item 19 , p < 0.01 ) . table 2 also shows statistically significant differences across students enrolled in the four kuhsc faculties for items 1 , 4 , 10 , 15 , 17 , and 19 . fifty - nine percent of the dental students were worried about visiting the dentist ( item 1 , p < 0.05 ) , and 24% were more likely to notice sticky deposits on their teeth ( item 4 , p < 0.05 ) , when compared to the students in the medical , pharmacy and allied health faculties . sixty - two percent of the pharmacy students were never taught professionally how to brush , a higher rate when compared to dental , medical , and allied health students ( item 10 , p < 0.01 ) . seventy - three percent of allied health students put off going to the dentist until they had a toothache , which was more often than that for students in the dental , medical , and pharmacy faculties ( item 15 , p < 0.01 ) . thirty - one percent of dental students used a toothbrush with hard bristles , which was significantly less than that of the students in the medical , pharmacy , and allied health faculties ( item 17 , p < 0.05 ) finally , 20% of the dental students felt that they took too much time to brush their teeth , a higher rate than that of the medical , pharmacy , or allied health students ( item 19 p < 0.05 ) . when comparing responses from students in all the four faculties based on gender , we found statistically significant differences for items 1 , 3 , 4 , 7 , 9 , 13 , 15 , and 19 [ table 2 ] . in general , female students displayed more interest in their overall oral health maintenance , and were more likely to worry about the color of their teeth ( item 3 , p < 0.05 ) , notice white sticky deposits on their teeth ( item 4 , p < 0.01 ) , be bothered by the color of their gums ( item 7 , p < 0.01 ) , brush each of their teeth carefully ( item 9 , p < 0.05 ) , worry about having bad breath ( item 13 , p < 0.05 ) , and felt that they take too much time to brush their teeth ( item 19 , p < 0.01 ) . male students tended to be less worried about visiting a dentist compared to female students ( item 1 , p < 0.01 ) and were more likely to put off going to the dentist until they had a toothache ( item 15 , p < 0.05 ) . because of the large amount of data obtained , a factor analysis test was utilized for data reduction , removal of redundancy , and to reveal any patterns that may exist . a total of four constructs were created based on questionnaire items grouped into descriptive and meaningful categories for additional data interpretation . questionnaire items 1 , 6 , 7 , and 13 were grouped into a construct representing students perceptions and cognitive thoughts about their own oral health and were termed cognitive effects . items 10 , 14 , and 20 were grouped into a construct representing the students previous exposure to dental care , and items 9 , 12 , and 15 were grouped into a construct representing the students practice of oral health and maintenance . the last construct , representing students brushing techniques , consisted of items 4 , 11 , 17 , and 18 . after identifying these four constructs , kuhsc students scored a weighted average of 0.64 within the oral health and maintenance construct , which was the highest among the four constructs . students scored a weighted average of 0.51 under the brushing techniques construct , and scored a 0.40 for questions representing the cognitive effects construct . finally , students scored a weighted average of 0.39 within the previous exposure to dental care construct . statistical summary of the four factor analysis constructs non - parametric test results testing gender against the four constructs testing academic level against the four constructs because the collected data did not follow a normal distribution , the mann whitney u and kruskal wallis tests were used to analyze the data under the four constructs . the mann whitney u test was used to identify differences when comparing students questionnaire responses within one faculty to the other three faculties [ table 4 ] . we found a statistically significant difference in the students responses when utilizing the cognitive effects construct , where medical students scored the highest mean score of 0.62 , followed by the dental students with a mean score of 0.48 . allied health and pharmacy students mean scores were comparable at 0.28 and 0.27 , respectively ( p = 0.05 ) . when comparing these students utilizing the oral health and maintenance construct , we found a statistically significant difference where dental students scored the highest mean ( 0.67 ) , followed by allied health students ( 0.65 ) . medical and pharmacy students both scored a mean of 0.63 ( p < 0.05 ) . there was no statistically significant difference in students among the four faculties within the constructs representing previous exposure to dental care or brushing techniques ( p = 0.07 and 0.16 , respectively ) . our data revealed that there were statistically significant differences between male and female students when analyzing their responses utilizing factor analysis [ table 5 ] . for questionnaire items pertaining to cognitive effects , males scored a mean of 0.37 whereas females scored a mean of 0.48 ( p < 0.05 ) . in addition , there exists a statistically significant difference between male and female students responses on questionnaire items pertaining to oral health and maintenance ( females = 0.65 , males = 0.63 , p < 0.05 ) . the kruskal wallis test was used to compare students questionnaire responses within each academic level to the four constructs and was found to be statistically significant [ table 6 ] . students within their clinical level of training demonstrated the most positive oral health attitudes and behavior on questionnaire items represented by the cognitive effects construct ( 0.47 ) followed by students in their pre - clinical years ( 0.38 ) , and finally by students in their basic sciences years ( 0.38 ) , and was statistically significant ( p < 0.05 ) . questionnaire items represented by the previous exposure to dental care construct showed a similar pattern , where clinical students scored the highest followed by the pre - clinical and basic sciences students ( p < 0.05 ) [ table 6 ] . students within the pre - clinical level of training scored the highest mean on questionnaire items represented by the oral health and maintenance construct , followed by the basic sciences students and the clinical students ( 0.65 , 0.64 , 0.60 , respectively ; p < 0.05 ) . pre - clinical students also scored the highest mean on items represented by the brushing techniques construct , followed by the clinical students and , finally , the basic sciences students ( 0.53 , 0.51 , 0.48 , respectively ; p < 0.05 ) . non - parametric test results testing gender against the four constructs testing academic level against the four constructs because the collected data did not follow a normal distribution , the mann whitney u and kruskal wallis tests were used to analyze the data under the four constructs . the mann whitney u test was used to identify differences when comparing students questionnaire responses within one faculty to the other three faculties [ table 4 ] . we found a statistically significant difference in the students responses when utilizing the cognitive effects construct , where medical students scored the highest mean score of 0.62 , followed by the dental students with a mean score of 0.48 . allied health and pharmacy students mean scores were comparable at 0.28 and 0.27 , respectively ( p = 0.05 ) . when comparing these students utilizing the oral health and maintenance construct , we found a statistically significant difference where dental students scored the highest mean ( 0.67 ) , followed by allied health students ( 0.65 ) . medical and pharmacy students both scored a mean of 0.63 ( p < 0.05 ) . there was no statistically significant difference in students among the four faculties within the constructs representing previous exposure to dental care or brushing techniques ( p = 0.07 and 0.16 , respectively ) . our data revealed that there were statistically significant differences between male and female students when analyzing their responses utilizing factor analysis [ table 5 ] . for questionnaire items pertaining to cognitive effects , males scored a mean of 0.37 whereas females scored a mean of 0.48 ( p < 0.05 ) . in addition , there exists a statistically significant difference between male and female students responses on questionnaire items pertaining to oral health and maintenance ( females = 0.65 , males = 0.63 , p < 0.05 ) . the kruskal wallis test was used to compare students questionnaire responses within each academic level to the four constructs and was found to be statistically significant [ table 6 ] . students within their clinical level of training demonstrated the most positive oral health attitudes and behavior on questionnaire items represented by the cognitive effects construct ( 0.47 ) followed by students in their pre - clinical years ( 0.38 ) , and finally by students in their basic sciences years ( 0.38 ) , and was statistically significant ( p < 0.05 ) . questionnaire items represented by the previous exposure to dental care construct showed a similar pattern , where clinical students scored the highest followed by the pre - clinical and basic sciences students ( p < 0.05 ) [ table 6 ] . students within the pre - clinical level of training scored the highest mean on questionnaire items represented by the oral health and maintenance construct , followed by the basic sciences students and the clinical students ( 0.65 , 0.64 , 0.60 , respectively ; p < 0.05 ) . pre - clinical students also scored the highest mean on items represented by the brushing techniques construct , followed by the clinical students and , finally , the basic sciences students ( 0.53 , 0.51 , 0.48 , respectively ; p < 0.05 ) . the study reports significant differences in the level of oral health attitudes and behavior among students of different academic levels in all four faculties . dental students showed that they have knowledge and attitude of oral health when compared to students from other kuhsc faculties . in addition , the results of this study demonstrated that female students had better overall hu - dbi scores and better oral health attitudes and behavior than male students , which agreed with previous studies that reported higher hu - dbi scores for female students than that of male students . moreover , students in higher academic levels ( e.g. pre - clinical vs basic sciences , and clinical vs pre - clinical or basic sciences ) generally had higher hu - dbi scores , representing better oral health attitudes and behavior as students progressed within their academic training , which is in agreement with prior studies . students within their clinical year also scored the highest mean within the cognitive effects and previous exposure to dental care constructs when compared to the pre - clinical and basic sciences students . these findings may be attributed not only to increase in the fund of knowledge as students advance through their training but also to their pre - clinical and clinical dental experience , which allows them to apply their knowledge outside the classroom . as expected , dental students demonstrated better oral health attitudes and behavior , scoring a mean score of 5.74 0.23 , the highest when compared to their nondental professional colleagues . this finding was observed by other studies when comparing dental students with their nondental professional student colleagues . reported that dental students demonstrated better hu - dbi scores than students from other faculties such as medicine and engineering . in addition , kumar et al . concluded that dental students had higher hu - dbi scores , and thus , possessed better oral health attitudes and behavior than that of pharmacy students . the dental students superior oral health attitudes and behavior may be attributed to their clinical exposure to oral health and preventative care courses as they advance through their dental training . the results of the study inferred that dental students enroll in courses such as cariology , periodontology , and dental public health at the beginning of their 5 year ( clinical ) , which is likely to have the most impact on students oral health knowledge , attitudes , and behavior . periodontology and dental public health courses continue through the 7 year of the dental students clinical training . therefore , it is hypothesized that , as students advance through their dental training , they are expected to develop better oral health attitudes and behavior . medical students had a mean score similar to that of dental students ( 5.43 0.47 ) , demonstrating oral health attitudes and behavior to a level that was almost as high as their dental colleagues . similar results were reported by doshi et al . who reported that dental and medical students have better attitudes toward oral health behavior than their counterparts in other faculties . this similarity may be explained by the nature of the clinical curriculum of the faculty of medicine . during their clinical years , medical students rotate through various clinical clerkships , including internal medicine , community medicine , and pediatrics , which exposes them to important oral health topics for children , adults , and the general community . although these clinical rotations do not train medical students on oral health maintenance and disease prevention to the same degree as dental students , the medical clinical rotations may be adequate in training medical students on topics related to oral health maintenance . students enrolled in the faculty of pharmacy or allied health , however , had considerably lower hu - dbi mean scores ( 4.73 0.33 and 4.55 0.24 , respectively ) when compared to their medical and dental colleagues . a likely reason for this discrepancy is attributed to the lack of clinical rotations that expose pharmacy and allied health students to topics on oral health maintenance . in addition , the length of training for students enrolled in the faculty of pharmacy or allied health is shorter in duration than that of those enrolled in the faculty of dentistry or medicine , which may impact the amount of oral healthcare exposure these students receive during training . because the faculty of dentistry at kuhsc is the sole dental school in the state of kuwait that is responsible for training the next generation of kuwaiti dentists , it is important to assess the effectiveness and identify areas requiring revision within the dental curriculum . although they had higher hu - dbi scores than that of their nondental colleagues , the dental students at kuhsc had one of the lowest hu - dbi mean scores ( 5.74 0.23 ) when compared to studies conducted in other countries such as croatia , turkey , and greece ( 6.62 1.54 , 6.53 1.99 , and 6.86 1.83 , respectively ) . countries such as britain , finland , and japan reported even higher hu - dbi scores among their dental students ( 7.33 , 7.15 1.13 , and 7.40 2.55 , respectively ) and oral health attitudes and behavior of dental students of kuhsc were more similar to that of dental students in jordan , india , and china ( 5.2 , 5.07 , and 6.06 respectively ) . this discrepancy maybe attributed to the lack of early exposure to oral health at younger age along with shortage from the government , represented by school health programs , to educate mothers , care givers , and young students to implant the basic knowledge of dental and oral health . in addition , this divergence may be attributed to cultural and regional differences found among these dental schools ; however , these differences would likely be best demonstrated by the hu - dbi scores of students just beginning their dental education . therefore , the hu - dbi scores of all dental students is more likely to be attributed to the direct effects of the dental students curriculum . the dental students exhibiting the highest hu - dbi scores were the students in their clinical years of dental school , followed by the pre - clinical students and those in the basic sciences ( 6.10 1.46 , 5.93 1.72 , and 5.20 1.85 , respectively ) . this observation can be attributed to the fact that dental students acquire knowledge regarding oral healthcare maintenance and disease prevention within the various dental courses as they progress in their dental training . the most improvement in the students knowledge occurred during the transition from the basic sciences to the pre - clinical level ( 5.20 1.85 to 5.93 1.72 , p < 0.05 ) . this is likely attributed to the fact that , as students progress in their education from the basic sciences to the pre - clinical level , they become more cognizant of their own oral health practices in preparation for their clinical years at the faculty of dentistry . there was less improvement in the oral health attitudes and behavior in students transitioning from their pre - clinical to clinical years ( 5.93 1.72 and 6.10 1.46 ) . dental students in their clinical years are offered courses that heavily emphasize oral health maintenance and disease prevention in advanced courses such as periodontology and dental public health . this finding suggests that an improvement in the coursework offered to students in their clinical years may be warranted because the dental students in their clinical years did not demonstrate much of an improvement in their oral health attitudes and behavior from their pre - clinical years when compared to the improvement seen in students from basic science to pre - clinical years . among the four constructs , the oral health and maintenance construct had the highest mean , suggesting that the questions under this construct had the greatest influence on students attitudes toward oral health . higher means were found among dental and medical students within the cognitive effects and oral health and maintenance constructs when compared to the pharmacy and allied health students , which may be attributed to the dental and medical curricula . dental and medical students may have an increased sense of awareness of their own oral health practices , secondary to their oral health training experience within their respective curricula . the results of this study indicate that female students had better overall hu - dbi scores , and therefore , better oral health attitudes and behavior than male students . previous studies have also reported higher hu - dbi scores for female students than male students , confirming that there is a significant relationship between gender and oral health attitude and behavior . al - ansari and honkala have found that female students practiced better oral hygiene and possessed more knowledge about oral health maintenance than male students . al - shammari et al . reported similar findings in their study among the general kuwaiti population , which concluded that females brush , floss , use mouth wash , and visit the dentist more often than males . these findings may be attributed by the fact that females , in general , have more concerns about their physical appearance , including oral health , and are more self - conscious to maintain their appearance for better social and occupational opportunities . strong knowledge base of oral health might be one of the foremost elements of oral health and attitude , however , there are other determinants that might also play a major role . the paradigm of human behavior can not be purely presented by one factor , instead , the essence of its complexity is represented in a complex interplay of multiple factors . determinants such as socioeconomic and sociodemographic factors could have a major influence on health behavior and attitude in addition to knowledge . a limitation of this study is the presence of diverse educational backgrounds among the students at kuhsc that may have led to variations in the students oral health maintenance practices . in addition , the sampled population was restricted to a specialized subset of the general kuwaiti population . future studies are warranted to investigate , as a continuation of this project , the nonhealthcare faculties of kuwait university . finally , is a generalized introduction of the hu - dbi questionnaire to the entire general population of kuwait and other countries within the region . students in higher academic levels tend to have higher hu - dbi scores , representing an increase in knowledge and improvement in oral health attitudes and behavior as students progress in their training . students in their clinical years were more cognizant about their own oral health and demonstrated a higher level of maintenance techniques . female students were more likely to demonstrate better oral health practices and perceptions than male students . dental students demonstrated the highest hu - dbi scores when compared to students enrolled in nondental faculties , exhibiting better oral health attitudes and behavior than their nondental colleagues . this finding has demonstrated the ongoing knowledge gap between oral health and general health within the healthcare professional curriculum . further studies assessing the adequacy of basic dental education within nondental health professional curricula may aid in bridging this knowledge gap . this issue is reflecting negatively on the healthcare system , and educators and policy makers need to collaborate to bridge the gap among healthcare providers for better oral health care . therefore , additional studies investigating the complex interplay of knowledge , attitude , and behavior of is indispensable .

aim : the aims of this study were to assess attitudes and behavior of oral health maintenance among students in four faculties ( medicine , dentistry , pharmacy , and allied health ) and to compare oral health attitudes and behavior of all students at kuwait university health sciences center ( kuhsc ) based on their academic level.materials and methods : students enrolled in the faculties of dentistry , medicine , pharmacy , and allied health at kuhsc were evaluated regarding their oral health attitudes and behavior by an e - mail invitation with a link to the hiroshima university dental behavior inventory survey that was sent to all 1802 students with kuwait university health sciences center e - mail addresses . the data were analyzed for frequency distributions , and differences among the groups were assessed using the mann whitney u test , chi - square test , and kruskal wallis test . p values less than 0.05 were considered to be statistically significant ( p < 0.05).results : the results of this study indicated that dental students achieved better oral health attitudes and behavior than that of their nondental professional fellow students ( p < 0.05 ) . students in advanced academic levels and female students demonstrated better oral health attitudes and behavior.conclusion:dental students and students who were in advanced levels of their training along with female students demonstrated better oral health practices and perceptions than students in lower academic levels and male students , respectively . additional studies for investigating the effectiveness and identifying areas requiring modification within the dental curriculum at kuhsc may be warranted .

INTRODUCTION MATERIALS AND METHODS RESULTS Differences within constructs created by factor analysis [Tables DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

assessing these patterns of oral health attitudes and behavior among healthcare professional students are of particular importance because the development of their own perceptions and practices of oral health maintenance have a direct impact on their ability to influence their patients perceptions and practice of oral health maintenance . kawamura ( 1988 ) developed the hiroshima university - dental behavioral inventory ( hu - dbi ) to assess patients attitudes , behavior , and perception of oral health which was eventually utilized within dental schools . prior studies have found that the students of kuwait university health sciences center ( kuhsc ) possessed limited knowledge regarding the etiology of dental diseases and the correct methods of maintaining oral health . this might be useful in assessing the existing differences in oral health practices and perceptions among dental and nondental students at kuhsc , as well as in assessing the students attitudes and behavior regarding oral health maintenance and oral disease prevention . thus , the aims of this study were to assess the attitudes and behavior of oral health maintenance among kuhsc students in four faculties ( medicine , dentistry , pharmacy , and allied health ) and to compare the oral health attitudes and behavior of all kuhsc students based on their academic level . students enrolled in the faculties of medicine , dentistry , pharmacy , and allied health of kuhsc were asked to participate in the study . the sample size covered the entire population of registered students at kuhsc , which was satisfactory for the aims of this project . an e - mail invitation with a link to the hu - dbi survey was sent to all 1802 students with kuhsc e - mail addresses via the qualtrics survey system to ensure anonymity and privacy . a numerical estimation of oral health attitudes and behavior was calculated based on the total agree / disagree responses from 12 scored items out of a total of 20 items in the hu - dbi [ appendix 1 ] . students enrolled in the faculty of pharmacy or allied health were also grouped into three academic levels , namely , a basic sciences group consisting of years 1 and 3 , a pre - clinical group consisting of years 3 and 4 , and a clinical group consisting of year 5 . the data were analyzed for frequency distributions , and differences among the groups were assessed by the mann a factor analysis test was conducted to reduce redundancy and cluster survey questions into broader categories and represented by a common factor . p values less than 0.05 were considered to be statistically significant ( p < 0.05 ) . of the 1387 participants , 397 ( 28.6% ) were from the faculty of medicine , 141 ( 10.2% ) were from the faculty of dentistry , 329 ( 23.7% ) were from the faculty of pharmacy , and 520 ( 37.5% ) were from the faculty of allied health . the mean hu - dbi score was 5.43 0.47 for the students enrolled in the faculty of medicine , 5.74 0.23 for the faculty of dentistry , 4.73 0.33 for the faculty of pharmacy , and 4.55 0.24 for the faculty of allied health ; all the scores were statistically significant at p < 0.05 . the results showed that there were statistically significant differences among the responses when students from all the four faculties were grouped by academic level ( basic , pre - clinical , or clinical ) for questionnaire items 3 , 9 , 10 , 17 , and 19 [ table 2 ] . thirty - two percent of pre - clinical students in all the four faculties were worried about the color of their teeth , which was lower than that of the students in the basic sciences and clinical years ( item 3 , p < 0.01 ) . forty - three percent of students in their clinical years responded that they were never taught professionally how to brush , which was higher than that of the students in the basic sciences and pre - clinical groups ( item 10 , p < 0.01 ) . questionnaire items and percentages of agree responses by academic level , faculty , and gender eighteen percent of the students in the basic sciences group reported that they brushed each of their teeth carefully , which was lower compared to the students in the pre - clinical and clinical groups ( item 9 , p < 0.05 ) . thirty - three percent of basic sciences students were more likely to use a toothbrush with hard bristles , a higher rate when compared to the pre - clinical and clinical students ( item 17 , p < 0.05 ) . thirty - one percent of students in the clinical years reported spending too much time brushing their teeth , which was a higher rate than that of the students in the basic sciences and pre - clinical years ( item 19 , p < 0.01 ) . fifty - nine percent of the dental students were worried about visiting the dentist ( item 1 , p < 0.05 ) , and 24% were more likely to notice sticky deposits on their teeth ( item 4 , p < 0.05 ) , when compared to the students in the medical , pharmacy and allied health faculties . sixty - two percent of the pharmacy students were never taught professionally how to brush , a higher rate when compared to dental , medical , and allied health students ( item 10 , p < 0.01 ) . seventy - three percent of allied health students put off going to the dentist until they had a toothache , which was more often than that for students in the dental , medical , and pharmacy faculties ( item 15 , p < 0.01 ) . thirty - one percent of dental students used a toothbrush with hard bristles , which was significantly less than that of the students in the medical , pharmacy , and allied health faculties ( item 17 , p < 0.05 ) finally , 20% of the dental students felt that they took too much time to brush their teeth , a higher rate than that of the medical , pharmacy , or allied health students ( item 19 p < 0.05 ) . when comparing responses from students in all the four faculties based on gender , we found statistically significant differences for items 1 , 3 , 4 , 7 , 9 , 13 , 15 , and 19 [ table 2 ] . in general , female students displayed more interest in their overall oral health maintenance , and were more likely to worry about the color of their teeth ( item 3 , p < 0.05 ) , notice white sticky deposits on their teeth ( item 4 , p < 0.01 ) , be bothered by the color of their gums ( item 7 , p < 0.01 ) , brush each of their teeth carefully ( item 9 , p < 0.05 ) , worry about having bad breath ( item 13 , p < 0.05 ) , and felt that they take too much time to brush their teeth ( item 19 , p < 0.01 ) . male students tended to be less worried about visiting a dentist compared to female students ( item 1 , p < 0.01 ) and were more likely to put off going to the dentist until they had a toothache ( item 15 , p < 0.05 ) . statistical summary of the four factor analysis constructs non - parametric test results testing gender against the four constructs testing academic level against the four constructs because the collected data did not follow a normal distribution , the mann whitney u and kruskal wallis tests were used to analyze the data under the four constructs . the mann whitney u test was used to identify differences when comparing students questionnaire responses within one faculty to the other three faculties [ table 4 ] . we found a statistically significant difference in the students responses when utilizing the cognitive effects construct , where medical students scored the highest mean score of 0.62 , followed by the dental students with a mean score of 0.48 . allied health and pharmacy students mean scores were comparable at 0.28 and 0.27 , respectively ( p = 0.05 ) . when comparing these students utilizing the oral health and maintenance construct , we found a statistically significant difference where dental students scored the highest mean ( 0.67 ) , followed by allied health students ( 0.65 ) . medical and pharmacy students both scored a mean of 0.63 ( p < 0.05 ) . there was no statistically significant difference in students among the four faculties within the constructs representing previous exposure to dental care or brushing techniques ( p = 0.07 and 0.16 , respectively ) . for questionnaire items pertaining to cognitive effects , males scored a mean of 0.37 whereas females scored a mean of 0.48 ( p < 0.05 ) . in addition , there exists a statistically significant difference between male and female students responses on questionnaire items pertaining to oral health and maintenance ( females = 0.65 , males = 0.63 , p < 0.05 ) . the kruskal wallis test was used to compare students questionnaire responses within each academic level to the four constructs and was found to be statistically significant [ table 6 ] . students within their clinical level of training demonstrated the most positive oral health attitudes and behavior on questionnaire items represented by the cognitive effects construct ( 0.47 ) followed by students in their pre - clinical years ( 0.38 ) , and finally by students in their basic sciences years ( 0.38 ) , and was statistically significant ( p < 0.05 ) . questionnaire items represented by the previous exposure to dental care construct showed a similar pattern , where clinical students scored the highest followed by the pre - clinical and basic sciences students ( p < 0.05 ) [ table 6 ] . students within the pre - clinical level of training scored the highest mean on questionnaire items represented by the oral health and maintenance construct , followed by the basic sciences students and the clinical students ( 0.65 , 0.64 , 0.60 , respectively ; p < 0.05 ) . pre - clinical students also scored the highest mean on items represented by the brushing techniques construct , followed by the clinical students and , finally , the basic sciences students ( 0.53 , 0.51 , 0.48 , respectively ; p < 0.05 ) . non - parametric test results testing gender against the four constructs testing academic level against the four constructs because the collected data did not follow a normal distribution , the mann whitney u and kruskal wallis tests were used to analyze the data under the four constructs . the mann whitney u test was used to identify differences when comparing students questionnaire responses within one faculty to the other three faculties [ table 4 ] . we found a statistically significant difference in the students responses when utilizing the cognitive effects construct , where medical students scored the highest mean score of 0.62 , followed by the dental students with a mean score of 0.48 . allied health and pharmacy students mean scores were comparable at 0.28 and 0.27 , respectively ( p = 0.05 ) . when comparing these students utilizing the oral health and maintenance construct , we found a statistically significant difference where dental students scored the highest mean ( 0.67 ) , followed by allied health students ( 0.65 ) . there was no statistically significant difference in students among the four faculties within the constructs representing previous exposure to dental care or brushing techniques ( p = 0.07 and 0.16 , respectively ) . for questionnaire items pertaining to cognitive effects , males scored a mean of 0.37 whereas females scored a mean of 0.48 ( p < 0.05 ) . in addition , there exists a statistically significant difference between male and female students responses on questionnaire items pertaining to oral health and maintenance ( females = 0.65 , males = 0.63 , p < 0.05 ) . the kruskal wallis test was used to compare students questionnaire responses within each academic level to the four constructs and was found to be statistically significant [ table 6 ] . students within their clinical level of training demonstrated the most positive oral health attitudes and behavior on questionnaire items represented by the cognitive effects construct ( 0.47 ) followed by students in their pre - clinical years ( 0.38 ) , and finally by students in their basic sciences years ( 0.38 ) , and was statistically significant ( p < 0.05 ) . questionnaire items represented by the previous exposure to dental care construct showed a similar pattern , where clinical students scored the highest followed by the pre - clinical and basic sciences students ( p < 0.05 ) [ table 6 ] . students within the pre - clinical level of training scored the highest mean on questionnaire items represented by the oral health and maintenance construct , followed by the basic sciences students and the clinical students ( 0.65 , 0.64 , 0.60 , respectively ; p < 0.05 ) . pre - clinical students also scored the highest mean on items represented by the brushing techniques construct , followed by the clinical students and , finally , the basic sciences students ( 0.53 , 0.51 , 0.48 , respectively ; p < 0.05 ) . the study reports significant differences in the level of oral health attitudes and behavior among students of different academic levels in all four faculties . in addition , the results of this study demonstrated that female students had better overall hu - dbi scores and better oral health attitudes and behavior than male students , which agreed with previous studies that reported higher hu - dbi scores for female students than that of male students . pre - clinical vs basic sciences , and clinical vs pre - clinical or basic sciences ) generally had higher hu - dbi scores , representing better oral health attitudes and behavior as students progressed within their academic training , which is in agreement with prior studies . as expected , dental students demonstrated better oral health attitudes and behavior , scoring a mean score of 5.74 0.23 , the highest when compared to their nondental professional colleagues . reported that dental students demonstrated better hu - dbi scores than students from other faculties such as medicine and engineering . concluded that dental students had higher hu - dbi scores , and thus , possessed better oral health attitudes and behavior than that of pharmacy students . the dental students superior oral health attitudes and behavior may be attributed to their clinical exposure to oral health and preventative care courses as they advance through their dental training . the results of the study inferred that dental students enroll in courses such as cariology , periodontology , and dental public health at the beginning of their 5 year ( clinical ) , which is likely to have the most impact on students oral health knowledge , attitudes , and behavior . therefore , it is hypothesized that , as students advance through their dental training , they are expected to develop better oral health attitudes and behavior . medical students had a mean score similar to that of dental students ( 5.43 0.47 ) , demonstrating oral health attitudes and behavior to a level that was almost as high as their dental colleagues . although these clinical rotations do not train medical students on oral health maintenance and disease prevention to the same degree as dental students , the medical clinical rotations may be adequate in training medical students on topics related to oral health maintenance . students enrolled in the faculty of pharmacy or allied health , however , had considerably lower hu - dbi mean scores ( 4.73 0.33 and 4.55 0.24 , respectively ) when compared to their medical and dental colleagues . a likely reason for this discrepancy is attributed to the lack of clinical rotations that expose pharmacy and allied health students to topics on oral health maintenance . in addition , the length of training for students enrolled in the faculty of pharmacy or allied health is shorter in duration than that of those enrolled in the faculty of dentistry or medicine , which may impact the amount of oral healthcare exposure these students receive during training . because the faculty of dentistry at kuhsc is the sole dental school in the state of kuwait that is responsible for training the next generation of kuwaiti dentists , it is important to assess the effectiveness and identify areas requiring revision within the dental curriculum . although they had higher hu - dbi scores than that of their nondental colleagues , the dental students at kuhsc had one of the lowest hu - dbi mean scores ( 5.74 0.23 ) when compared to studies conducted in other countries such as croatia , turkey , and greece ( 6.62 1.54 , 6.53 1.99 , and 6.86 1.83 , respectively ) . countries such as britain , finland , and japan reported even higher hu - dbi scores among their dental students ( 7.33 , 7.15 1.13 , and 7.40 2.55 , respectively ) and oral health attitudes and behavior of dental students of kuhsc were more similar to that of dental students in jordan , india , and china ( 5.2 , 5.07 , and 6.06 respectively ) . this discrepancy maybe attributed to the lack of early exposure to oral health at younger age along with shortage from the government , represented by school health programs , to educate mothers , care givers , and young students to implant the basic knowledge of dental and oral health . therefore , the hu - dbi scores of all dental students is more likely to be attributed to the direct effects of the dental students curriculum . the dental students exhibiting the highest hu - dbi scores were the students in their clinical years of dental school , followed by the pre - clinical students and those in the basic sciences ( 6.10 1.46 , 5.93 1.72 , and 5.20 1.85 , respectively ) . the most improvement in the students knowledge occurred during the transition from the basic sciences to the pre - clinical level ( 5.20 1.85 to 5.93 1.72 , p < 0.05 ) . this is likely attributed to the fact that , as students progress in their education from the basic sciences to the pre - clinical level , they become more cognizant of their own oral health practices in preparation for their clinical years at the faculty of dentistry . there was less improvement in the oral health attitudes and behavior in students transitioning from their pre - clinical to clinical years ( 5.93 1.72 and 6.10 1.46 ) . dental students in their clinical years are offered courses that heavily emphasize oral health maintenance and disease prevention in advanced courses such as periodontology and dental public health . this finding suggests that an improvement in the coursework offered to students in their clinical years may be warranted because the dental students in their clinical years did not demonstrate much of an improvement in their oral health attitudes and behavior from their pre - clinical years when compared to the improvement seen in students from basic science to pre - clinical years . higher means were found among dental and medical students within the cognitive effects and oral health and maintenance constructs when compared to the pharmacy and allied health students , which may be attributed to the dental and medical curricula . the results of this study indicate that female students had better overall hu - dbi scores , and therefore , better oral health attitudes and behavior than male students . previous studies have also reported higher hu - dbi scores for female students than male students , confirming that there is a significant relationship between gender and oral health attitude and behavior . al - ansari and honkala have found that female students practiced better oral hygiene and possessed more knowledge about oral health maintenance than male students . a limitation of this study is the presence of diverse educational backgrounds among the students at kuhsc that may have led to variations in the students oral health maintenance practices . students in higher academic levels tend to have higher hu - dbi scores , representing an increase in knowledge and improvement in oral health attitudes and behavior as students progress in their training . female students were more likely to demonstrate better oral health practices and perceptions than male students . dental students demonstrated the highest hu - dbi scores when compared to students enrolled in nondental faculties , exhibiting better oral health attitudes and behavior than their nondental colleagues . therefore , additional studies investigating the complex interplay of knowledge , attitude , and behavior of is indispensable .

guideline - based management results in significant improvement in health - related quality of life in most patients.1 although the goal of therapy is to control asthma by reducing impairment and risk , many patients seem to continue to have some asthma symptoms , such as cough , phlegm , or dyspnea , when asked about their symptoms on a questionnaire.2 some important complications that occur with asthma , such as rhinitis,3 sinusitis,4,5 and gastroesophageal reflux disease ( gerd),6 are known to affect asthma symptoms . these complications may make bronchial inflammation worse , because asthma symptoms are mainly caused by airway inflammation . whether these complications directly affect the intensity of bronchial inflammation continues to be an unanswered question . the frequency scale for the symptoms of gerd ( f scale ) is the standard questionnaire used in japan for the diagnosis of gerd and assessment of the response to treatment.7 recently , the f scale was modified by adding two questions on interdigestive and postprandial epigastric pain.8 we imitated the f scale and developed a new questionnaire , the frequency scale for the symptoms of asthma and rhinosinusitis developed in gunma ( g scale ) , to assess the symptoms of asthma and rhinosinusitis in adult patients with asthma . the relationships between asthma symptoms and the symptoms that come from the complications were investigated using both the modified f scale and the g scale . measurement of fractional exhaled nitric oxide ( feno ) , a surrogate marker of eosinophilic airway inflammation,9 is slowly becoming part of the routine clinical evaluation of asthmatic patients in japan . in this study , whether the symptoms of rhinosinusitis or upper abdominal symptoms were related to eosinophilic airway inflammation , and which symptoms in asthmatic patients were related to eosinophilic inflammation during treatment for asthma , were also investigated . a total of 252 patients diagnosed as having asthma by medical specialists ( certified by either the japanese respiratory society or the japanese allergology society ) participated in this prospective , observational study to investigate the correlations between symptoms and eosinophilic airway inflammation . their written , informed consent , the patients answered two kinds of questionnaire : the modified f scale and the g scale ( table 1 ) . in developing the g scale , the intention was not to use it for the diagnosis of asthma or rhinosinusitis . the goal was to evaluate chronic rhinosinusitis ; but symptoms of acute rhinosinusitis and those of chronic rhinosinusitis can not be distinguished using the g scale . complications associated with asthma , such as rhinosinusitis , gerd , or dyspepsia , were not diagnosed by objective findings in all patients in the present study , although some patients were diagnosed , based on the findings of endoscopy or computerized tomography ( ct ) , as part of their routine medical care . feno was measured in all participants , using the niox mino ( aerocrine ab , solna , sweden ) , according to the manufacturer s instructions . of the 252 patients , 2 were found not to have asthma , by a physician in charge , after they had provided their consent . two patients with asthma , who agreed to participate in this observational study , did not take any medicine for asthma , because their asthmatic symptoms were stable without treatment . therefore , the data obtained from 248 patients who took controller medications for asthma were analyzed in this prospective , observational study . these 248 patients ( 97 males , 151 females ; age range : 1688 years ; mean standard deviation ( sd ) : 58.616.3 years ) received sufficient treatment for asthma . treatments for rhinorrhea , sneezing , nasal obstruction , olfactory dysfunction , or upper abdominal symptoms were prescribed at the physician s discretion . of the 248 patients , 246 had used inhaled corticosteroids ( ics ) : 92 used low - dose ics , 106 used medium - dose ics , and 48 used high - dose ics . in addition , 50 patients were treated according to treatment step 2 of the global initiative for asthma ( gina ) guidelines , 47 patients were treated according to treatment step 3 , 120 were treated according to treatment step 4 , and 31 were treated according to treatment step 5 . long - acting beta - agonists ( laba ) were used by 170 patients , leukotriene receptor antagonists were used by 86 patients , sustained release theophylline was used by 44 patients , and oral corticosteroids were used by 31 patients for asthma control . proton pump inhibitors ( ppis ) were used by 61 patients , and 6 patients took histamine h2 blockers . forty - eight patients took histamine h1 blockers , and 31 patients used nasal corticosteroids . this prospective , observational study ( umin 000007762 ) was approved by the ethics committees of gunma university faculty of medical science ( maebashi , japan ) , national numata hospital ( numata , japan ) , maebashi kyoritsu hospital ( maebashi , japan ) , and heisei hidaka clinic ( takasaki , japan ) . this study was started on april 10 , 2012 and completed on november 30 , 2012 . the retrospective analysis for the relationship between the asthma control test ( act ) and the g scale was performed as follows . both the act and the g scale had been used at the same time in the daily medical treatment of outpatients with asthma since october 2012 . the data on the act and the g scale obtained from 128 patients ( 42 males , 86 females ; age range : 2288 years ; mean sd : 60.316.9 years ) who had been treated by controller medications for asthma from october 2012 to december 2013 ( in gunma university hospital , heisei hidaka clinic , maebashi kyoritsu hospital , university of fukui hospital , and fukui sogo clinic ) , were analyzed retrospectively . of these 128 patients , 125 had used ics ( 53 patients had used low - dose ics , 37 had used medium - dose ics , and 35 had used high - dose ics ) . labas were used by 104 patients , leukotriene receptor antagonists by 40 patients , sustained release theophylline by 22 patients , and oral corticosteroids by 24 patients , for asthma control . in addition , 20 patients were treated according to treatment step 2 in the gina guidelines , 31 according to treatment step 3 , 53 according to treatment step 4 , and 24 according to treatment step 5 . ppis were used by 30 patients , and 1 patient took a histamine h2 blocker . thirty - one patients took histamine h1 blockers , and nasal corticosteroids were used by 22 patients . when data were repeatedly obtained from the same patient , the latest data were used in the analysis . the data were analyzed using spss software ( ibm japan , tokyo , japan ) . the differences between the mean feno values of two categorical independent groups were determined using an unpaired t - test ( student s t - test or welch s t - test ) , after the equality of variances was assessed by levene s test . correlations of two variables were calculated using spearman s rank correlation coefficient , because the variables did not show a normal distribution and some of them were discrete . multiple regression analysis was performed to determine the multiple regression function , and the standardized partial regression coefficients were calculated to compare the strengths of two factors that affected a third factor . a total of 252 patients diagnosed as having asthma by medical specialists ( certified by either the japanese respiratory society or the japanese allergology society ) participated in this prospective , observational study to investigate the correlations between symptoms and eosinophilic airway inflammation . their written , informed consent , the patients answered two kinds of questionnaire : the modified f scale and the g scale ( table 1 ) . in developing the g scale , the intention was not to use it for the diagnosis of asthma or rhinosinusitis . the goal was to evaluate chronic rhinosinusitis ; but symptoms of acute rhinosinusitis and those of chronic rhinosinusitis can not be distinguished using the g scale . complications associated with asthma , such as rhinosinusitis , gerd , or dyspepsia , were not diagnosed by objective findings in all patients in the present study , although some patients were diagnosed , based on the findings of endoscopy or computerized tomography ( ct ) , as part of their routine medical care . feno was measured in all participants , using the niox mino ( aerocrine ab , solna , sweden ) , according to the manufacturer s instructions . of the 252 patients , 2 were found not to have asthma , by a physician in charge , after they had provided their consent . two patients with asthma , who agreed to participate in this observational study , did not take any medicine for asthma , because their asthmatic symptoms were stable without treatment . therefore , the data obtained from 248 patients who took controller medications for asthma were analyzed in this prospective , observational study . these 248 patients ( 97 males , 151 females ; age range : 1688 years ; mean standard deviation ( sd ) : 58.616.3 years ) received sufficient treatment for asthma . treatments for rhinorrhea , sneezing , nasal obstruction , olfactory dysfunction , or upper abdominal symptoms were prescribed at the physician s discretion . of the 248 patients , 246 had used inhaled corticosteroids ( ics ) : 92 used low - dose ics , 106 used medium - dose ics , and 48 used high - dose ics . in addition , 50 patients were treated according to treatment step 2 of the global initiative for asthma ( gina ) guidelines , 47 patients were treated according to treatment step 3 , 120 were treated according to treatment step 4 , and 31 were treated according to treatment step 5 . long - acting beta - agonists ( laba ) were used by 170 patients , leukotriene receptor antagonists were used by 86 patients , sustained release theophylline was used by 44 patients , and oral corticosteroids were used by 31 patients for asthma control . proton pump inhibitors ( ppis ) were used by 61 patients , and 6 patients took histamine h2 blockers . forty - eight patients took histamine h1 blockers , and 31 patients used nasal corticosteroids . this prospective , observational study ( umin 000007762 ) was approved by the ethics committees of gunma university faculty of medical science ( maebashi , japan ) , national numata hospital ( numata , japan ) , maebashi kyoritsu hospital ( maebashi , japan ) , and heisei hidaka clinic ( takasaki , japan ) . this study was started on april 10 , 2012 and completed on november 30 , 2012 . the retrospective analysis for the relationship between the asthma control test ( act ) and the g scale was performed as follows . both the act and the g scale had been used at the same time in the daily medical treatment of outpatients with asthma since october 2012 . the data on the act and the g scale obtained from 128 patients ( 42 males , 86 females ; age range : 2288 years ; mean sd : 60.316.9 years ) who had been treated by controller medications for asthma from october 2012 to december 2013 ( in gunma university hospital , heisei hidaka clinic , maebashi kyoritsu hospital , university of fukui hospital , and fukui sogo clinic ) , were analyzed retrospectively . of these 128 patients , 125 had used ics ( 53 patients had used low - dose ics , 37 had used medium - dose ics , and 35 had used high - dose ics ) . labas were used by 104 patients , leukotriene receptor antagonists by 40 patients , sustained release theophylline by 22 patients , and oral corticosteroids by 24 patients , for asthma control . in addition , 20 patients were treated according to treatment step 2 in the gina guidelines , 31 according to treatment step 3 , 53 according to treatment step 4 , and 24 according to treatment step 5 . ppis were used by 30 patients , and 1 patient took a histamine h2 blocker . thirty - one patients took histamine h1 blockers , and nasal corticosteroids were used by 22 patients . when data were repeatedly obtained from the same patient , the latest data were used in the analysis . the data were analyzed using spss software ( ibm japan , tokyo , japan ) . the differences between the mean feno values of two categorical independent groups were determined using an unpaired t - test ( student s t - test or welch s t - test ) , after the equality of variances was assessed by levene s test . correlations of two variables were calculated using spearman s rank correlation coefficient , because the variables did not show a normal distribution and some of them were discrete . multiple regression analysis was performed to determine the multiple regression function , and the standardized partial regression coefficients were calculated to compare the strengths of two factors that affected a third factor . the data on the act and the g scale obtained from 128 asthmatic patients who received controller medication continuously were analyzed . the asthma symptoms score was calculated as the sum of the first eight questions on asthma symptoms of the g scale . the asthma symptoms score was strongly but negatively correlated with the act sum score ( figure 1a ) . act sum scores of 20 and 19 are useful for identifying patients with controlled and uncontrolled asthma , respectively . when an act cut - off point of 19 was established to screen for uncontrolled asthma , a cut - off point of 6 for the asthma symptoms score was suitable for identifying uncontrolled asthma . the sensitivity and specificity for identifying uncontrolled asthma using this cut - off value were 89.3% and 84.0% , respectively ( figure 1b ) . asthma was considered uncontrolled in 41 ( 32% ) of the 128 patients because their asthma symptoms scores were 6 or greater . the data on the g scale obtained from the 248 patients with asthma who participated were analyzed . the percentages of respondents who answered sometimes , often , or always for each question of the g scale were 28% ( g1 ) , 27% ( g2 ) , 25% ( g3 ) , 28% ( g4 ) , 15% ( g5 ) , 15% ( g6 ) , 13% ( g7 ) , 16% ( g8 ) , 31% ( g9 ) , 31% ( g10 ) , 29% ( g11 ) , and 25% ( g12 ) . the frequencies of cough and phlegm were higher than those of wheeze and difficulty breathing . g3 and g4 did not refer to the features of phlegm , although it was important to explore the cause of phlegm . the frequencies of symptoms of rhinosinusitis ( g9g12 ) were relatively higher than those of symptoms of asthma ( g1g8 ) . the question to which patients most often answered always ( 14% ) was the question on losing the sense of smell ( g12 ) . a diagnosis of allergic rhinitis or sinusitis can not be made using the g scale . although post nasal drip was also one of the important findings in patients with sinusitis , there was no question that referred directly to post nasal drip , because the g scale consisted of patients subjective symptoms . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g3 , the question on daytime phlegm ( 47.036.6 parts per billion [ ppb ] , n=104 ) , than in patients who answered never the frequency of losing the sense of smell was also significantly correlated with feno ( figure 2c ) . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g12 , the question on losing the sense of smell ( 50.840.1 ppb , n=84 ) , than in patients who answered never although the frequency of losing the sense of smell was well correlated with feno , the frequencies of runny nose , sneezing , and nasal congestion were not ( data not shown ) . in contrast , the scores for runny nose , sneezing , or nasal congestion , but not that for losing the sense of smell , were significantly correlated with the asthma symptoms score ( figure 3a d ) . the sum scores for upper abdominal symptoms ( upper abdominal score ) , gerd symptoms ( gerd score ) , and dyspepsia symptoms ( dyspepsia score ) were calculated using the modified f scale.8 specifically , the upper abdominal score was calculated as the sum of 14 questions of the modified f scale . the gerd score or dyspepsia score was calculated as the sum of seven questions , each on gerd symptoms or dyspepsia symptoms . the upper abdominal score , gerd score , and dyspepsia score were well correlated with the asthma symptoms score ( figure 4a c ) . although symptoms of both gerd and dyspepsia seemed to affect asthma symptoms , gerd symptoms had a greater contribution to asthma symptoms than dyspepsia symptoms . the multiple regression function was as follows : asthma symptoms score=0.790gerd score+0.259dyspepsia score+2.924 the standardized partial regression coefficients were 0.419 for gerd symptoms and the upper abdominal score and the dyspepsia score were negatively correlated with feno ( figure 5a and c ) . the gerd score also tended to be negatively correlated with feno , though this correlation was not significant ( p=0.097 ) ( figure 5b ) . the data on the act and the g scale obtained from 128 asthmatic patients who received controller medication continuously were analyzed . the asthma symptoms score was calculated as the sum of the first eight questions on asthma symptoms of the g scale . the asthma symptoms score was strongly but negatively correlated with the act sum score ( figure 1a ) . act sum scores of 20 and 19 are useful for identifying patients with controlled and uncontrolled asthma , respectively . when an act cut - off point of 19 was established to screen for uncontrolled asthma , a cut - off point of 6 for the asthma symptoms score was suitable for identifying uncontrolled asthma . the sensitivity and specificity for identifying uncontrolled asthma using this cut - off value were 89.3% and 84.0% , respectively ( figure 1b ) . asthma was considered uncontrolled in 41 ( 32% ) of the 128 patients because their asthma symptoms scores were 6 or greater . the data on the g scale obtained from the 248 patients with asthma who participated were analyzed . the percentages of respondents who answered sometimes , often , or always for each question of the g scale were 28% ( g1 ) , 27% ( g2 ) , 25% ( g3 ) , 28% ( g4 ) , 15% ( g5 ) , 15% ( g6 ) , 13% ( g7 ) , 16% ( g8 ) , 31% ( g9 ) , 31% ( g10 ) , 29% ( g11 ) , and 25% ( g12 ) . the frequencies of cough and phlegm were higher than those of wheeze and difficulty breathing . g3 and g4 did not refer to the features of phlegm , although it was important to explore the cause of phlegm . the frequencies of symptoms of rhinosinusitis ( g9g12 ) were relatively higher than those of symptoms of asthma ( g1g8 ) . the question to which patients most often answered always ( 14% ) was the question on losing the sense of smell ( g12 ) . a diagnosis of allergic rhinitis or sinusitis can not be made using the g scale . although post nasal drip was also one of the important findings in patients with sinusitis , there was no question that referred directly to post nasal drip , because the g scale consisted of patients subjective symptoms . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g3 , the question on daytime phlegm ( 47.036.6 parts per billion [ ppb ] , n=104 ) , than in patients who answered never the frequency of losing the sense of smell was also significantly correlated with feno ( figure 2c ) . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g12 , the question on losing the sense of smell ( 50.840.1 ppb , n=84 ) , than in patients who answered never although the frequency of losing the sense of smell was well correlated with feno , the frequencies of runny nose , sneezing , and nasal congestion were not ( data not shown ) . in contrast , the scores for runny nose , sneezing , or nasal congestion , but not that for losing the sense of smell , were significantly correlated with the asthma symptoms score ( figure 3a d ) . the sum scores for upper abdominal symptoms ( upper abdominal score ) , gerd symptoms ( gerd score ) , and dyspepsia symptoms ( dyspepsia score ) were calculated using the modified f scale.8 specifically , the upper abdominal score was calculated as the sum of 14 questions of the modified f scale . the gerd score or dyspepsia score was calculated as the sum of seven questions , each on gerd symptoms or dyspepsia symptoms . the upper abdominal score , gerd score , and dyspepsia score were well correlated with the asthma symptoms score ( figure 4a c ) . although symptoms of both gerd and dyspepsia seemed to affect asthma symptoms , gerd symptoms had a greater contribution to asthma symptoms than dyspepsia symptoms . the multiple regression function was as follows : asthma symptoms score=0.790gerd score+0.259dyspepsia score+2.924 the standardized partial regression coefficients were 0.419 for gerd symptoms and the upper abdominal score and the dyspepsia score were negatively correlated with feno ( figure 5a and c ) . the gerd score also tended to be negatively correlated with feno , though this correlation was not significant ( p=0.097 ) ( figure 5b ) . asthma control is assessed by daytime symptoms , limitation of activities , nocturnal symptoms / awakening , need for reliever / rescue treatment , and lung function . several composite control measures , such as act10 and the asthma control questionnaire,11 have been developed and are being validated . we have developed a new questionnaire , the g scale , to evaluate the symptoms of rhinosinusitis , as well as the symptoms of asthma , in asthmatic patients . unexpectedly , asthma symptoms were not well controlled in about one - third of patients , despite taking controller medications . this result suggests that asthma symptoms might be underestimated by doctors in patients treated continuously . evaluation of asthma control and rhinosinusitis symptoms using the g scale was considered not always superior to a combination of act with the sacra questionnaire , which was designed to reflect asthma control and the condition of allergic rhinitis . however , the g scale might be convenient for screening the concomitant sinusitis , because it contains a question on olfactory dysfunction , which suggests the presence of sinusitis . certainly , imaging with ct or magnetic resonance imaging ( mri ) is much more useful for an accurate and objective diagnosis of sinusitis . it is known that the prevalence of allergic rhinitis as a complication of asthma is 67.3% , and the percentage of patients whose asthma is well controlled is lower in those with allergic rhinitis than those without allergic rhinitis , suggesting that allergic rhinitis may negatively affect asthma control or asthma symptoms.3 the results of the present study , based on patients reports of symptom frequencies , support this theory , because the symptoms of rhinitis , such as runny nose , sneezing , and nasal congestion were well correlated with asthma symptoms , such as cough , phlegm , wheeze , and difficulty breathing . the presence of sinusitis , as well as rhinitis , in adult asthmatic patients is a very serious problem , because rhinosinusitis , in many cases of adult asthma , is expected to be eosinophilic chronic rhinosinusitis , which is characterized by bilateral nasal polyps , ethmoidal antrum - dominant lesions , and losing the sense of smell.12 this type of rhinosinusitis , which occurs mainly in adults , is resistant to macrolide therapy , and is sometimes accompanied by sinus and peripheral blood eosinophilia.13,14 according to the retrospective data on feno measured in gunma university hospital , by the american thoracic society / european respiratory society - recommended online method , feno levels in 12 patients , who complained of olfactory dysfunction and who showed abnormal shadows in bilateral ethmoid sinuses on ct or mri , were higher than those of asthmatic patients who had not complained of olfactory dysfunction , although both groups had been treated by controller medications for asthma ( 74.535.4 ppb versus 37.324.9 ppb ; n=12 versus n=58 ) . this observation motivated us to develop the g scale containing the question on olfactory dysfunction . as expected , feno was significantly more elevated in asthmatic patients with olfactory dysfunction than in other asthmatic patients , even if their asthma had been treated adequately by standard therapy . because the diagnosis of rhinosinusitis was not made by ct , mri imaging , or rhinoscopy in the present study , one can only conclude that olfactory dysfunction , as one of the subjective symptoms , was related to feno levels . although olfactory dysfunction is a representative symptom of eosinophilic sinusitis or nasal polyps , this symptom is not specific for eosinophilic sinusitis or nasal polyps . among the symptoms of asthma , interleukin 13 , a pleiotropic th2 cytokine that has been shown to be central to the pathogenesis of asthma , induces goblet cell hyperplasia and increased mucus secretion.15 it also induces nitric oxide synthase in bronchial epithelium,16 resulting in increased breath no levels . although increased airway secretion is not a specific phenomenon for th2-dominant or eosinophilic inflammation , persistent hypersecretion resistant to pharmacotherapy in asthmatic patients may be a key sign that suggests intense eosinophilic inflammation remaining in the lower airways . epidemiologic evidence suggests that 30%90% of asthmatic patients have gerd,17,18 and respiratory symptoms associated with asthma are increased among patients with gerd.19 it is suggested that esophageal acid may produce bronchoconstriction and , therefore , exacerbate airflow obstruction in asthmatic patients.2022 however , the impact of gerd therapy on objective outcome measures of asthma control has been variable.2326 in patients with no evidence of organic disease , the modified f scale was useful to distinguish functional dyspepsia from nonerosive reflux disease , and to assess dyspeptic symptoms.8 dyspepsia is defined as one or more of the following symptoms : postprandial fullness , early satiation , and epigastric pain or burning.27 up to 75% of patients have functional dyspepsia with no underlying cause on diagnostic evaluation.2830 upper abdominal symptoms , including both gerd and dyspepsia , were well correlated with the degree of asthma symptoms , without enhancing the intensity of eosinophilic inflammation . on the other hand , the coexistence of upper abdominal symptoms , especially dyspepsia , may suggest decreased eosinophilic inflammation . the presence of gerd contributes more to respiratory symptoms associated with asthma than does dyspepsia , although the coexistence of gerd itself never makes eosinophilic inflammation worse . in this study , the goal was to investigate only whether upper abdominal symptoms or rhinosinusitis symptoms affect eosinophilic inflammation of the lower respiratory tract in asthmatic patients sufficiently treated by standard asthma therapy . the present results do not necessarily suggest that some factors of the g scale , which do not affect feno levels , do not reflect airway inflammation of asthma . the possibility that some factors of the g scale or modified f scale may affect airway inflammation , especially noneosinophilic inflammation , under sufficient treatment for asthma , was not ruled out . the presence of rhinitis and gerd may affect asthma control independent of eosinophilic inflammation , and loss of smell or persistent daytime sputum may suggest persistent eosinophilic inflammation in asthmatic patients taking guideline - recommended therapy .

backgroundlosing the sense of smell , which suggests eosinophilic rhinosinusitis , is a subjective symptom , sometimes reported in asthmatic patients taking controller medication . upper abdominal symptoms , suggesting gastroesophageal reflux disease ( gerd ) or functional dyspepsia , occur also in these patients . however , the relationship between these symptoms , concomitant with asthma , and the intensity of eosinophilic airway inflammation remains obscure.objectiveto assess the symptoms of asthma and rhinosinusitis , and to examine the relationship between the symptoms and bronchial inflammation , a new questionnaire , the g scale , was developed . to investigate the effects of gerd , dyspepsia , and rhinosinusitis on asthma symptoms and bronchial inflammation , the symptoms of asthma and rhinosinusitis obtained by the g scale , upper abdominal symptoms obtained by the modified f scale , a questionnaire for gerd and dyspepsia , and fractional exhaled nitric oxide ( feno ) were analyzed.methodsa prospective , observational study was performed in four hospitals in gunma prefecture , and a retrospective analysis was done using data obtained from five hospitals in gunma prefecture and fukui prefecture , japan . a total of 252 patients diagnosed as having asthma participated in the prospective study.resultsthe frequency of daytime phlegm or losing the sense of smell had a positive correlation with feno levels in asthmatic patients taking controller medication . upper abdominal symptoms , as well as symptoms suggesting rhinitis , were well correlated with asthma symptoms . however , neither upper abdominal symptoms nor rhinitis symptoms increased feno levels , which reflect eosinophilic airway inflammation during treatment for asthma . on the other hand , the degree of upper abdominal symptoms or dyspepsia symptoms had a weak but significant negative correlation with feno levels.conclusiondaytime phlegm and losing the sense of smell suggest that eosinophilic airway inflammation persists , despite anti - inflammatory therapy , in patients with asthma . although rhinitis and gerd made the subjective symptoms of asthma worse , they did not seem to enhance eosinophilic airway inflammation .

Introduction Methods Study design and patients Statistical analysis Results The relationship between the asthma symptoms score in the G scale and ACT sum score G scale score in asthmatic patients continuously treated by controller medications The relationship between FeNO and symptoms The relationships between upper abdominal symptoms, GERD symptoms, dyspepsia symptoms, and asthma symptoms The relationships between upper abdominal symptoms, GERD symptoms, dyspepsia symptoms, and FeNO Discussion Conclusion

guideline - based management results in significant improvement in health - related quality of life in most patients.1 although the goal of therapy is to control asthma by reducing impairment and risk , many patients seem to continue to have some asthma symptoms , such as cough , phlegm , or dyspnea , when asked about their symptoms on a questionnaire.2 some important complications that occur with asthma , such as rhinitis,3 sinusitis,4,5 and gastroesophageal reflux disease ( gerd),6 are known to affect asthma symptoms . these complications may make bronchial inflammation worse , because asthma symptoms are mainly caused by airway inflammation . whether these complications directly affect the intensity of bronchial inflammation continues to be an unanswered question . the frequency scale for the symptoms of gerd ( f scale ) is the standard questionnaire used in japan for the diagnosis of gerd and assessment of the response to treatment.7 recently , the f scale was modified by adding two questions on interdigestive and postprandial epigastric pain.8 we imitated the f scale and developed a new questionnaire , the frequency scale for the symptoms of asthma and rhinosinusitis developed in gunma ( g scale ) , to assess the symptoms of asthma and rhinosinusitis in adult patients with asthma . the relationships between asthma symptoms and the symptoms that come from the complications were investigated using both the modified f scale and the g scale . measurement of fractional exhaled nitric oxide ( feno ) , a surrogate marker of eosinophilic airway inflammation,9 is slowly becoming part of the routine clinical evaluation of asthmatic patients in japan . in this study , whether the symptoms of rhinosinusitis or upper abdominal symptoms were related to eosinophilic airway inflammation , and which symptoms in asthmatic patients were related to eosinophilic inflammation during treatment for asthma , were also investigated . a total of 252 patients diagnosed as having asthma by medical specialists ( certified by either the japanese respiratory society or the japanese allergology society ) participated in this prospective , observational study to investigate the correlations between symptoms and eosinophilic airway inflammation . their written , informed consent , the patients answered two kinds of questionnaire : the modified f scale and the g scale ( table 1 ) . in developing the g scale , the intention was not to use it for the diagnosis of asthma or rhinosinusitis . the goal was to evaluate chronic rhinosinusitis ; but symptoms of acute rhinosinusitis and those of chronic rhinosinusitis can not be distinguished using the g scale . complications associated with asthma , such as rhinosinusitis , gerd , or dyspepsia , were not diagnosed by objective findings in all patients in the present study , although some patients were diagnosed , based on the findings of endoscopy or computerized tomography ( ct ) , as part of their routine medical care . two patients with asthma , who agreed to participate in this observational study , did not take any medicine for asthma , because their asthmatic symptoms were stable without treatment . therefore , the data obtained from 248 patients who took controller medications for asthma were analyzed in this prospective , observational study . this prospective , observational study ( umin 000007762 ) was approved by the ethics committees of gunma university faculty of medical science ( maebashi , japan ) , national numata hospital ( numata , japan ) , maebashi kyoritsu hospital ( maebashi , japan ) , and heisei hidaka clinic ( takasaki , japan ) . the retrospective analysis for the relationship between the asthma control test ( act ) and the g scale was performed as follows . both the act and the g scale had been used at the same time in the daily medical treatment of outpatients with asthma since october 2012 . the data on the act and the g scale obtained from 128 patients ( 42 males , 86 females ; age range : 2288 years ; mean sd : 60.316.9 years ) who had been treated by controller medications for asthma from october 2012 to december 2013 ( in gunma university hospital , heisei hidaka clinic , maebashi kyoritsu hospital , university of fukui hospital , and fukui sogo clinic ) , were analyzed retrospectively . multiple regression analysis was performed to determine the multiple regression function , and the standardized partial regression coefficients were calculated to compare the strengths of two factors that affected a third factor . a total of 252 patients diagnosed as having asthma by medical specialists ( certified by either the japanese respiratory society or the japanese allergology society ) participated in this prospective , observational study to investigate the correlations between symptoms and eosinophilic airway inflammation . their written , informed consent , the patients answered two kinds of questionnaire : the modified f scale and the g scale ( table 1 ) . in developing the g scale , the intention was not to use it for the diagnosis of asthma or rhinosinusitis . complications associated with asthma , such as rhinosinusitis , gerd , or dyspepsia , were not diagnosed by objective findings in all patients in the present study , although some patients were diagnosed , based on the findings of endoscopy or computerized tomography ( ct ) , as part of their routine medical care . two patients with asthma , who agreed to participate in this observational study , did not take any medicine for asthma , because their asthmatic symptoms were stable without treatment . therefore , the data obtained from 248 patients who took controller medications for asthma were analyzed in this prospective , observational study . long - acting beta - agonists ( laba ) were used by 170 patients , leukotriene receptor antagonists were used by 86 patients , sustained release theophylline was used by 44 patients , and oral corticosteroids were used by 31 patients for asthma control . this prospective , observational study ( umin 000007762 ) was approved by the ethics committees of gunma university faculty of medical science ( maebashi , japan ) , national numata hospital ( numata , japan ) , maebashi kyoritsu hospital ( maebashi , japan ) , and heisei hidaka clinic ( takasaki , japan ) . the retrospective analysis for the relationship between the asthma control test ( act ) and the g scale was performed as follows . both the act and the g scale had been used at the same time in the daily medical treatment of outpatients with asthma since october 2012 . the data on the act and the g scale obtained from 128 patients ( 42 males , 86 females ; age range : 2288 years ; mean sd : 60.316.9 years ) who had been treated by controller medications for asthma from october 2012 to december 2013 ( in gunma university hospital , heisei hidaka clinic , maebashi kyoritsu hospital , university of fukui hospital , and fukui sogo clinic ) , were analyzed retrospectively . when data were repeatedly obtained from the same patient , the latest data were used in the analysis . multiple regression analysis was performed to determine the multiple regression function , and the standardized partial regression coefficients were calculated to compare the strengths of two factors that affected a third factor . the data on the act and the g scale obtained from 128 asthmatic patients who received controller medication continuously were analyzed . the asthma symptoms score was calculated as the sum of the first eight questions on asthma symptoms of the g scale . when an act cut - off point of 19 was established to screen for uncontrolled asthma , a cut - off point of 6 for the asthma symptoms score was suitable for identifying uncontrolled asthma . the data on the g scale obtained from the 248 patients with asthma who participated were analyzed . the percentages of respondents who answered sometimes , often , or always for each question of the g scale were 28% ( g1 ) , 27% ( g2 ) , 25% ( g3 ) , 28% ( g4 ) , 15% ( g5 ) , 15% ( g6 ) , 13% ( g7 ) , 16% ( g8 ) , 31% ( g9 ) , 31% ( g10 ) , 29% ( g11 ) , and 25% ( g12 ) . the frequencies of symptoms of rhinosinusitis ( g9g12 ) were relatively higher than those of symptoms of asthma ( g1g8 ) . the question to which patients most often answered always ( 14% ) was the question on losing the sense of smell ( g12 ) . although post nasal drip was also one of the important findings in patients with sinusitis , there was no question that referred directly to post nasal drip , because the g scale consisted of patients subjective symptoms . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g3 , the question on daytime phlegm ( 47.036.6 parts per billion [ ppb ] , n=104 ) , than in patients who answered never the frequency of losing the sense of smell was also significantly correlated with feno ( figure 2c ) . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g12 , the question on losing the sense of smell ( 50.840.1 ppb , n=84 ) , than in patients who answered never although the frequency of losing the sense of smell was well correlated with feno , the frequencies of runny nose , sneezing , and nasal congestion were not ( data not shown ) . in contrast , the scores for runny nose , sneezing , or nasal congestion , but not that for losing the sense of smell , were significantly correlated with the asthma symptoms score ( figure 3a d ) . the sum scores for upper abdominal symptoms ( upper abdominal score ) , gerd symptoms ( gerd score ) , and dyspepsia symptoms ( dyspepsia score ) were calculated using the modified f scale.8 specifically , the upper abdominal score was calculated as the sum of 14 questions of the modified f scale . the gerd score or dyspepsia score was calculated as the sum of seven questions , each on gerd symptoms or dyspepsia symptoms . the upper abdominal score , gerd score , and dyspepsia score were well correlated with the asthma symptoms score ( figure 4a c ) . although symptoms of both gerd and dyspepsia seemed to affect asthma symptoms , gerd symptoms had a greater contribution to asthma symptoms than dyspepsia symptoms . the multiple regression function was as follows : asthma symptoms score=0.790gerd score+0.259dyspepsia score+2.924 the standardized partial regression coefficients were 0.419 for gerd symptoms and the upper abdominal score and the dyspepsia score were negatively correlated with feno ( figure 5a and c ) . the data on the act and the g scale obtained from 128 asthmatic patients who received controller medication continuously were analyzed . the asthma symptoms score was calculated as the sum of the first eight questions on asthma symptoms of the g scale . when an act cut - off point of 19 was established to screen for uncontrolled asthma , a cut - off point of 6 for the asthma symptoms score was suitable for identifying uncontrolled asthma . the data on the g scale obtained from the 248 patients with asthma who participated were analyzed . the frequencies of symptoms of rhinosinusitis ( g9g12 ) were relatively higher than those of symptoms of asthma ( g1g8 ) . the question to which patients most often answered always ( 14% ) was the question on losing the sense of smell ( g12 ) . although post nasal drip was also one of the important findings in patients with sinusitis , there was no question that referred directly to post nasal drip , because the g scale consisted of patients subjective symptoms . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g3 , the question on daytime phlegm ( 47.036.6 parts per billion [ ppb ] , n=104 ) , than in patients who answered never the frequency of losing the sense of smell was also significantly correlated with feno ( figure 2c ) . feno was significantly higher in patients who answered occasionally , sometimes , often , or always for g12 , the question on losing the sense of smell ( 50.840.1 ppb , n=84 ) , than in patients who answered never although the frequency of losing the sense of smell was well correlated with feno , the frequencies of runny nose , sneezing , and nasal congestion were not ( data not shown ) . in contrast , the scores for runny nose , sneezing , or nasal congestion , but not that for losing the sense of smell , were significantly correlated with the asthma symptoms score ( figure 3a d ) . the sum scores for upper abdominal symptoms ( upper abdominal score ) , gerd symptoms ( gerd score ) , and dyspepsia symptoms ( dyspepsia score ) were calculated using the modified f scale.8 specifically , the upper abdominal score was calculated as the sum of 14 questions of the modified f scale . the gerd score or dyspepsia score was calculated as the sum of seven questions , each on gerd symptoms or dyspepsia symptoms . the upper abdominal score , gerd score , and dyspepsia score were well correlated with the asthma symptoms score ( figure 4a c ) . although symptoms of both gerd and dyspepsia seemed to affect asthma symptoms , gerd symptoms had a greater contribution to asthma symptoms than dyspepsia symptoms . the multiple regression function was as follows : asthma symptoms score=0.790gerd score+0.259dyspepsia score+2.924 the standardized partial regression coefficients were 0.419 for gerd symptoms and the upper abdominal score and the dyspepsia score were negatively correlated with feno ( figure 5a and c ) . we have developed a new questionnaire , the g scale , to evaluate the symptoms of rhinosinusitis , as well as the symptoms of asthma , in asthmatic patients . evaluation of asthma control and rhinosinusitis symptoms using the g scale was considered not always superior to a combination of act with the sacra questionnaire , which was designed to reflect asthma control and the condition of allergic rhinitis . however , the g scale might be convenient for screening the concomitant sinusitis , because it contains a question on olfactory dysfunction , which suggests the presence of sinusitis . it is known that the prevalence of allergic rhinitis as a complication of asthma is 67.3% , and the percentage of patients whose asthma is well controlled is lower in those with allergic rhinitis than those without allergic rhinitis , suggesting that allergic rhinitis may negatively affect asthma control or asthma symptoms.3 the results of the present study , based on patients reports of symptom frequencies , support this theory , because the symptoms of rhinitis , such as runny nose , sneezing , and nasal congestion were well correlated with asthma symptoms , such as cough , phlegm , wheeze , and difficulty breathing . the presence of sinusitis , as well as rhinitis , in adult asthmatic patients is a very serious problem , because rhinosinusitis , in many cases of adult asthma , is expected to be eosinophilic chronic rhinosinusitis , which is characterized by bilateral nasal polyps , ethmoidal antrum - dominant lesions , and losing the sense of smell.12 this type of rhinosinusitis , which occurs mainly in adults , is resistant to macrolide therapy , and is sometimes accompanied by sinus and peripheral blood eosinophilia.13,14 according to the retrospective data on feno measured in gunma university hospital , by the american thoracic society / european respiratory society - recommended online method , feno levels in 12 patients , who complained of olfactory dysfunction and who showed abnormal shadows in bilateral ethmoid sinuses on ct or mri , were higher than those of asthmatic patients who had not complained of olfactory dysfunction , although both groups had been treated by controller medications for asthma ( 74.535.4 ppb versus 37.324.9 ppb ; n=12 versus n=58 ) . as expected , feno was significantly more elevated in asthmatic patients with olfactory dysfunction than in other asthmatic patients , even if their asthma had been treated adequately by standard therapy . because the diagnosis of rhinosinusitis was not made by ct , mri imaging , or rhinoscopy in the present study , one can only conclude that olfactory dysfunction , as one of the subjective symptoms , was related to feno levels . among the symptoms of asthma , interleukin 13 , a pleiotropic th2 cytokine that has been shown to be central to the pathogenesis of asthma , induces goblet cell hyperplasia and increased mucus secretion.15 it also induces nitric oxide synthase in bronchial epithelium,16 resulting in increased breath no levels . although increased airway secretion is not a specific phenomenon for th2-dominant or eosinophilic inflammation , persistent hypersecretion resistant to pharmacotherapy in asthmatic patients may be a key sign that suggests intense eosinophilic inflammation remaining in the lower airways . epidemiologic evidence suggests that 30%90% of asthmatic patients have gerd,17,18 and respiratory symptoms associated with asthma are increased among patients with gerd.19 it is suggested that esophageal acid may produce bronchoconstriction and , therefore , exacerbate airflow obstruction in asthmatic patients.2022 however , the impact of gerd therapy on objective outcome measures of asthma control has been variable.2326 in patients with no evidence of organic disease , the modified f scale was useful to distinguish functional dyspepsia from nonerosive reflux disease , and to assess dyspeptic symptoms.8 dyspepsia is defined as one or more of the following symptoms : postprandial fullness , early satiation , and epigastric pain or burning.27 up to 75% of patients have functional dyspepsia with no underlying cause on diagnostic evaluation.2830 upper abdominal symptoms , including both gerd and dyspepsia , were well correlated with the degree of asthma symptoms , without enhancing the intensity of eosinophilic inflammation . on the other hand , the coexistence of upper abdominal symptoms , especially dyspepsia , may suggest decreased eosinophilic inflammation . the presence of gerd contributes more to respiratory symptoms associated with asthma than does dyspepsia , although the coexistence of gerd itself never makes eosinophilic inflammation worse . in this study , the goal was to investigate only whether upper abdominal symptoms or rhinosinusitis symptoms affect eosinophilic inflammation of the lower respiratory tract in asthmatic patients sufficiently treated by standard asthma therapy . the present results do not necessarily suggest that some factors of the g scale , which do not affect feno levels , do not reflect airway inflammation of asthma . the possibility that some factors of the g scale or modified f scale may affect airway inflammation , especially noneosinophilic inflammation , under sufficient treatment for asthma , was not ruled out . the presence of rhinitis and gerd may affect asthma control independent of eosinophilic inflammation , and loss of smell or persistent daytime sputum may suggest persistent eosinophilic inflammation in asthmatic patients taking guideline - recommended therapy .

colon cancer is the second leading cause of cancer deaths among men and women combined in the united states , australia , and europe and is a major problem in many other countries . a typical western diet , high in fat and low in fiber , has been shown to contribute to the development of colon cancer in epidemiologic and animal studies . bile acids / salts , present in high concentration in the feces of patients on a high fat / low fiber diet , have been associated with colon cancer risk . the most common bile acid present in the human feces is deoxycholic acid ( doc ) , a hydrophobic bile acid . doc is a promoter of colon cancer , and also a genotoxic carcinogen [ 68 ] , and may be responsible for initiating gastrointestinal cancers ( reviewed by bernstein et al . ) . however , the mechanism by which hydrophobic bile acids act in progression to colon cancer is unclear . hydrophobic bile acids are known inducers of at least five stress - response pathways in gastrointestinal cells , including er stress , oxidative stress [ 6 , 1113 ] , nitrosative stress [ 14 , 15 ] , mitochondrial stress [ 1013 , 16 ] , and dna damage [ 6 , 1719 ] . some of these bile acid - induced cellular stresses may ultimately lead to cell death by mechanisms that include both apoptosis [ 20 , 21 ] and necrosis . in addition , they may act as carcinogens [ 6 , 7 ] and/or select for outgrowth of clones of mutant cells resistant to bile acid - induced cell death . one of the cell survival pathways activated in response to bile acid exposure is the nf-b stress - response pathway [ 11 , 20 , 22 , 23 ] . persistent activation of nf-b causes cells to become apoptosis - resistant , and such cells tend to acquire mutations , some of which may contribute to colon cancer . autophagy ( greek for the eating of oneself ) is an evolutionarily conserved lysosomal pathway that allows eukaryotic cells ( yeast to mammals ) to survive under nutrient starvation conditions [ 2426 ] . macroautophagy ( herein referred to as autophagy ) involves the bulk lysosomal turnover of long - lived proteins , protein aggregates , and organelles such as damaged mitochondria , damaged endoplasmic reticulum ( er ) , and ribosomes . the autophagic process occurs in several stages that begin with the formation of a crescent - shaped isolation membrane ( phagophore ) that sequesters organelles , matures into an autophagosome that surrounds the organelle , followed by the fusion of the autophagosome with a lysosome to form an autophagolysosome [ 30 , 31 ] . hydrolytic enzymes within the acid ph of the interior of the autophagolysosome , then act to degrade macromolecules , thereby providing nutrients for the survival of the eukaryotic cell . an analysis of this morphologic process at the molecular level reveals a complex series of biochemical events involving the products of numerous autophagy - related genes [ 24 , 26 , 3235 ] . the autophagy pathway is becoming increasingly recognized as an important mechanism of tumor cell survival and drug resistance in cancer chemotherapy [ 3638 ] . a recent study indicated that autophagy is activated in colorectal cancer cells and contributes to tolerance to nutrient deprivation . we now show , for the first time , that deoxycholate ( doc ) activates the autophagic pathway in noncancer colon epithelial cells ( ncm-460 ) , and that this activation contributes to cell survival . we also show that the constitutive activation of autophagy contributes to the survival of apoptosis - resistant colon cancer cells ( hct-116rc ) that were developed in our laboratory by repeated exposure to increasing concentrations of doc . the present findings , coupled with findings from our in vivo animal model of deoxycholate - induced colonic inflammation and from our in vitro apoptosis - resistant colon cancer cell lines , implicate autophagy in colon carcinogenesis and suggest an additional mechanism by which hydrophobic bile acids contribute to colon carcinogenesis . these studies may aid in the identification of potential biomarkers of the autophagy pathway in the nonneoplastic colonic mucosa of patients at risk for colon cancer . in addition , combining inhibitors of autophagy with chemotherapeutic agents commonly used to treat colon cancer may lead to an improved clinical outcome . sodium deoxycholate ( doc ) , 3-methyladenine ( 3-ma ) , rapamycin ( rapa ) ( derived from streptomyces hygroscopicus ) , cudips [ copper(ii ) 3,5-diisopropylsalicylate hydrate ) , and catalase ( 10 00040 000 u / mg protein ) were obtained from sigma - aldrich ( st . louis , mo , usa ) . bafilomycin a1 , mntbap [ mn ( iii ) tetrakis ( 4-benzoic acid ) porphyrin chloride ] , hbed [ n , n-di-(2-hydroxybenzyl)ethylenediamine - n , n-diacetic acid ) ] , pepstatin a , and e-64d were from biomol research laboratories ( plymouth meeting , pa , usa ) , hydroxychloroquine sulfate ( hcs ) was from acros organics ( morris plains , nj ) , and trypan blue was from gibco brl life technologies ( grand island , ny , usa ) . the concentrations used to modulate autophagy and cell death in the cell culture experiments are provided below . in all instances , the specific concentrations that were reported in the literature were tested for their effect on the viability of ncm-460 and hct-116rc cells used in the present study . in some instances dose - response curves that assess the concentration of the drug and its effect on cell viability were performed to ensure that the concentration of the chemical itself was not too toxic to be used in the proposed experiments . this was especially important for 3-ma , which is used at a wide range of millimolar concentrations in published reports . the three antioxidants ( hbed , mntbap , cudips ) used to evaluate the doc - induced increase in beclin-1 expression were used at the same concentration ( 100 m ) so that direct comparisons can be made . the concentration chosen was well within the range of concentrations reported in the literature for use with cultured cells ( see cited articles below ) , and this concentration did not induce any cytotoxicity in the sensitive ncm-460 cells . cudips : 100 m ; bafilomycin a1 : 1 nm [ 41 , 42 ] ; catalase : 1000 u / mg ; e-64d : 10 g / ml ; 3-ma : 4 mm ; hbed : 100 m [ 44 , 45 ] ; hcs : 10 m ; mntbap : 100 m ; pepstatin a : 10 g / ml ; rapa : 100 m . the ncm-460 colon cell line was obtained from incell corporation , llc ( san antonio , tex , usa ) and cultured in m3:10tm medium to insure that it maintains its characteristic features . this cell line was not obtained from a colon cancer and is considered to be a noncancerous colonic epithelial cell line . hct-116rc is a stable apoptosis - resistant colon cancer cell line that was developed in our laboratory after persistent exposure to increasing concentrations of nadoc . this cell line was maintained in dmem media supplemented with 10% heat - inactivated fetal calf serum ( omega scientific , tarzana , calif , usa ) , 1% mem nonessential amino acids , 100 g / ml streptomycin , 100 u / ml penicillin , and 3.44 mg / ml l - glutamine . media components were from gibco brl life technologies ( grand island , ny , usa ) . all treatments utilizing ncm-460 cells were performed with the cells in approximate mid - logarithmic phase to avoid inconsistencies in cellular responses based on growth phase , as previously described . starvation experiments were performed by incubation cells in hank 's balanced salt solution ( hbss ) , as previously described for colon epithelial cells . hbss was obtained from gibco / invitrogen corp . , carlsbad , calif , usa . ncm460 cells were plated on a fibronectin - coated costar ( fisher scientific , pittsburg , pa , usa ) 96 well black plate at 2 10 cells / ml . after treatments , cells were spun in an allegra 6r centrifuge ( beckman coulter , fullerton , calif , usa ) at 1000 rpm for 5 minutes and the fluorescent dyes added as described below . triplicate wells were plated for each experimental protocol , and the intensity values were normalized for each well using a nucleic acid stain . mean fold changes in intensity values ( doc versus control untreated cells ) were statistically compared using student 's t - test . louis , mo , usa ) was used at a final concentration of 200 m , and cells were incubated for 30 minutes at 37c . mdc was removed and sytox green ( molecular probes / invitrogen , carlsbad , calif , usa ) was added at a final concentration of 2.5 m ; cells were incubated for 20 minutes at room temperature . fluorescence was assessed immediately using an optima fluostar plate reader ( bmg labtech , durham , nc , usa ) . mdc ( excitation 335 nm ; emission 508 nm ) and sytox green ( excitation 504 nm ; emission 523 nm ) fluorescence were assessed using appropriate filters . all fluorescence values were normalized to the amount of cells in individual wells by obtaining the ratio of mdc fluorescence ( assessment of acid vesicles ) to sytox green fluorescence ( nucleic acid stain ) . lysotracker red ( molecular probes / invitrogen , carlsbad , calif , usa ) was added to the cells at a final concentration of 100 nm and incubated for 30 minutes at 37c . lysotracker red was removed and hoechst # 33342 dye ( molecular probes / invitrogen , carlsbad , calif , usa ) , made up in tissue culture media at a final concentration of 10 g / ml , was added to the cells and incubated for 10 minutes at 37c . cells were spun at 1000 rpm for 10 minutes , the supernatant removed , and the pelleted cells were fixed with 4% formaldehyde in pbs for 20 minutes at room temperature . fluorescence at 30 minutes and 1 hour was assessed immediately using an optima fluostar plate reader ( bmg labtech , durham , nc , usa ) . lysotracker red ( excitation 577 nm ; emission 590 nm ) and hoechst ( excitation 350 nm ; emission 461 nm ) fluorescence were assessed using appropriate filters . all fluorescence values were normalized to the amount of cells in individual wells by obtaining the ratio of lysotracker red fluorescence ( assessment of acid vesicles ) to hoechst fluorescence ( nucleic acid stain ) . cells were grown in 20 100 mm falcon polystyrene tissue culture dishes ( fisher scientific , pittsburgh , pa , usa ) . cultures treated with doc or incubated in control media were disrupted in lysis buffer ( 50 mm tris ph 8 , 5 mm edta , 150 mm nacl , 0.5% np-40 ) supplemented with 1 mm phenylmethylsulfonyl fluoride ( pmsf ) , leupeptin ( 1 g / ml ) , and aprotinin ( 0.01 u / ml ) . cell lysates were prepared at a concentration of 2 g/l of protein , and 10 g of protein were added to each well of a 15% criterion tris - hcl gel ( biorad , hercules , calif , usa ) for size fractionation by electrophoresis . the proteins were blotted onto immobilon - p pvdf transfer membrane ( millipore , bedford , mass , usa ) . the membranes were incubated with a mouse antibeclin monoclonal antibody ( bd transduction laboratories , san diego , calif , usa ) at a dilution of 1 : 1000 or rabbit anti - lc3b polyclonal antibody ( cell signaling technology , inc . , the membranes were then incubated with goat antimouse or goat antirabbit secondary antibodies conjugated to horseradish peroxidase ( pierce , rockford , ill , usa ) . antibody complexes were detected using the supersignal west pico chemiluminescence detection system ( pierce , rockford , ill , usa ) . finally , the membranes were stained for 20 minutes with brilliant blue g dye ( sigma - aldrich , st . we have chosen to use the staining of the membranes with brilliant blue g dye rather than a specific protein as a loading control , since the former looks at numerous bands , whereas the latter can be misleading . this is based on work published from our laboratory using gapdh and g3pd , and those of others who screened 22 housekeeping genes and found a large number to be modulated by various experimental conditions . all western blot experiments were repeated at least three times ; in the repeats , separate cultures were treated , and cell lysates were separately prepared . the band intensities after doc treatments or starvation conditions ( incubation in hbss ) were then statistically compared using automated densitometry ( quantiscan imaging analysis , biology software net , uk ) and the values normalized to the control values . cells were grown in 20 100 mm falcon polystyrene tissue culture dishes . control ( untreated ) and doc - treated cells were trypsinized , washed with pbs and fixed with 4% formaldehyde overnight . the details of the immunohistochemical procedures have been previously described . briefly , after deparaffinization , rehydration , and incubation in 3% hydrogen peroxide in methanol , sections were blocked with 1.5% goat serum ( vector laboratories , burlingame ) and immunostained using a polyclonal antibeclin-1 antibody from prosci inc . ( poway , calif , usa ) at a concentration of 1 g / ml . sections were then incubated using a biotinylated antirabbit secondary antibody ( vector laboratories ) , vectastain elite abc ( avidin biotin complex ) reagent ( vector laboratories ) , and dab ( 3 - 3diaminobenzidine ) activated by hydrogen peroxide . apoptosis - resistant hct-116rc cells were plated at 2 10 cells / ml in a 24 well falcon plate . after treatments , the supernatants containing floaters were removed , adherent cells were trypsinized and added to the floaters . an equal volume of trypan blue solution was added to a 50 l aliquot of the supernatant containing adherent cells and floaters . a minimum of 100 cells were counted on a hemacytometer slide under the 10x objective of a brightfield microscope . the percentage of cells that were stained with trypan blue was determined for each treatment . cells were spun onto glass slides using the cytospin 3 ( shandon , pittsburg , pa , usa ) and then were fixed in 100% methanol for 3 minutes . to stain the slides , 10% giemsa stain ( sigma ) cells were examined under a 100 x oil immersion lens and evaluated for apoptosis and necrosis , using criteria previously reported for brightfield microscopy . all cell death experiments were repeated at least twice with similar results . to evaluate statistical significance , 100 cells were scored from 5 different areas of the slide , and a mean s.d . was obtained for each experimental group . the difference between groups was considered significant at the 95% probability level using student 's t - test . cells were grown in 10 35 mm falcon polystyrene tissue culture dishes ( fisher scientific , pittsburgh , pa , usa ) . all cells were pretreated with protease inhibitors ( 10 g / ml e-64d , 10 g / ml pepstatin a ) to enhance the identification of cellular organelles and debris in autophagic vacuoles . control cells were incubated in the absence of doc for the same period of time . a total of at least 3 10 cells from the two kinds of treatment groups were rinsed in pbs and fixed in situ with 1% glutaraldehyde made up in 0.1 m cacodylate buffer ( ph 7.2 ) for 30 minutes . cells were then scraped from the surface of the tissue culture plates using a rubber policeman , pelleted , and then resuspended in 3% glutaraldehyde made up in 0.1 m cacodylate buffer ( ph 7.2 ) for 2 hours at 4c . cells were washed in pbs , postfixed in 2% osmium tetroxide , dehydrated in a graded series of ethanols , and embedded in spurr 's epoxy resin . ultrathin sections were stained with uranyl acetate and lead citrate and photographed using a philips cm12s transmission electron microscope operating at 80 kev . since ncm-460 cells are very sensitive to doc - induced cytotoxicity , only 13 hour experiments were performed using 0.4 mm doc ; longer treatment times with this concentration of doc resulted in significant apoptosis and necrosis . treatment of ncm-460 cells with 0.4 mm doc for 13 hours resulted in the appearance of autophagic vacuoles , assessed using tem ( figure 1 ) . these ultrastructural findings are considered one of the gold standards for identifying the activation of autophagy . another major finding was the increase in expression of microtubule - associated proteinlight chain 3 ( lc3 ) , the mammalian homologue of the yeast atg8 autophagic protein . the appearance of cytosolic lc3-i by posttranslational modification of pro - lc3 by hatg4b and the dynamic increase in the formation of lc3-ii ( a lc3-phospholipid conjugate ) and localization to autophagosomal membranes over time in the presence of protease inhibitors ( e-64d and pepstatin a ) are considered another excellent indication for the activation of the autophagic pathway . the use of protease inhibitors prevents the degradation of lc3-ii which is membrane - bound and subject to proteolytic degradation in mature autophagolysosomes . we preincubated ncm-460 cells in media containing 10 g / ml e-64d and 10 g / ml pepstatin a for 24 hours and then exposed cells to 0.4 mm doc for 0 - 1 hour , a time period that we determined to have abundant autophagic vacuoles by tem and prior to the appearance of apoptotic or necrotic cells . figure 2 shows western blots and densitometric analysis of doc - treated ncm-460 cells ( figures 2(a)2(d ) ) and starved cells ( incubation in hbss ) as a positive control ( figures 2(e)2(h ) ) , indicating the dynamics associated with the appearance of lc3-i and lc-3-ii . it can be seen that both lc3-i and lc3-ii were increased by doc and starvation conditions at the 1 hour time point compared to control untreated cells , indicating the activation of the early cytosolic form of lc3 ( lc3-i ) and the late membrane - bound form of lc3 ( lc3-ii ) . in different experiments the reason for this is not clear , but may relate to different number of cell passages . in all cases , the basal levels of both lc3-i and lc3-ii were increased by both doc and starvation conditions ; however , the actual fold increase can not be directly compared because of this inherent variability . as shown in figures 2(e)2(h ) , the increase of lc3-i and lc3-ii over time in the presence of the protease inhibitors was observed under starvation conditions as was observed after incubation in 0.4 mm doc ( figures 2(a)2(d ) ) . this increase in lc3-i and lc3-ii levels after doc treatment was similar to the findings of ellington et al . who studied soybean b - group triterpenoid saponin - induced autophagy in a colonic adenocarcinoma cell line ( hct-15 ) . in the present study and that of ellington et al . , this increase in expression of lc3-i and lc3-ii was accompanied by the presence of autophagic vacuoles assessed by tem , the classic gold standard for the activation of the autophagic pathway . an early step in the autophagic process is the acidification of cytoplasmic vesicles , which provides the acidic milieu necessary for the optimal activity of digestive enzymes contained within lysosomes . we were able to demonstrate the acidification of vesicles within 30 to 60 minutes after doc treatment by assessing either the increase in fluorescence of mdc or lysotracker red ( figure 3 ) , two dyes that target acid vesicles [ 57 , 58 ] . the tem studies coupled with the lc3 results and the vesicular acidification assays strongly indicate that hydrophobic bile acids can activate autophagy as an early stress - response pathway . ncm-460 cells were exposed to 0.2 mm doc for 24 hours , and beclin-1 expression was assessed using immunohistochemical ( figure 4(a ) ) and western blot ( figures 4(b)4(d ) ) analysis . this concentration of doc did not induce appreciable apoptosis during a 24-hour period and was , therefore , chosen for this experiment . treatment with 0.2 mm doc induced a dramatic increase in the protein levels of beclin-1 using both techniques . since doc induces a significant amount of oxidative / nitrosative stress [ 6 , 1115 ] , we determined if the doc - induced increase in beclin-1 expression was mediated , in part , through an oxidative mechanism . we pretreated ncm-460 cells for 2 hours with 4 different agents that reduce oxygen - free radicals through different mechanisms , followed by a 24-hour incubation with 0.2 mm doc . the 4 agents used were catalase , hbed , mntbap , and cudips . catalase catalytically breaks down hydrogen peroxide to water and oxygen ; hbed is an iron chelator and inhibits ferric ion catalyzed formation of hydroxyl radicals ; mntbap is a cell permeable superoxide dismutase mimetic ( sod ) and peroxynitrite scavenger [ 61 , 62 ] ; cudips is a cell permeable sod mimetic . all 4 agents had a marked effect on preventing the doc - induced increase in beclin-1 expression , although catalase was the most effective ( figure 5 ) . in addition , it was determined that the constitutive levels of beclin-1 are also highly dependent on endogenous oxidative stress levels in the cell . as shown in figure 5 , all 4 antioxidants decreased the constitutive levels of beclin-1 , with catalase being the most effective . to determine whether the activation of autophagy contributes to doc - induced cell death or is a prosurvival stress - response pathway , ncm-460 cells were pretreated with rapamycin , an agent that activates autophagy , and 3-methyladenine ( 3-ma ) , an agent that inhibits the autophagic process . ncm-460 cells were pretreated with 100 m rapamycin ( figure 6(a ) ) or 4 mm 3-ma ( figure 6(b ) ) for 24 hours and then incubated with 0.4 mm doc for 4 hours . total cell number and the trypan blue exclusion assay were used as measures of cell growth and viability . doc treatment , alone , resulted in a significant ( p < .05 ) decrease in cell counts compared to untreated control cells . rapamycin pretreatment significantly ( p < .05 ) decreased trypan blue uptake and prevented the cell loss caused by doc treatment ( figure 6(a ) ) . the significant decrease in cell counts in the absence of significant trypan blue uptake by 100 m rapamycin , alone ( figure 6(a ) ) , is most probably a reflection of a decrease in cell proliferation caused by the activation of autophagy . opposite to the effects of rapamycin , pretreatment with 3-ma significantly ( p < .05 ) increased trypan blue uptake and increased the cell loss caused by doc ( figure 6(b ) ) . we have previously reported that persistent exposure of hct-116 apoptosis - competent colon cancer cells to increasing concentrations of doc resulted in the development of stable apoptosis - resistant cell populations in which several stress - response pathways were upregulated [ 40 , 66 ] . it was determined that the autophagic activity was constitutively upregulated in each of the apoptosis - resistant cell lines ( hct-116rb , hct-116rc , hct-116rd cells ) . increased autophagy was indicated by the presence of numerous late - stage autophagolysosomes in the cytoplasm of the resistant cells , identified in some cases by the presence of numerous whorls of digested material . to evaluate whether the constitutive upregulation of the autophagic pathway has a survival function in these apoptosis - resistant cells or is merely an epiphenomenon , we exposed hct-116rc cells to various agents that modulate the autophagic process . since the hct-116rc cells are resistant to cell death , all experiments requiring bile acid treatment were performed using 0.5 mm doc , and cells were treated in late log phase of growth . these conditions were necessary to elicit a cellular response to autophagy inhibitors / inducers , as described below . hct-116rc cells were pretreated with 100 m rapamycin or 4 mm 3-ma for 24 hours and then incubated with 0.5 mm doc for an additional 24 hours . total cell number and the trypan blue exclusion assay were used as measures of cell growth and viability . similar to the results with the noncancer cell line , ncm-460 , rapamycin pretreatment of the apoptosis - resistant cancer cell line , hct-116rc , followed by 24 hours of treatment with 0.5 mm doc , resulted in a significant ( p < .05 ) increase in cell number and a significant ( p < .05 ) decrease in trypan blue uptake ( i.e. , increase in viable cells ) compared to doc treatment , alone ( figure 7(a ) ) . the significant decrease in cell counts in the absence of significant trypan blue uptake by 100 m rapamycin , alone ( figure 7(a ) ) , is most probably a reflection of a decrease in cell proliferation caused by the activation of autophagy . on the other hand , pretreatment of hct-116rc cells with 4 mm 3-ma had no effect on increasing cell death induced by 0.5 mm doc ( figure 7(b ) ) . the significant decrease in cell counts in the absence of significant trypan blue uptake by 4 mm 3-ma , alone , is most probably a reflection of a decrease in cell proliferation ( figure 7(b ) ) . since we have previously shown that autophagy is constitutively expressed in these cells and rapamycin had a significant effect on cell survival , the negative results obtained with the combination of 3-ma and doc can not be taken as conclusive evidence of lack of involvement of autophagy . since 3-ma inhibits autophagy at an early stage by preventing the formation of autophagosomes , it has been reported that in order to adequately assess the modulation of the autophagy process , inhibitors that act at a different stages of the autophagy process should also be tested . therefore , hct-116rc cells were exposed to 2 different inhibitors of the autophagic process , bafilomycin a1 and hydroxychloroquine , which act at the level of acid vesicles / lysosomes . bafilomycin a1 appears to block the fusion of autophagosomes and lysosomes , and hydroxychloroquine ( an amine ) diffuses into acid vesicles / lysosomes and raises the intraorganellar ph . bafilomycin a1 also raises the ph of acid vesicles / lysosomes by inhibiting the proton - translocating atpase ( h - atpase ) . hct-116rc cells were pretreated with 1 nm bafilomycin a1 for 24 hours and then incubated with 0.5 mm doc for an additional 24 hours . bafilomycin a1 pretreatment followed by doc treatment increased the percentage of apoptotic cells 4-fold over the level of apoptosis induced when doc was used alone ( table 1 ) . there was no increase in the percentage of doc - induced necrotic cells by bafilomycin a1 pretreatment . hct-116rc cells were pretreated with 10 m hydroxychloroquine for 24 hours and then incubated with 0.5 mm doc for an additional 24 hours . hydroxychloroquine pretreatment followed by doc treatment increased the percentage of apoptotic cells 4-fold over the level of apoptosis induced by doc , alone ( table 1(b ) ) . there was no increase in the percentage of doc - induced necrotic cells by hydroxychloroquine pretreatment . in summary , the collective data indicate that autophagy has a survival value for both noncancerous and cancerous colon cells when exposed to hydrophobic bile acids in a nutrient - rich environment . high concentrations of hydrophobic bile acids , associated with a high - fat diet , induce proapoptotic and prosurvival stress - response pathways . the ultimate fate of the cell depends upon the balance of proapoptotic and antiapoptotic proteins activated or synthesized in response to bile acid exposure , and the level of energy demands placed upon the stressed cell . we hypothesized that persistent cellular stress induced by bile acids , such as er stress , dna damage , and mitochondrial stress , will lead to the clonal selection of apoptosis - resistant cells and the constitutive activation of cell survival pathways ( figure 8) . we tested this hypothesis by generating apoptosis - resistant colon cells by repeated exposure of apoptosis - sensitive hct-116 cells in vitro to increasing concentrations of the hydrophobic bile acid , doc , and evaluating stress - induced cell death and apoptosis - related gene expression at the molecular and cellular levels [ 40 , 66 ] . nf-b and many proteins that protect against oxidative stress were constitutively upregulated in these apoptosis - resistant cells . in addition , the development of apoptosis resistance was accompanied by the modulation of genes associated with the autophagy pathway . the autophagy - related genes that exhibit increased expression include six rab genes involved in vesicle transport , a rab interacting lysosomal protein - like 2 protein ( rilpl2 ) , pi(3)k , 2 subunits of the lysosomal proton ( h)-translocating atpase , cathepsin d , lysosomal - associated membrane protein 1 ( lamp-1 ) , a multipass membrane transporter protein ( mfsd8/cln7 ) , and prenylcysteine lyase , a lysosomal enzyme involved in the degradation of prenylated proteins . we also found that chronic feeding of wild - type b6.129 mice with doc added to the diet results in an increase in apg4 , a cysteine protease that acts during the formation of autophagosomes and whose activity is regulated by reactive oxygen species ( ros ) . the functional role of autophagy in colon carcinogenesis , however , was not determined from these in vitro microarray and in vivo animal studies . in the present study , we first evaluated the ability of doc to activate autophagy in ncm-460 cells , and then determined whether autophagy has a prosurvival function in this noncancerous colon epithelial cell line . we demonstrated that doc activated autophagy using different methods of detection , and that this activation contributed to cell survival . we next determined that the constitutive upregulation of autophagy also has a prosurvival function in hct-116rc apoptosis - resistant colon cancer epithelial cells . this is based on the experiments with bafilomycin a1/hydroxychloroquine , in which we showed that autophagy prevented cells from undergoing doc - induced apoptosis , but not from doc - induced necrosis . the possible roles of autophagy in colon carcinogenesis based on published results and present findings are shown schematically in figure 8 . the cellular stresses induced by hydrophobic bile acids ( e.g. , er stress , dna damage , mitochondrial stress ) are also inducers of the autophagic pathway [ 7275 ] , most probably mediated through the generation of ros . evidence that doc induces the autophagic pathway through an oxidative / nitrosative mechanism was provided in the present study using 3 different antioxidants in addition to catalase . these antioxidant conditions dramatically reduced the level of doc - induced beclin-1 protein expression , a major protein involved in the mammalian autophagic pathway . the rationale for choosing beclin-1 ( homologue of the yeast autophagy gene apg6/vps30 ) to assess the effects of antioxidants on the doc - induced increase in autophagy was based on ( 1 ) its dramatic increase in expression in noncancer cells by doc , a known inducer of oxidative stress , compared to other autophagy - related proteins ( data not shown ) , ( 2 ) its critical involvement in the initial step of autophagosome formation [ 7880 ] , ( 3 ) the documented importance of an increase in beclin-1 at the premetastasis stage of colon cancer development , ( 4 ) , its role in tumorigenesis , in general [ 82 , 83 ] , ( 5 ) its function as an antiapoptotic protein [ 8487 ] , and ( 6 ) its potential as a possible biomarker to assess colon cancer risk . although the oxidative mechanism by which doc increases beclin-1 protein expression is most probably multifactorial , we suggest that an important signaling pathway may involve the generation of ceramide . this is based on the fact that ( 1 ) ceramide is an important sphingolipid molecule involved in the increase in beclin-1 in ht-29 colon epithelial cells and other cell types , doc is known to generate ceramide through several mechanisms [ 8092 ] , ( 2 ) ceramide treatment decreases catalase enzymatic activity and expression , a possible link to the present findings indicating that catalase reduces the doc - induced increase in beclin-1 expression , and ( 3 ) ceramide can damage mitochondria [ 9498 ] , a known inducer of the autophagic process . other mechanisms that may be responsible for doc - induced increase in beclin-1 expression may involve alterations of lipid trafficking , a process known to induce beclin-1 expression in other cell types . although we focussed on the role of oxidative stress in the doc - induced modulation of beclin-1 , other aspects of the autophagic process that are now known to be regulated by ros / rns [ 102105 ] may also be modulated by doc . since we have shown that autophagy is a survival pathway for apoptosis - resistant colon cancer cells , the constitutive activation of autophagy and the activation of autophagy induced by cancer chemotherapeutic agents should , therefore , be taken into consideration when designing effective clinical treatment regimens for cancer . we plan to determine the effectiveness of modulators of autophagy in combination with cytotoxic drugs , such as 5-fluorouracil , oxaliplatin , and irenotecan [ 107 , 108 ] , in enhancing cell death in vitro in our apoptosis - resistant colon cancer cell lines . the precedence for combining inhibitors of autophagy with chemotherapeutic agents for the treatment of colon was recently established . li et al . reported that 3-ma enhanced the effect of 5-fluorouracil in inducing apoptosis of colo26 and ht-29 colon cancer cells . it is also anticipated that a better understanding of the mechanisms of autophagy in colon cells , their modulation by dietary factors , and aberrant expression of autophagic proteins during colon carcinogenesis will contribute to the important field of hypothesis - driven biomarker development to assess colon cancer risk .

we report that deoxycholate ( doc ) , a hydrophobic bile acid associated with a high - fat diet , activates the autophagic pathway in non - cancer colon epithelial cells ( ncm-460 ) , and that this activation contributes to cell survival . the doc - induced increase in autophagy was documented by an increase in autophagic vacuoles ( detected using transmission electron microscopy , increased levels of lc3-i and lc3-ii ( western blotting ) , an increase in acidic vesicles ( fluorescence spectroscopy of monodansycadaverine and lysotracker red probes ) , and increased expression of the autophagic protein , beclin-1 ( immunohistochemistry / western blotting ) . the doc - induced increase in beclin-1 expression was ros - dependent . rapamycin ( activator of autophagy ) pre - treatment of ncm-460 cells significantly ( p < .05 ) decreased , and 3-ma ( inhibitor of autophagy ) significantly ( p < .05 ) increased the cell loss caused by doc treatment , alone . rapamycin pre - treatment of the apoptosis - resistant colon cancer cell line , hct-116rc ( developed in our laboratory ) , resulted in a significant decrease in doc - induced cell death . bafilomycin a1 and hydroxychloroquine ( inhibitors of the autophagic process ) increased the doc - induced percentage of apoptotic cells in hct-116rc cells . it was concluded that the activation of autophagy by doc has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

a typical western diet , high in fat and low in fiber , has been shown to contribute to the development of colon cancer in epidemiologic and animal studies . bile acids / salts , present in high concentration in the feces of patients on a high fat / low fiber diet , have been associated with colon cancer risk . the most common bile acid present in the human feces is deoxycholic acid ( doc ) , a hydrophobic bile acid . some of these bile acid - induced cellular stresses may ultimately lead to cell death by mechanisms that include both apoptosis [ 20 , 21 ] and necrosis . in addition , they may act as carcinogens [ 6 , 7 ] and/or select for outgrowth of clones of mutant cells resistant to bile acid - induced cell death . one of the cell survival pathways activated in response to bile acid exposure is the nf-b stress - response pathway [ 11 , 20 , 22 , 23 ] . persistent activation of nf-b causes cells to become apoptosis - resistant , and such cells tend to acquire mutations , some of which may contribute to colon cancer . the autophagic process occurs in several stages that begin with the formation of a crescent - shaped isolation membrane ( phagophore ) that sequesters organelles , matures into an autophagosome that surrounds the organelle , followed by the fusion of the autophagosome with a lysosome to form an autophagolysosome [ 30 , 31 ] . we now show , for the first time , that deoxycholate ( doc ) activates the autophagic pathway in noncancer colon epithelial cells ( ncm-460 ) , and that this activation contributes to cell survival . we also show that the constitutive activation of autophagy contributes to the survival of apoptosis - resistant colon cancer cells ( hct-116rc ) that were developed in our laboratory by repeated exposure to increasing concentrations of doc . the present findings , coupled with findings from our in vivo animal model of deoxycholate - induced colonic inflammation and from our in vitro apoptosis - resistant colon cancer cell lines , implicate autophagy in colon carcinogenesis and suggest an additional mechanism by which hydrophobic bile acids contribute to colon carcinogenesis . these studies may aid in the identification of potential biomarkers of the autophagy pathway in the nonneoplastic colonic mucosa of patients at risk for colon cancer . in addition , combining inhibitors of autophagy with chemotherapeutic agents commonly used to treat colon cancer may lead to an improved clinical outcome . sodium deoxycholate ( doc ) , 3-methyladenine ( 3-ma ) , rapamycin ( rapa ) ( derived from streptomyces hygroscopicus ) , cudips [ copper(ii ) 3,5-diisopropylsalicylate hydrate ) , and catalase ( 10 00040 000 u / mg protein ) were obtained from sigma - aldrich ( st . bafilomycin a1 , mntbap [ mn ( iii ) tetrakis ( 4-benzoic acid ) porphyrin chloride ] , hbed [ n , n-di-(2-hydroxybenzyl)ethylenediamine - n , n-diacetic acid ) ] , pepstatin a , and e-64d were from biomol research laboratories ( plymouth meeting , pa , usa ) , hydroxychloroquine sulfate ( hcs ) was from acros organics ( morris plains , nj ) , and trypan blue was from gibco brl life technologies ( grand island , ny , usa ) . the three antioxidants ( hbed , mntbap , cudips ) used to evaluate the doc - induced increase in beclin-1 expression were used at the same concentration ( 100 m ) so that direct comparisons can be made . the concentration chosen was well within the range of concentrations reported in the literature for use with cultured cells ( see cited articles below ) , and this concentration did not induce any cytotoxicity in the sensitive ncm-460 cells . hct-116rc is a stable apoptosis - resistant colon cancer cell line that was developed in our laboratory after persistent exposure to increasing concentrations of nadoc . starvation experiments were performed by incubation cells in hank 's balanced salt solution ( hbss ) , as previously described for colon epithelial cells . apoptosis - resistant hct-116rc cells were plated at 2 10 cells / ml in a 24 well falcon plate . since ncm-460 cells are very sensitive to doc - induced cytotoxicity , only 13 hour experiments were performed using 0.4 mm doc ; longer treatment times with this concentration of doc resulted in significant apoptosis and necrosis . treatment of ncm-460 cells with 0.4 mm doc for 13 hours resulted in the appearance of autophagic vacuoles , assessed using tem ( figure 1 ) . these ultrastructural findings are considered one of the gold standards for identifying the activation of autophagy . another major finding was the increase in expression of microtubule - associated proteinlight chain 3 ( lc3 ) , the mammalian homologue of the yeast atg8 autophagic protein . the appearance of cytosolic lc3-i by posttranslational modification of pro - lc3 by hatg4b and the dynamic increase in the formation of lc3-ii ( a lc3-phospholipid conjugate ) and localization to autophagosomal membranes over time in the presence of protease inhibitors ( e-64d and pepstatin a ) are considered another excellent indication for the activation of the autophagic pathway . we preincubated ncm-460 cells in media containing 10 g / ml e-64d and 10 g / ml pepstatin a for 24 hours and then exposed cells to 0.4 mm doc for 0 - 1 hour , a time period that we determined to have abundant autophagic vacuoles by tem and prior to the appearance of apoptotic or necrotic cells . figure 2 shows western blots and densitometric analysis of doc - treated ncm-460 cells ( figures 2(a)2(d ) ) and starved cells ( incubation in hbss ) as a positive control ( figures 2(e)2(h ) ) , indicating the dynamics associated with the appearance of lc3-i and lc-3-ii . it can be seen that both lc3-i and lc3-ii were increased by doc and starvation conditions at the 1 hour time point compared to control untreated cells , indicating the activation of the early cytosolic form of lc3 ( lc3-i ) and the late membrane - bound form of lc3 ( lc3-ii ) . as shown in figures 2(e)2(h ) , the increase of lc3-i and lc3-ii over time in the presence of the protease inhibitors was observed under starvation conditions as was observed after incubation in 0.4 mm doc ( figures 2(a)2(d ) ) . this increase in lc3-i and lc3-ii levels after doc treatment was similar to the findings of ellington et al . , this increase in expression of lc3-i and lc3-ii was accompanied by the presence of autophagic vacuoles assessed by tem , the classic gold standard for the activation of the autophagic pathway . we were able to demonstrate the acidification of vesicles within 30 to 60 minutes after doc treatment by assessing either the increase in fluorescence of mdc or lysotracker red ( figure 3 ) , two dyes that target acid vesicles [ 57 , 58 ] . ncm-460 cells were exposed to 0.2 mm doc for 24 hours , and beclin-1 expression was assessed using immunohistochemical ( figure 4(a ) ) and western blot ( figures 4(b)4(d ) ) analysis . since doc induces a significant amount of oxidative / nitrosative stress [ 6 , 1115 ] , we determined if the doc - induced increase in beclin-1 expression was mediated , in part , through an oxidative mechanism . all 4 agents had a marked effect on preventing the doc - induced increase in beclin-1 expression , although catalase was the most effective ( figure 5 ) . in addition , it was determined that the constitutive levels of beclin-1 are also highly dependent on endogenous oxidative stress levels in the cell . to determine whether the activation of autophagy contributes to doc - induced cell death or is a prosurvival stress - response pathway , ncm-460 cells were pretreated with rapamycin , an agent that activates autophagy , and 3-methyladenine ( 3-ma ) , an agent that inhibits the autophagic process . doc treatment , alone , resulted in a significant ( p < .05 ) decrease in cell counts compared to untreated control cells . rapamycin pretreatment significantly ( p < .05 ) decreased trypan blue uptake and prevented the cell loss caused by doc treatment ( figure 6(a ) ) . the significant decrease in cell counts in the absence of significant trypan blue uptake by 100 m rapamycin , alone ( figure 6(a ) ) , is most probably a reflection of a decrease in cell proliferation caused by the activation of autophagy . opposite to the effects of rapamycin , pretreatment with 3-ma significantly ( p < .05 ) increased trypan blue uptake and increased the cell loss caused by doc ( figure 6(b ) ) . we have previously reported that persistent exposure of hct-116 apoptosis - competent colon cancer cells to increasing concentrations of doc resulted in the development of stable apoptosis - resistant cell populations in which several stress - response pathways were upregulated [ 40 , 66 ] . it was determined that the autophagic activity was constitutively upregulated in each of the apoptosis - resistant cell lines ( hct-116rb , hct-116rc , hct-116rd cells ) . to evaluate whether the constitutive upregulation of the autophagic pathway has a survival function in these apoptosis - resistant cells or is merely an epiphenomenon , we exposed hct-116rc cells to various agents that modulate the autophagic process . since the hct-116rc cells are resistant to cell death , all experiments requiring bile acid treatment were performed using 0.5 mm doc , and cells were treated in late log phase of growth . similar to the results with the noncancer cell line , ncm-460 , rapamycin pretreatment of the apoptosis - resistant cancer cell line , hct-116rc , followed by 24 hours of treatment with 0.5 mm doc , resulted in a significant ( p < .05 ) increase in cell number and a significant ( p < .05 ) decrease in trypan blue uptake ( i.e. , increase in viable cells ) compared to doc treatment , alone ( figure 7(a ) ) . the significant decrease in cell counts in the absence of significant trypan blue uptake by 100 m rapamycin , alone ( figure 7(a ) ) , is most probably a reflection of a decrease in cell proliferation caused by the activation of autophagy . therefore , hct-116rc cells were exposed to 2 different inhibitors of the autophagic process , bafilomycin a1 and hydroxychloroquine , which act at the level of acid vesicles / lysosomes . bafilomycin a1 appears to block the fusion of autophagosomes and lysosomes , and hydroxychloroquine ( an amine ) diffuses into acid vesicles / lysosomes and raises the intraorganellar ph . bafilomycin a1 pretreatment followed by doc treatment increased the percentage of apoptotic cells 4-fold over the level of apoptosis induced when doc was used alone ( table 1 ) . there was no increase in the percentage of doc - induced necrotic cells by bafilomycin a1 pretreatment . hydroxychloroquine pretreatment followed by doc treatment increased the percentage of apoptotic cells 4-fold over the level of apoptosis induced by doc , alone ( table 1(b ) ) . there was no increase in the percentage of doc - induced necrotic cells by hydroxychloroquine pretreatment . high concentrations of hydrophobic bile acids , associated with a high - fat diet , induce proapoptotic and prosurvival stress - response pathways . the ultimate fate of the cell depends upon the balance of proapoptotic and antiapoptotic proteins activated or synthesized in response to bile acid exposure , and the level of energy demands placed upon the stressed cell . we hypothesized that persistent cellular stress induced by bile acids , such as er stress , dna damage , and mitochondrial stress , will lead to the clonal selection of apoptosis - resistant cells and the constitutive activation of cell survival pathways ( figure 8) . we tested this hypothesis by generating apoptosis - resistant colon cells by repeated exposure of apoptosis - sensitive hct-116 cells in vitro to increasing concentrations of the hydrophobic bile acid , doc , and evaluating stress - induced cell death and apoptosis - related gene expression at the molecular and cellular levels [ 40 , 66 ] . the autophagy - related genes that exhibit increased expression include six rab genes involved in vesicle transport , a rab interacting lysosomal protein - like 2 protein ( rilpl2 ) , pi(3)k , 2 subunits of the lysosomal proton ( h)-translocating atpase , cathepsin d , lysosomal - associated membrane protein 1 ( lamp-1 ) , a multipass membrane transporter protein ( mfsd8/cln7 ) , and prenylcysteine lyase , a lysosomal enzyme involved in the degradation of prenylated proteins . in the present study , we first evaluated the ability of doc to activate autophagy in ncm-460 cells , and then determined whether autophagy has a prosurvival function in this noncancerous colon epithelial cell line . we demonstrated that doc activated autophagy using different methods of detection , and that this activation contributed to cell survival . we next determined that the constitutive upregulation of autophagy also has a prosurvival function in hct-116rc apoptosis - resistant colon cancer epithelial cells . these antioxidant conditions dramatically reduced the level of doc - induced beclin-1 protein expression , a major protein involved in the mammalian autophagic pathway . the rationale for choosing beclin-1 ( homologue of the yeast autophagy gene apg6/vps30 ) to assess the effects of antioxidants on the doc - induced increase in autophagy was based on ( 1 ) its dramatic increase in expression in noncancer cells by doc , a known inducer of oxidative stress , compared to other autophagy - related proteins ( data not shown ) , ( 2 ) its critical involvement in the initial step of autophagosome formation [ 7880 ] , ( 3 ) the documented importance of an increase in beclin-1 at the premetastasis stage of colon cancer development , ( 4 ) , its role in tumorigenesis , in general [ 82 , 83 ] , ( 5 ) its function as an antiapoptotic protein [ 8487 ] , and ( 6 ) its potential as a possible biomarker to assess colon cancer risk . this is based on the fact that ( 1 ) ceramide is an important sphingolipid molecule involved in the increase in beclin-1 in ht-29 colon epithelial cells and other cell types , doc is known to generate ceramide through several mechanisms [ 8092 ] , ( 2 ) ceramide treatment decreases catalase enzymatic activity and expression , a possible link to the present findings indicating that catalase reduces the doc - induced increase in beclin-1 expression , and ( 3 ) ceramide can damage mitochondria [ 9498 ] , a known inducer of the autophagic process . other mechanisms that may be responsible for doc - induced increase in beclin-1 expression may involve alterations of lipid trafficking , a process known to induce beclin-1 expression in other cell types . although we focussed on the role of oxidative stress in the doc - induced modulation of beclin-1 , other aspects of the autophagic process that are now known to be regulated by ros / rns [ 102105 ] may also be modulated by doc . since we have shown that autophagy is a survival pathway for apoptosis - resistant colon cancer cells , the constitutive activation of autophagy and the activation of autophagy induced by cancer chemotherapeutic agents should , therefore , be taken into consideration when designing effective clinical treatment regimens for cancer . we plan to determine the effectiveness of modulators of autophagy in combination with cytotoxic drugs , such as 5-fluorouracil , oxaliplatin , and irenotecan [ 107 , 108 ] , in enhancing cell death in vitro in our apoptosis - resistant colon cancer cell lines . the precedence for combining inhibitors of autophagy with chemotherapeutic agents for the treatment of colon was recently established . it is also anticipated that a better understanding of the mechanisms of autophagy in colon cells , their modulation by dietary factors , and aberrant expression of autophagic proteins during colon carcinogenesis will contribute to the important field of hypothesis - driven biomarker development to assess colon cancer risk .

influenza has a significant clinical impact in immunocompromised patients , including adults with cancer , and can cause severe complications . in particular , patients after hematopoietic stem cell transplantation and those undergoing chemotherapy for leukemia are at highest risk , with high morbidity from complications , such as pneumonia , and a clinically relevant mortality rate [ 1 - 3 ] . although the immunogenicity of influenza vaccine varies annually , it is well accepted that an influenza vaccine can reduce morbidity and disease severity . despite the vaccine having weaker immunogenicity in immunocompromised patients than in healthy subjects , it is still recommended for use in such patients for the prevention of severe influenza or superimposed bacterial infections . in korea , administration of an influenza vaccine to immunocompromised patients , including adult cancerpatients , is recommended prior to the winter seasons . however , no mass medical survey of vaccine coverage in immunocompromised patients has been conducted , and it is presumed that the actual vaccine coverage remains poor in high - risk patients , as is the case in other countries [ 6 - 9 ] . reported that the main reasons for low vaccine coverage included lack of promotion by the treating physician ( 72% ) , fear of side - effects ( 33% ) , and concerns regarding the vaccination efficacy ( 10% ) . among medical oncologists , the leading self - reported reason for the lack of vaccination was due to minimal awareness of recommendations . colorectal cancer is estimated as the third most frequent cancer ( second in male and third in female ) in korea according to 2012 's report . due to a currently rapid growing incidence , the importance about colorectal cancer is also growing in korea ; also because , different from other cancers , the chemotherapy regimen for colorectal cancer was well established , it is easy to identify the influences of chemotherapy on the immunogenicity of influenza vaccine . further , because colorectal cancer develops more frequently in older age , both aging and immune - compromising factors are supposed to act as the reducing factors in the immunogenicity of influenza . this study aims to answer the question : if older colorectal patients were vaccinated , what the immunogenicity will be like ? the primary aim of this study is to evaluate the influenza vaccine immunogenicity in a group of high - risk subjects , colorectal cancer patients , from the korea cancer center hospital ( kcch ) , which is a single institute experience , and to offer a reference for immunization guidelines . the secondary aim of this study is to investigate the factors ( immune status , duration of chemotherapy , chemo regimen , etc . ) influencing the immunogenicity of influenza vaccine in colorectal patients . during the 2009 - 2010 and 2010 - 2011 influenza seasons , patients with colorectal cancer , under treatment at kcch , were invited to participate in this study . institutional review board 's approval was obtained , and the subjects gave written informed consent for blood collection for hemagglutination inhibition ( hi ) assays and 1 - 2 months post - vaccination to assess the immune response to influenza vaccination . the baseline patient demographics , including age , sex , tumor stage , surgery history , and type of chemotherapy , were collected . subjects with severe allergy to an influenza vaccine or egg protein , with acute febrile illness at the time of vaccination , who were receiving treatment with corticosteroids for other reasons except for the purpose of anticancer drug with a history of transfusion within 6 months , or with any other condition that might interfere with the evaluation of the study were excluded . paired blood samples were obtained for immunogenicity analysis from all subjects . on day 0 and days 30 - 60 after the first vaccination subjects were excluded from an immunogenicity analysis if they were found to be non - compliant with the immunization or blood sampling schedule . sk influenza ix vaccine ( trivalent split influenza vaccine ; sk chemicals , seongnam , korea ) was administered intramuscularly to all subjects during the 2009 - 2010 and 2010 - 2011 influenza seasons . in the 2009 - 2010 influenza season , sk influenza ix vaccinecontained 15 g of each hemagglutinin from a / brisbane/59/2007 ( h1n1 ) strain ( ivr-148 ) , a / uruguay/716/2007 ( h3n2 ) strain ( nymcx-175c ) , and b / brisbane/60/2008 strain . in the 2010 - 2011 influenza season , sk influenza ix vaccine contained 15 g of each hemagglutinin from a / california/7/2009 ( reassortant nymc x-181 ; pandemic h1n1 , ph1n1 ) strain , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) strain , and b / brisbane/60/2008 strain . the hi test , using the chicken red blood cells , was performed to determine the anti - hemagglutinin antibody titers . anti - hemagglutinin titers to h1n1 , h3n2 , and influenza b were measured using a / brisbane/59/2007 ivr-148 ( h1n1 ) , a / uruguay/716/2007 nymcx-175c ( h3n2 ) , and b / brisbane/60/2008 in the 2009 - 2010 season , and a / california/7/2009 ( reassortant nymc x-181 ; ph1n1 ) , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) , and b / brisbane/60/2008 in the 2010 - 2011 season . seroconversion was defined as a change from the baseline titer < 1:10 to a post - vaccination titer1:40 or a 4-fold or greater rise in titer in those with the initial titer1:10 . immunogenicity of the vaccine was assessed based on these findings : seroprotection rates on days 0 and 30 , seroconversion rate on day 30 , and mean fold increase ( mfi ) of geometric mean titer ( gmt ) of the hi assay between days 0 and 30 . in order to confirm the protective immunogenicity of the vaccine based on the european medicines agency criteria , one of the following criteria needed to be met : seroprotection rate>70% for subjects aged 18 - 60 years and>60% for subjects aged>60 years , seroconversion rate>40% for subjects aged 18 - 60 years and>30% for subjects aged>60 years , or mfi>2.5 for subjects aged 18 - 60 years and>2.0 for subjects aged>60 years . we defined an acceptable but limited immune response at the seroprotection rate of 30 - 70% . one - sample t - test , independent - sample t - test , one - way analysis of variance ( anova ) , kruskal - wallis test , and levene 's test were used for the assessment of vaccine immunogenicity . multivariate logistic regression analysis was used for identifying various factors influencing on the immunogenicity of influenza vaccine for colorectal cancer patients . during the 2009 - 2010 and 2010 - 2011 influenza seasons , patients with colorectal cancer , under treatment at kcch , were invited to participate in this study . institutional review board 's approval was obtained , and the subjects gave written informed consent for blood collection for hemagglutination inhibition ( hi ) assays and 1 - 2 months post - vaccination to assess the immune response to influenza vaccination . the baseline patient demographics , including age , sex , tumor stage , surgery history , and type of chemotherapy , were collected . subjects with severe allergy to an influenza vaccine or egg protein , with acute febrile illness at the time of vaccination , who were receiving treatment with corticosteroids for other reasons except for the purpose of anticancer drug with a history of transfusion within 6 months , or with any other condition that might interfere with the evaluation of the study were excluded . paired blood samples were obtained for immunogenicity analysis from all subjects . on day 0 and days 30 - 60 after the first vaccination subjects were excluded from an immunogenicity analysis if they were found to be non - compliant with the immunization or blood sampling schedule . sk influenza ix vaccine ( trivalent split influenza vaccine ; sk chemicals , seongnam , korea ) was administered intramuscularly to all subjects during the 2009 - 2010 and 2010 - 2011 influenza seasons . in the 2009 - 2010 influenza season , sk influenza ix vaccinecontained 15 g of each hemagglutinin from a / brisbane/59/2007 ( h1n1 ) strain ( ivr-148 ) , a / uruguay/716/2007 ( h3n2 ) strain ( nymcx-175c ) , and b / brisbane/60/2008 strain . in the 2010 - 2011 influenza season , sk influenza ix vaccine contained 15 g of each hemagglutinin from a / california/7/2009 ( reassortant nymc x-181 ; pandemic h1n1 , ph1n1 ) strain , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) strain , and b / brisbane/60/2008 strain . the hi test , using the chicken red blood cells , was performed to determine the anti - hemagglutinin antibody titers . anti - hemagglutinin titers to h1n1 , h3n2 , and influenza b were measured using a / brisbane/59/2007 ivr-148 ( h1n1 ) , a / uruguay/716/2007 nymcx-175c ( h3n2 ) , and b / brisbane/60/2008 in the 2009 - 2010 season , and a / california/7/2009 ( reassortant nymc x-181 ; ph1n1 ) , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) , and b / brisbane/60/2008 in the 2010 - 2011 season . seroconversion was defined as a change from the baseline titer < 1:10 to a post - vaccination titer1:40 or a 4-fold or greater rise in titer in those with the initial titer1:10 . immunogenicity of the vaccine was assessed based on these findings : seroprotection rates on days 0 and 30 , seroconversion rate on day 30 , and mean fold increase ( mfi ) of geometric mean titer ( gmt ) of the hi assay between days 0 and 30 . in order to confirm the protective immunogenicity of the vaccine based on the european medicines agency criteria , one of the following criteria needed to be met : seroprotection rate>70% for subjects aged 18 - 60 years and>60% for subjects aged>60 years , seroconversion rate>40% for subjects aged 18 - 60 years and>30% for subjects aged>60 years , or mfi>2.5 for subjects aged 18 - 60 years and>2.0 for subjects aged>60 years . we defined an acceptable but limited immune response at the seroprotection rate of 30 - 70% . chicago , il ) was used for all analyses . one - sample t - test , independent - sample t - test , one - way analysis of variance ( anova ) , kruskal - wallis test , and levene 's test were used for the assessment of vaccine immunogenicity . multivariate logistic regression analysis was used for identifying various factors influencing on the immunogenicity of influenza vaccine for colorectal cancer patients . a total of 62 patients with colorectal cancer at kcch were enrolled in this study from october 2009 to december 2010 . we excluded 22 patients because paired samples were not available , leaving 40 patients with colorectal cancer available for evaluation . the mean age was 61.1 years ( range , 26.52 to 88.68 years ; 66.0 years in the 2009 - 2010 season and 56.70 years in the 2010 - 2011 seasons ) . the male / female ratio was 26/14 ( 11/8 in the 2009 - 2010 seasons and 15/6 in the 2010 - 2011 seasons ) . table 1 shows the characteristics of the patients . in total , 17 of 19 patients with colorectal cancer enrolled in 2009 were treated with surgery and chemotherapy , and the remaining 2 patients received only surgery . all subjects received influenza vaccination when their absolute neutrophil count ( ancs ) was over 1,000 during the bone marrow recovery phase after previous chemotherapy . the average white blood cell ( wbc ) count was 5,476/l , and the average anc was 3,627 on the day of influenza vaccination . the average interval between influenza vaccination and post - vaccination sampling was 33.2 days in the 2009 - 2010 season and 44.15 days in the 2010 - 2011 seasons ( table 1 ) . before vaccination , 68.4% ( n=13 ) , 31.6% ( n=6 ) , and 10.5% ( n=2 ) of 19 patients enrolled in the 2009 - 2010 season had seroprotective antibody titers ( 1:40 ) against h1n1 , h3n2 , and b , respectively . after vaccination , 94.7% ( n=18 ) , 42.1% ( n=8 ) , and 47.3% ( n=9 ) of patients had seroprotective titers against h1n1 , h3n2 , and b , respectively . seroconversion was observed in 52.6% ( n=10 ) , 26.3% ( n=5 ) , and 36.8% ( n=7 ) of patients against h1n1 , h3n2 , and b , respectively . gmt fold increase was 3.86 , 1.49 , and 3.33 against h1n1 , h3n2 , and b , respectively ( table 2 ) . these seroprotection and seroconversion rates indicated good immunogenicity ( seroprotection rate70% , seroconversion40% ) against h1n1 , and an acceptable but limited immune response against influenza h3n2 and b. the gmt fold increase indicated good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2009 - 2010 season . before vaccination , 4.8% ( n=1 ) , 4.8% ( n=1 ) , and 4.8% ( n=1 ) of 21 patients enrolled in the 2010 - 2011 season had seroprotective antibody titers ( 1:40 ) against ph1n1 , h3n2 , and influenza b , respectively . after vaccination , the seroprotection rates were 57.1% ( n=12 ) , 52.4% ( n=11 ) , and 38.1% ( n=8 ) , respectively , against these strains . seroconversion was observed for 52.4% ( n=11 ) , 47.6% ( n=10 ) , and 33.3% ( n=7 ) of patients against ph1n1 , h3n2 , and influenza b , respectively . gmt fold increase was 12.29 , 3.62 , and 4.27 against the respective strains ( table 2 ) . this seroprotection rate indicated an acceptable but limited immune response against all vaccine strains in the 2010 - 2011 seasons . however , the seroconversion rate indicated a good immunogenicity ( seroconversion 40% ) against ph1n1 and h3n2 strains , while there was an acceptable but limited immune response against the influenza b strain . the gmt fold increase indicated a good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2010 - 2011 seasons . for an assessment of the effects of age on immunogenicity , linear regression analysis between the age of subjects and their hi titer was applied for all subjects . for each h1n1 and b , there was a significant reverse linear correlation between the age of subjects and their hi titer . ( r=0.143 , p=0.016 and r=0.20 , p=0.004 for each h1n1 and b ) ( fig . 1 ) . we applied a cut - off value of 65 years to subjects for further analysis , because it was well known that an individual of>65 year of age has poor immunogenicity to an influenza vaccine . for assessment , subjects were divided into 2 age groups ( < 65 years and65 years ) . in the 2009 - 2010 season , the seroprotection rates against h1n1 , h3n2 , and influenza b were 100.0% , 28.6% , and 71.4% , respectively , in those aged < 65 years ; these values were 91.7% , 50.0% , and 33.3% , respectively , in those aged65 years . the seroconversion rates against h1n1 , h3n2 , and influenza b were 57.1% , 14.3% , and 57.1% , respectively , in those aged < 65 years ; these values were 50.0% , 33.3% , and 25.0% , respectively , in those aged65 years . gmt fold increases against h1n1 , h3n2 , and influenza b were , 5.38 , 1.00 , and 5.94 , respectively , in those aged < 65 years ; these values were 6.56 , 2.97 , and 4.00 , respectively , in those aged65 years . there were no statistically significant differences in the immune responses between the 2 age groups in the 2009 - 2010 seasons , except in post - vaccination gmt against h3n2 ( table 3 ) . in the 2010 - 2011 season , the seroprotection rates against ph1n1 , h3n2 , and influenza b were 60.0% , 46.7% , and 26.7% , respectively , for those aged < 65 years ; these values were 50.0% , 66.7% , and 66.7% , respectively , for those aged65 years . seroconversion rates against ph1n1 , h3n2 , and influenza b were 53.3% , 40.0% , and 26.7% , respectively , in those aged < 65 years ; these values were 50.0% , 66.7% , and 50.0% , respectively , in those aged65 years . gmt fold increases against ph1n1 , h3n2 , and influenza b were 4.88 , 2.00 , and 1.81 , respectively , in those aged < 65 years ; these values were 6.35 , 7.13 , and 7.13 , respectively , in those aged65 years . for all strains , there were no statistically significant differences in the immune responses between the 2 age groups in the 2010 - 2011 seasons ( table 4 ) . for an assessment regarding the effects of chemotherapy on the influenza vaccine immunogenicity , linear regression analysis between recent and total chemotherapy duration , chemotherapy types ( adjuvant , palliative , adjuvant , and palliative combined ) and their hi titer was applied for all subjects without the respect of different influenza seasons . for h3n2 , there was a significant linear correlation between total chemotherapy duration and its hi titer ( r=0.313 , p=0.000 ) . however , there were no correlations between total chemotherapy duration and its hi titer for h1n1and b ( fig . we applied a cut - off value for each 2 , 4 , 6 , 8 , 10 , and 12 months of recent chemotherapy duration to subjects for further analysis . there were no differences in the seroprotection rates to influenza viruses between the two groups , according to the cut - off value . for an assessment regarding the effects of immune status on the influenza vaccine immunogenicity , linear regression analysis between wbc count , anc , absolute lymphocyte count ( alc ) and their hi titer was applied for all subjects without the respect of different influenza seasons . for b , there was a significant linear correlation between anc and its hi titer ( r=0.148 , p=0.017 ) . however , there were no correlations between anc and its hi titer for h1n1and h3n2 . there were no correlations between wnc count , alc and its hi titer for all strains . when applied the cut - off value 1,000 for anc and alc , there were no differences of the immune response to influenza vaccine between subjects with1,000 and subjects with<1,000 . on further analyses , stage of colorectal cancer and recurrence state at vaccination did not affect the immune response to influenza antigens . on day 30 post - vaccination , participants were asked to report any adverse effects of the vaccination . reported side effects included some local adverse reactions ; however , serious adverse effects , such as guillain - barre syndrome , were not reported . a total of 62 patients with colorectal cancer at kcch were enrolled in this study from october 2009 to december 2010 . we excluded 22 patients because paired samples were not available , leaving 40 patients with colorectal cancer available for evaluation . the mean age was 61.1 years ( range , 26.52 to 88.68 years ; 66.0 years in the 2009 - 2010 season and 56.70 years in the 2010 - 2011 seasons ) . the male / female ratio was 26/14 ( 11/8 in the 2009 - 2010 seasons and 15/6 in the 2010 - 2011 seasons ) . table 1 shows the characteristics of the patients . in total , 17 of 19 patients with colorectal cancer enrolled in 2009 were treated with surgery and chemotherapy , and the remaining 2 patients received only surgery . all subjects received influenza vaccination when their absolute neutrophil count ( ancs ) was over 1,000 during the bone marrow recovery phase after previous chemotherapy . the average white blood cell ( wbc ) count was 5,476/l , and the average anc was 3,627 on the day of influenza vaccination . the average interval between influenza vaccination and post - vaccination sampling was 33.2 days in the 2009 - 2010 season and 44.15 days in the 2010 - 2011 seasons ( table 1 ) . before vaccination , 68.4% ( n=13 ) , 31.6% ( n=6 ) , and 10.5% ( n=2 ) of 19 patients enrolled in the 2009 - 2010 season had seroprotective antibody titers ( 1:40 ) against h1n1 , h3n2 , and b , respectively . after vaccination , 94.7% ( n=18 ) , 42.1% ( n=8 ) , and 47.3% ( n=9 ) of patients had seroprotective titers against h1n1 , h3n2 , and b , respectively . seroconversion was observed in 52.6% ( n=10 ) , 26.3% ( n=5 ) , and 36.8% ( n=7 ) of patients against h1n1 , h3n2 , and b , respectively . gmt fold increase was 3.86 , 1.49 , and 3.33 against h1n1 , h3n2 , and b , respectively ( table 2 ) . these seroprotection and seroconversion rates indicated good immunogenicity ( seroprotection rate70% , seroconversion40% ) against h1n1 , and an acceptable but limited immune response against influenza h3n2 and b. the gmt fold increase indicated good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2009 - 2010 season . before vaccination , 4.8% ( n=1 ) , 4.8% ( n=1 ) , and 4.8% ( n=1 ) of 21 patients enrolled in the 2010 - 2011 season had seroprotective antibody titers ( 1:40 ) against ph1n1 , h3n2 , and influenza b , respectively . after vaccination , the seroprotection rates were 57.1% ( n=12 ) , 52.4% ( n=11 ) , and 38.1% ( n=8 ) , respectively , against these strains . seroconversion was observed for 52.4% ( n=11 ) , 47.6% ( n=10 ) , and 33.3% ( n=7 ) of patients against ph1n1 , h3n2 , and influenza b , respectively . gmt fold increase was 12.29 , 3.62 , and 4.27 against the respective strains ( table 2 ) . this seroprotection rate indicated an acceptable but limited immune response against all vaccine strains in the 2010 - 2011 seasons . however , the seroconversion rate indicated a good immunogenicity ( seroconversion 40% ) against ph1n1 and h3n2 strains , while there was an acceptable but limited immune response against the influenza b strain . the gmt fold increase indicated a good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2010 - 2011 seasons . for an assessment of the effects of age on immunogenicity , linear regression analysis between the age of subjects and their hi titer was applied for all subjects . for each h1n1 and b , there was a significant reverse linear correlation between the age of subjects and their hi titer . ( r=0.143 , p=0.016 and r=0.20 , p=0.004 for each h1n1 and b ) ( fig . 1 ) . we applied a cut - off value of 65 years to subjects for further analysis , because it was well known that an individual of>65 year of age has poor immunogenicity to an influenza vaccine . for assessment , subjects were divided into 2 age groups ( < 65 years and65 years ) . in the 2009 - 2010 season , the seroprotection rates against h1n1 , h3n2 , and influenza b were 100.0% , 28.6% , and 71.4% , respectively , in those aged < 65 years ; these values were 91.7% , 50.0% , and 33.3% , respectively , in those aged65 years . the seroconversion rates against h1n1 , h3n2 , and influenza b were 57.1% , 14.3% , and 57.1% , respectively , in those aged < 65 years ; these values were 50.0% , 33.3% , and 25.0% , respectively , in those aged65 years . gmt fold increases against h1n1 , h3n2 , and influenza b were , 5.38 , 1.00 , and 5.94 , respectively , in those aged < 65 years ; these values were 6.56 , 2.97 , and 4.00 , respectively , in those aged65 years . there were no statistically significant differences in the immune responses between the 2 age groups in the 2009 - 2010 seasons , except in post - vaccination gmt against h3n2 ( table 3 ) . in the 2010 - 2011 season , the seroprotection rates against ph1n1 , h3n2 , and influenza b were 60.0% , 46.7% , and 26.7% , respectively , for those aged < 65 years ; these values were 50.0% , 66.7% , and 66.7% , respectively , for those aged65 years . seroconversion rates against ph1n1 , h3n2 , and influenza b were 53.3% , 40.0% , and 26.7% , respectively , in those aged < 65 years ; these values were 50.0% , 66.7% , and 50.0% , respectively , in those aged65 years . gmt fold increases against ph1n1 , h3n2 , and influenza b were 4.88 , 2.00 , and 1.81 , respectively , in those aged < 65 years ; these values were 6.35 , 7.13 , and 7.13 , respectively , in those aged65 years . for all strains , there were no statistically significant differences in the immune responses between the 2 age groups in the 2010 - 2011 seasons ( table 4 ) . for an assessment regarding the effects of chemotherapy on the influenza vaccine immunogenicity , linear regression analysis between recent and total chemotherapy duration , chemotherapy types ( adjuvant , palliative , adjuvant , and palliative combined ) and their hi titer was applied for all subjects without the respect of different influenza seasons . for h3n2 , there was a significant linear correlation between total chemotherapy duration and its hi titer ( r=0.313 , p=0.000 ) . however , there were no correlations between total chemotherapy duration and its hi titer for h1n1and b ( fig . we applied a cut - off value for each 2 , 4 , 6 , 8 , 10 , and 12 months of recent chemotherapy duration to subjects for further analysis . there were no differences in the seroprotection rates to influenza viruses between the two groups , according to the cut - off value . for an assessment regarding the effects of immune status on the influenza vaccine immunogenicity , linear regression analysis between wbc count , anc , absolute lymphocyte count ( alc ) and their hi titer was applied for all subjects without the respect of different influenza seasons . for b , there was a significant linear correlation between anc and its hi titer ( r=0.148 , p=0.017 ) . however , there were no correlations between anc and its hi titer for h1n1and h3n2 . there were no correlations between wnc count , alc and its hi titer for all strains . when applied the cut - off value 1,000 for anc and alc , there were no differences of the immune response to influenza vaccine between subjects with1,000 and subjects with<1,000 . on further analyses , stage of colorectal cancer and recurrence state at vaccination did not affect the immune response to influenza antigens . on day 30 post - vaccination , participants were asked to report any adverse effects of the vaccination . reported side effects included some local adverse reactions ; however , serious adverse effects , such as guillain - barre syndrome , were not reported . this study demonstrated the safety of the trivalent , an inactivated influenza vaccine in colorectal cancer patients , with an acceptable but limited immune response . this result matched well with the results of previous studies in immunocompromised patients , especially in colorectal cancer patients [ 13 - 16 ] . reported that 70.6% of 85 colorectal cancer patients vaccinated with an influenza vaccine showed an immune response during the 2006 - 2007 influenza seasons . in the present study , the seroprotection rate was 94.7% for h1n1 , 42.1% for h3n2 , and 47.4% for influenza b in the 2009 - 2010 seasons . the seroprotection rate against h1n1 was significantly higher than that against h3n2 and influenza b in the 2009 - 2010 seasons . the high seroprotection rate against h1n1 was probably a result of a high pre - seroprotection rate against that strain . the cause of the high pre - seroprotection rate against h1n1 might have been the long - term homologous h1n1 vaccine antigen stimuli . in the 2007 - 2008 influenza seasons , there was some mismatch between the circulating wild strain and the vaccine strain , and the vaccine strain was subsequently changed from a / wisconsin/67/2005 to a / brisbane/10/2007 ( h3n2 ) . it was supposed that a short - term h3n2 vaccine antigen stimulus was unable to induce a good immune response , especially in immunocompromised patients . regarding the relatively poor immune response to b antigen , our results were similar to the results of the study performed by xie et al . . they suggested that the low immunogenicity of b / brisbane/60/2008 was probably due to the fact that it was a new strain , unlike the a / brisbane/59/2007 ( h1n1 ) strain , which had been a vaccine component since the 2008 - 2009 season . compared with the control group ( diabetes mellitus patients , n=15 ) vaccinated in the 2009 - 2010 season , there were no differences in the immunogenicity , except against the influenza b strain . in the control group , the pre - seroprotection rate and gmt were statistically significantly higher against influenza b. therefore , direct comparisons between the study and control groups for the vaccine immunogenicity against influenza b strains were not possible ( table 5 ) . age , type of malignancy , wbc count , lymphocyte count , serum immunoglobulin g ( igg ) level , and status of cancer therapy are well known factors affecting the immune response after an influenza vaccination . in this study , age and anc showed a correlation with the immune response for some strains , but status of cancer therapy , wbc count , and lymphocyte count did not show a significant correlation with the immune response . furthermore , in this study , total chemotherapy duration showed a correlation with the immune response for some strains . adults aged65 years produce weaker immune response to vaccination than adults aged < 65 years . a number of protective immune functions decline with age , and along with physiological and anatomical changes , this contributes to increased susceptibility to infectious diseases and suboptimal immune responses to vaccination . in the present study , the relatively good immune response in subjects aged65 years is probably related to a higher rate of influenza vaccination during the previous epidemic season . 's study , influenza vaccination coverage among subjects aged 65 years in south korea , during the 2004 - 2005 influenza seasons , was 77.2% . data on the correlations between the immunogenicity of influenza vaccines and wbc count or lymphocyte count are often conflicting . reported that a response to influenza antigen was not affected by the total wbc count at immunization . other reports have demonstrated a positive correlation between the response to influenza antigen and total numbers of circulating lymphocytes or neutrophils on the day of immunization . in this study , the wbc and lymphocyte counts did not affect the response to influenza antigen , but anc affected the response to b influenza antigen . in this study , total chemotherapy duration showed not a negative , but a positive correlation with the immune response for some strains . although the reason why it showed a positive correlation between total chemotherapy duration and immune response will be further investigated or verified , the fact that at least the long term chemotherapy in colorectal cancer subjects did not impair an immune response of influenza vaccination , probably due to relatively weaker chemo intensity of colorectal cancer than other cancer , offer a good reason to recommend influenza vaccination to colorectal cancer patients under chemotherapy . in the present study , therefore , other strategies are needed to reinforce the efficacy of influenza vaccination in immunocompromised patients . family member vaccination , healthcare worker vaccination , 2-dose vaccination , and avoidance of immunization at times of leukopenia or lymphopenia , might be effective . because children play an important role in the transmission of influenza virus , all children in a cancer patient 's family should be vaccinated . the lack of an appropriate control group is a limitation of this study ; hence , comparison with diabetes mellitus patients was conducted ( table 5 ) . further studies with larger sample sizes that assess the correlation between the immunogenicity of influenza vaccines and chemotherapy state , chemotherapy regimen , and time from cessation of chemotherapy are necessary . in view of the observed acceptable but limited immune response and no serious side effects , annual influenza vaccination is strongly recommended for colorectal cancer patients .

purposealthough influenza is regarded as a major cause of morbidity and mortality in immunocompromised patients , vaccine coverage remains poor . we evaluated the immunogenicity of influenza vaccines in colorectal cancer patients.materials and methodsin this study , 40 colorectal cancer patients who received an influenza vaccine at the korea cancer center hospital during the 2009 - 2010 and 2010 - 2011 influenza seasons were analyzed . the blood samples were collected at prevaccination and 30 days post vaccination , and antibody titers were measured using the hemagglutination - inhibition tests.resultsin the 2009 - 2011 season , the seroprotection rate for h1n1 ( 94.7% ) was significantly higher than that for h3n2 ( 42.1% ) and b ( 47.3% ) . the seroconversion rate was 52.6% , 26.3% , and 36.8% for h1n1 , h3n2 , and b , respectively . fold increase of geometric mean titer ( mfi ) was 3.86 , 1.49 , and 3.33 for h1n1 , h3n2 , and b , respectively . in the 2010 - 2011 season , the seroprotection rate for h1n1 ( 57.1% ) was significantly higher than that for h3n2 ( 52.4% ) and b ( 38.1% ) . the seroconversion rate was 52.4% , 47.6% and 33.3% for h1n1 , h3n2 , and b , respectively . mfi was 12.29 , 3.62 and 4.27 for h1n1 , h3n2 , and b , respectively.conclusionour study cohort showed an acceptable immune response to an influenza vaccine without significant adverse effects , supporting the recommendation for annual influenza vaccination in colorectal cancer patients .

Introduction Materials and Methods 1. Study design 2. Vaccines 3. Antibody studies 4. Immunogenicity assessment 5. Statistical analysis Results 1. Study subjects 2. Overall immunogenicity in colorectal cancer patients 3. Immunogenicity according to age 4. Immunogenicity according to various factors influencing immune state 5. Adverse effects of vaccination Discussion Conclusion

although the immunogenicity of influenza vaccine varies annually , it is well accepted that an influenza vaccine can reduce morbidity and disease severity . however , no mass medical survey of vaccine coverage in immunocompromised patients has been conducted , and it is presumed that the actual vaccine coverage remains poor in high - risk patients , as is the case in other countries [ 6 - 9 ] . due to a currently rapid growing incidence , the importance about colorectal cancer is also growing in korea ; also because , different from other cancers , the chemotherapy regimen for colorectal cancer was well established , it is easy to identify the influences of chemotherapy on the immunogenicity of influenza vaccine . further , because colorectal cancer develops more frequently in older age , both aging and immune - compromising factors are supposed to act as the reducing factors in the immunogenicity of influenza . the primary aim of this study is to evaluate the influenza vaccine immunogenicity in a group of high - risk subjects , colorectal cancer patients , from the korea cancer center hospital ( kcch ) , which is a single institute experience , and to offer a reference for immunization guidelines . influencing the immunogenicity of influenza vaccine in colorectal patients . during the 2009 - 2010 and 2010 - 2011 influenza seasons , patients with colorectal cancer , under treatment at kcch , were invited to participate in this study . subjects with severe allergy to an influenza vaccine or egg protein , with acute febrile illness at the time of vaccination , who were receiving treatment with corticosteroids for other reasons except for the purpose of anticancer drug with a history of transfusion within 6 months , or with any other condition that might interfere with the evaluation of the study were excluded . sk influenza ix vaccine ( trivalent split influenza vaccine ; sk chemicals , seongnam , korea ) was administered intramuscularly to all subjects during the 2009 - 2010 and 2010 - 2011 influenza seasons . in the 2009 - 2010 influenza season , sk influenza ix vaccinecontained 15 g of each hemagglutinin from a / brisbane/59/2007 ( h1n1 ) strain ( ivr-148 ) , a / uruguay/716/2007 ( h3n2 ) strain ( nymcx-175c ) , and b / brisbane/60/2008 strain . in the 2010 - 2011 influenza season , sk influenza ix vaccine contained 15 g of each hemagglutinin from a / california/7/2009 ( reassortant nymc x-181 ; pandemic h1n1 , ph1n1 ) strain , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) strain , and b / brisbane/60/2008 strain . anti - hemagglutinin titers to h1n1 , h3n2 , and influenza b were measured using a / brisbane/59/2007 ivr-148 ( h1n1 ) , a / uruguay/716/2007 nymcx-175c ( h3n2 ) , and b / brisbane/60/2008 in the 2009 - 2010 season , and a / california/7/2009 ( reassortant nymc x-181 ; ph1n1 ) , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) , and b / brisbane/60/2008 in the 2010 - 2011 season . immunogenicity of the vaccine was assessed based on these findings : seroprotection rates on days 0 and 30 , seroconversion rate on day 30 , and mean fold increase ( mfi ) of geometric mean titer ( gmt ) of the hi assay between days 0 and 30 . we defined an acceptable but limited immune response at the seroprotection rate of 30 - 70% . multivariate logistic regression analysis was used for identifying various factors influencing on the immunogenicity of influenza vaccine for colorectal cancer patients . during the 2009 - 2010 and 2010 - 2011 influenza seasons , patients with colorectal cancer , under treatment at kcch , were invited to participate in this study . subjects with severe allergy to an influenza vaccine or egg protein , with acute febrile illness at the time of vaccination , who were receiving treatment with corticosteroids for other reasons except for the purpose of anticancer drug with a history of transfusion within 6 months , or with any other condition that might interfere with the evaluation of the study were excluded . sk influenza ix vaccine ( trivalent split influenza vaccine ; sk chemicals , seongnam , korea ) was administered intramuscularly to all subjects during the 2009 - 2010 and 2010 - 2011 influenza seasons . in the 2009 - 2010 influenza season , sk influenza ix vaccinecontained 15 g of each hemagglutinin from a / brisbane/59/2007 ( h1n1 ) strain ( ivr-148 ) , a / uruguay/716/2007 ( h3n2 ) strain ( nymcx-175c ) , and b / brisbane/60/2008 strain . in the 2010 - 2011 influenza season , sk influenza ix vaccine contained 15 g of each hemagglutinin from a / california/7/2009 ( reassortant nymc x-181 ; pandemic h1n1 , ph1n1 ) strain , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) strain , and b / brisbane/60/2008 strain . anti - hemagglutinin titers to h1n1 , h3n2 , and influenza b were measured using a / brisbane/59/2007 ivr-148 ( h1n1 ) , a / uruguay/716/2007 nymcx-175c ( h3n2 ) , and b / brisbane/60/2008 in the 2009 - 2010 season , and a / california/7/2009 ( reassortant nymc x-181 ; ph1n1 ) , a / victoria/210/2009 ( reassortant nymc x-187 ; h3n2 ) , and b / brisbane/60/2008 in the 2010 - 2011 season . immunogenicity of the vaccine was assessed based on these findings : seroprotection rates on days 0 and 30 , seroconversion rate on day 30 , and mean fold increase ( mfi ) of geometric mean titer ( gmt ) of the hi assay between days 0 and 30 . we defined an acceptable but limited immune response at the seroprotection rate of 30 - 70% . multivariate logistic regression analysis was used for identifying various factors influencing on the immunogenicity of influenza vaccine for colorectal cancer patients . the mean age was 61.1 years ( range , 26.52 to 88.68 years ; 66.0 years in the 2009 - 2010 season and 56.70 years in the 2010 - 2011 seasons ) . the male / female ratio was 26/14 ( 11/8 in the 2009 - 2010 seasons and 15/6 in the 2010 - 2011 seasons ) . the average interval between influenza vaccination and post - vaccination sampling was 33.2 days in the 2009 - 2010 season and 44.15 days in the 2010 - 2011 seasons ( table 1 ) . before vaccination , 68.4% ( n=13 ) , 31.6% ( n=6 ) , and 10.5% ( n=2 ) of 19 patients enrolled in the 2009 - 2010 season had seroprotective antibody titers ( 1:40 ) against h1n1 , h3n2 , and b , respectively . after vaccination , 94.7% ( n=18 ) , 42.1% ( n=8 ) , and 47.3% ( n=9 ) of patients had seroprotective titers against h1n1 , h3n2 , and b , respectively . seroconversion was observed in 52.6% ( n=10 ) , 26.3% ( n=5 ) , and 36.8% ( n=7 ) of patients against h1n1 , h3n2 , and b , respectively . gmt fold increase was 3.86 , 1.49 , and 3.33 against h1n1 , h3n2 , and b , respectively ( table 2 ) . these seroprotection and seroconversion rates indicated good immunogenicity ( seroprotection rate70% , seroconversion40% ) against h1n1 , and an acceptable but limited immune response against influenza h3n2 and b. the gmt fold increase indicated good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2009 - 2010 season . before vaccination , 4.8% ( n=1 ) , 4.8% ( n=1 ) , and 4.8% ( n=1 ) of 21 patients enrolled in the 2010 - 2011 season had seroprotective antibody titers ( 1:40 ) against ph1n1 , h3n2 , and influenza b , respectively . after vaccination , the seroprotection rates were 57.1% ( n=12 ) , 52.4% ( n=11 ) , and 38.1% ( n=8 ) , respectively , against these strains . seroconversion was observed for 52.4% ( n=11 ) , 47.6% ( n=10 ) , and 33.3% ( n=7 ) of patients against ph1n1 , h3n2 , and influenza b , respectively . gmt fold increase was 12.29 , 3.62 , and 4.27 against the respective strains ( table 2 ) . this seroprotection rate indicated an acceptable but limited immune response against all vaccine strains in the 2010 - 2011 seasons . however , the seroconversion rate indicated a good immunogenicity ( seroconversion 40% ) against ph1n1 and h3n2 strains , while there was an acceptable but limited immune response against the influenza b strain . the gmt fold increase indicated a good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2010 - 2011 seasons . in the 2009 - 2010 season , the seroprotection rates against h1n1 , h3n2 , and influenza b were 100.0% , 28.6% , and 71.4% , respectively , in those aged < 65 years ; these values were 91.7% , 50.0% , and 33.3% , respectively , in those aged65 years . the seroconversion rates against h1n1 , h3n2 , and influenza b were 57.1% , 14.3% , and 57.1% , respectively , in those aged < 65 years ; these values were 50.0% , 33.3% , and 25.0% , respectively , in those aged65 years . gmt fold increases against h1n1 , h3n2 , and influenza b were , 5.38 , 1.00 , and 5.94 , respectively , in those aged < 65 years ; these values were 6.56 , 2.97 , and 4.00 , respectively , in those aged65 years . in the 2010 - 2011 season , the seroprotection rates against ph1n1 , h3n2 , and influenza b were 60.0% , 46.7% , and 26.7% , respectively , for those aged < 65 years ; these values were 50.0% , 66.7% , and 66.7% , respectively , for those aged65 years . seroconversion rates against ph1n1 , h3n2 , and influenza b were 53.3% , 40.0% , and 26.7% , respectively , in those aged < 65 years ; these values were 50.0% , 66.7% , and 50.0% , respectively , in those aged65 years . the mean age was 61.1 years ( range , 26.52 to 88.68 years ; 66.0 years in the 2009 - 2010 season and 56.70 years in the 2010 - 2011 seasons ) . the male / female ratio was 26/14 ( 11/8 in the 2009 - 2010 seasons and 15/6 in the 2010 - 2011 seasons ) . the average interval between influenza vaccination and post - vaccination sampling was 33.2 days in the 2009 - 2010 season and 44.15 days in the 2010 - 2011 seasons ( table 1 ) . before vaccination , 68.4% ( n=13 ) , 31.6% ( n=6 ) , and 10.5% ( n=2 ) of 19 patients enrolled in the 2009 - 2010 season had seroprotective antibody titers ( 1:40 ) against h1n1 , h3n2 , and b , respectively . after vaccination , 94.7% ( n=18 ) , 42.1% ( n=8 ) , and 47.3% ( n=9 ) of patients had seroprotective titers against h1n1 , h3n2 , and b , respectively . seroconversion was observed in 52.6% ( n=10 ) , 26.3% ( n=5 ) , and 36.8% ( n=7 ) of patients against h1n1 , h3n2 , and b , respectively . gmt fold increase was 3.86 , 1.49 , and 3.33 against h1n1 , h3n2 , and b , respectively ( table 2 ) . these seroprotection and seroconversion rates indicated good immunogenicity ( seroprotection rate70% , seroconversion40% ) against h1n1 , and an acceptable but limited immune response against influenza h3n2 and b. the gmt fold increase indicated good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2009 - 2010 season . before vaccination , 4.8% ( n=1 ) , 4.8% ( n=1 ) , and 4.8% ( n=1 ) of 21 patients enrolled in the 2010 - 2011 season had seroprotective antibody titers ( 1:40 ) against ph1n1 , h3n2 , and influenza b , respectively . after vaccination , the seroprotection rates were 57.1% ( n=12 ) , 52.4% ( n=11 ) , and 38.1% ( n=8 ) , respectively , against these strains . seroconversion was observed for 52.4% ( n=11 ) , 47.6% ( n=10 ) , and 33.3% ( n=7 ) of patients against ph1n1 , h3n2 , and influenza b , respectively . gmt fold increase was 12.29 , 3.62 , and 4.27 against the respective strains ( table 2 ) . this seroprotection rate indicated an acceptable but limited immune response against all vaccine strains in the 2010 - 2011 seasons . however , the seroconversion rate indicated a good immunogenicity ( seroconversion 40% ) against ph1n1 and h3n2 strains , while there was an acceptable but limited immune response against the influenza b strain . the gmt fold increase indicated a good immunogenicity ( gmt fold increase2.5 ) against all vaccine strains in the 2010 - 2011 seasons . in the 2009 - 2010 season , the seroprotection rates against h1n1 , h3n2 , and influenza b were 100.0% , 28.6% , and 71.4% , respectively , in those aged < 65 years ; these values were 91.7% , 50.0% , and 33.3% , respectively , in those aged65 years . the seroconversion rates against h1n1 , h3n2 , and influenza b were 57.1% , 14.3% , and 57.1% , respectively , in those aged < 65 years ; these values were 50.0% , 33.3% , and 25.0% , respectively , in those aged65 years . gmt fold increases against h1n1 , h3n2 , and influenza b were , 5.38 , 1.00 , and 5.94 , respectively , in those aged < 65 years ; these values were 6.56 , 2.97 , and 4.00 , respectively , in those aged65 years . there were no statistically significant differences in the immune responses between the 2 age groups in the 2009 - 2010 seasons , except in post - vaccination gmt against h3n2 ( table 3 ) . in the 2010 - 2011 season , the seroprotection rates against ph1n1 , h3n2 , and influenza b were 60.0% , 46.7% , and 26.7% , respectively , for those aged < 65 years ; these values were 50.0% , 66.7% , and 66.7% , respectively , for those aged65 years . seroconversion rates against ph1n1 , h3n2 , and influenza b were 53.3% , 40.0% , and 26.7% , respectively , in those aged < 65 years ; these values were 50.0% , 66.7% , and 50.0% , respectively , in those aged65 years . this study demonstrated the safety of the trivalent , an inactivated influenza vaccine in colorectal cancer patients , with an acceptable but limited immune response . this result matched well with the results of previous studies in immunocompromised patients , especially in colorectal cancer patients [ 13 - 16 ] . reported that 70.6% of 85 colorectal cancer patients vaccinated with an influenza vaccine showed an immune response during the 2006 - 2007 influenza seasons . in the present study , the seroprotection rate was 94.7% for h1n1 , 42.1% for h3n2 , and 47.4% for influenza b in the 2009 - 2010 seasons . the seroprotection rate against h1n1 was significantly higher than that against h3n2 and influenza b in the 2009 - 2010 seasons . compared with the control group ( diabetes mellitus patients , n=15 ) vaccinated in the 2009 - 2010 season , there were no differences in the immunogenicity , except against the influenza b strain . in the present study , the relatively good immune response in subjects aged65 years is probably related to a higher rate of influenza vaccination during the previous epidemic season . although the reason why it showed a positive correlation between total chemotherapy duration and immune response will be further investigated or verified , the fact that at least the long term chemotherapy in colorectal cancer subjects did not impair an immune response of influenza vaccination , probably due to relatively weaker chemo intensity of colorectal cancer than other cancer , offer a good reason to recommend influenza vaccination to colorectal cancer patients under chemotherapy . in the present study , therefore , other strategies are needed to reinforce the efficacy of influenza vaccination in immunocompromised patients . further studies with larger sample sizes that assess the correlation between the immunogenicity of influenza vaccines and chemotherapy state , chemotherapy regimen , and time from cessation of chemotherapy are necessary . in view of the observed acceptable but limited immune response and no serious side effects , annual influenza vaccination is strongly recommended for colorectal cancer patients .

there are implications of recent global financial crises on financial and economic stability , and hence on food security because the global food system is very vulnerable . world economic forum report 2013 stated : global food and nutrition security is a major global concern as the world prepares to feed a growing population on a dwindling resource base , in an era of increased volatility and uncertainty . thus , measures to improve food security have never been more urgently needed ( 2 ) . the main task for every government starts to be how to provide sustainable and healthy food supply , and protect its population against foodborne diseases . marc danzon stated that one of the most important steps is the coordination of policy making to ensure that the food policies of all sectors give the proper priority to public health ( 3 ) . many organizations are involved in the area of providing food security since the world food conference in 1974 defined food security in terms of food supply - assuring availability and price stability of basic foodstuffs at the international and national level ( 4 ) . food safety and food security are two highly interrelated subjects with similar and common fields of activities in terms and references . the boundaries between theory and practice may sometimes be blurred , and there are also various definitions of terms - this all results in quite versatile terminology which may also sometimes be confusing . food safety is concerned with all aspects , whether immediate or long - term , that may make food unsafe for the consumer . therefore , safe foods or foodstuffs contain nothing that is hazardous or injurious ( 5 ) . in many countries there exists some kind of expert bodies to avoid duplicating of efforts and decreasing unnecessary costs in the field of food security . therefore , since the safe food is a precondition for the protection and promotion of health , many organizations were and are involved in the efforts to mitigate the effects of foodborne illnesses on public health . the most influential is who . who works closely with the food and agriculture organization of the united nations ( fao ) , who for animal health ( oie ) , and other international organizations to address food safety issues along the entire food chain . the department of food safety and zoonoses ( fos ) provides leadership in its efforts to lower the burden of diseases from food and animals across the globe ( 6 ) . in 2004 , who launched the international food safety authorities network ( infosan ) as an early warning , communication and prevention system which enables rapid access to information during the food safety emergencies ( 7 ) . in the european region there are a lot of documents that outline the addressed policies about food supplies , food safety and nutrition , and also about mobilization resources for the activities on poverty and health ( 8 , 9 ) . across the ocean , in the usa the food safety legislation has recently been changed and that action initiated numerous discussions ( 10 - 12 ) . in the united states of america ( usa ) there are a great number of federal , state , and local agencies which share responsibilities for regulating the safety of the food supply . the combined efforts of the food industry and government regulatory agencies are often credited with making the us food supply among the safest in the world , but despite all efforts the centre for disease control and prevention ( cdc ) reports that each year an estimated one in six americans - a total of 48 million people - become sick from food borne illnesses through contaminated food ( 13 ) . the usa 111 congress passed comprehensive food safety legislation on december 2010 , authorizing the additional appropriations and staff for the us food and drug administration ( fda ) future food safety activities pathogens ( 14 ) . the republic of serbia ( rs ) has experienced important changes on its way to reach full membership in the european union . the impacts of global financial crisis made situation in the country more serious and its effects spilled over to the entire society . the number of poor and unemployed population has been increasing since then ( 15 ) . despite numerous efforts to provide adequate level of food safety the latest data have confirmed that legal framework is not the firm base for prevention of violations of current regulations in the area of food safety . therefore , the urgent needs for changes are recognized among stakeholders and are expected after the new government following the recent elections is established . the stakeholders involved in the implementation of actions are financed mainly from the state budget ; the finances are divided among all organisational units and provide the required number of staff performing official controls and cover costs for the implementation of such controls . a small portion is provided by international sources through donations and different projects , mostly from the european union . considering the above mentioned , the authors decided to analyze in this study how it is possible to improve the food safety system in serbia and avoid suffering of population as well as the health system obviously overwhelmed by numerous problems . adapting to the additional threats to food safety requires an integrated food system approach which will make the food system less vulnerable to attacks . the authors proved their hypothesis that response to food outbreak could be more effective after carefully conducted examination of literature , reports and modern practice . the purpose of this study is to address a current state in the food safety area in the republic of serbia and its inseparable connection with the public health . the methodology used in the study is the most appropriate for the social science . in the introduction the authors present the importance of food in human civilization , and the information needed for understanding the scope of the research problem . the second chapter is devoted to the organization of food safety system and health care in serbia regarding the european regulations . the last chapter presents the authors recommendation to the policy makers about the development of epidemiological investigation capacities as a tool for providing better food safety in serbia . among many other needs of innovation , there is one in particular to be highlighted and that is a change in conducting the epidemiological investigation by join efforts of both public health workforce and law enforcement in the food safety area . the main objective of this study is to consider current state in the food safety system in serbia . the additional objective is to present the need for improvement of epidemiological investigation in the food outbreak , which could be useful in the improvement of the system performances and these facts can be used to punish the violators . the study demonstrates an urgent need for the implementation of specific food safety policies in practice . because the activities in the food security area in serbia have been characterized by fragmented response , this approach will help to overcome and to develop joint activities which will be more successful . strengthening food safety policies and health system at the same time will form solid basis for future adequate response to the risks in food safety area . the authors use preparation methodology suitable for social sciences and the objectives of the study . the official publications of the relevant authorities in serbia and from the european union , and broader international community were examined . relevant scientific literature was also searched from numerous libraries like wageningen and in a few serbian libraries and trough different websites . the documents were also collected from electronic sources : literature resource centre like go gale group , online research database ebsco - host , academic onefile , e library , and printed material ( books , journals , official documents ) . this research is related to the events and changes in the area of health , food safety and in social sphere which occurred in serbia during the period 2008 - 2013 . serbia is a part of south - eastern europe ( see ) and today it is an eu candidate country . to facilitate the process of legal approximation , the serbian parliament adopted the national plan for the adoption of the acquis 2013 - 2016 ( npaa ) ( 16 ) therefore , acquis communautaire in chapter 12 - food safety , veterinary and phytosanitary policy has been transposed into serbian national legislation . competencies in the field of food safety have been divided between the ministry of agriculture , forestry and water management ( mafwm ) and the ministry of health ( mh ) . the similar organization is accepted in almost all countries in the region of west balkans following the recommendation of the european union ( eu ) , croatia , macedonia , montenegro etc . the ministry of internal and external trade and teleco - mmu - nication ( mott ) is responsible for the implementation of the law on standardization , and the market inspection department is responsible for inspecting food quality at the retail level . there are also a significant number of academic institutions departments at universities which undertake research contributing to the area of food safety . hence , the influential organizations are also the serbian chamber of commerce , and the national statistical office . mafwm is responsible for veterinary , phytosanitary and food safety policies ( the safety of food of animal origin , composite food , food of plant origin and feed ) . the mafwm supervises the legality of work through its four directorates : veterinary directorate ; plant protection directorate ; general inspectorate and directorate for national reference laboratories ( dnrl ) . the mafwm is central competent authority responsible for the organizations of official control and for ensuring efficient and effective coordination among all authorities and their directorates responsible for carrying out official controls of food . veterinary , phytosanitary and agricultural inspections are managed centrally but distributed territorially , so as to cover the entire territory of the rs . in the autonomous province of vojvodina ( apv ) , the tasks related to food safety that fall under the competency of the mh have been conferred to the secretary of the health of the province . the flow diagram presenting the serbian food safety system is given in fig.1 and it is useful for understanding the responsibilities of different sectors , inspection units and other stakeholders . it is clear that due to numerous organisational units the communication among stakeholders is not always simple and easy to achieve , especially in the case of emergency . competence of authorities in charge of food safety , veterinary and phytosanitary policies ( source : serbian government , 2011 ) the surveillance of foodborne diseases in the rs is a part of the communicable disease information system . the provisions for surveillance of foodborne diseases are based on the law on protection of population from communicable diseases ( 17 ) , under which the first contact physician is obliged to report an infectious disease or epidemic to the epidemiological service in charge . a national communication centre for surveillance is established , based on the european centre for disease prevention and control ( ecdc ) methodology , and capable to provide on - line communication in the case of pandemics in order to coordinate the national health care network and be in contact with the ecdc ( 18 ) . this part is done by the serbian national institute of public health ( sniph ) and through the network of regional public health institutes . pursuant to the open competition conducted by the general inspectorate , the authorities established a list of laboratories for laboratory tests in the field of food and feed safety and the implementation of monitoring programme . with entry into force of the rulebook on general and specific requirements for food hygiene in all stages of production , processing and circulation governing the microbiological criteria for foodstuffs , as of 1 june 2011 all food testing laboratories will apply test methods in accordance with the ec ( 19 ) . according to the food safety law as from 2009 food business operators should implement hazard analysis and critical control points ( haccp ) principles in all establishments involved in the production of animal and non animal food . serbia uses a lot of sources from international community for the purposes of food safety . under the 2003 community assistance for reconstruction , development and stability in the balkans programme , technical assistance was provided to two ministries to strengthen the protection of food safety . the health care system in serbia is characterized by a well - developed network of health institutions , with predominantly state - owned health care facilities . their work is financed from the budget of the government and the republic fund of health insurance ( rfhi ) . serbia allocated roughly the same percentage of its gdp to health care as the eu average ( 10.4% - serbia ; 9.5% eu average in 2010 ) . however , in terms of total money allocated , serbia allocates less than half of the eu average . due to the economic crisis , public health expenditure is decreasing while private expenditure for health increases ( 20 ) . therefore , the serbian health care system is facing serious problems due to political circumstances and financial constraints . democratic change brought expectations for a better future of the health system , and the first recovery signs were visible thanks to enormous number of international donations ( 21 ) . in serbia , the most important institution of public health is the national institute of public health ( sniph ) dr milan jovanovi - batut . the structural characteristics and the functioning of the sniph in serbia are regulated by a separate law on public health adopted in 2009 ( 22 ) . the sniph is financed from the governmental budget and by the republic fund of health insurance ( rhif ) . in the recently published report - european health consumer index ( ehci ) -serbian health system was listed as the last in the group of 35 eu countries ( 23 ) . the public recognized the health care system in serbia as the least efficient and the most corrupted and inefficient . therefore , the government and mh undertook many activities in order to improve the status of the health care system ( 24 ) . many countries are applying different measures in the area of protection of their population from foodborne diseases . the iranian ministry of health and medical education has developed a method for the disinfection of raw eaten fruits and vegetables ( 25 ) . serbia is developing the rapid alert system for food and feed ( rasff ) and in the last year the products from serbia have been the subject of notifications in the european rasff system , particularly related to fruit and vegetables and most recently ( 2013 ) related to norovirus contamination of frozen raspberries and aflatoxin contamination of maize . the products originating from serbia have been the subject of 48 notifications from 20082013 due to the data presented on the rasff portal database . in 2013 , the serbian public focused on food outbreak in smederevo , and aflatoxin affair in maize , and due to that in milk and other dairy products , etc . each of these events caused a great odium in the public due to the mutual accusations between the competent authorities . the first event happened in the town of smederevo during the famous tourist and business manifestation the institute of public health in pozarevac announced the epidemic of diarrhea and gastroenteritis because according to the smederevo general hospital , in two days medical advice was sought by about 310 patients , including 220 children and 90 adults . they undertook the necessary measures : epidemiological investigation , curing , health education work and continual cooperation with sanitary and veterinary inspections of podunavski county and the epidemic was recalled on 1 october . the institute stated that the source of disease was not recognized but they think there is no evidence of the connection between food poisoning and what was happening in smederevo . they also witnessed that during the epidemic they took a small number of laboratory samples and could not have any clue about the route of the foodborne outbreak . this event would not be different from other similar food outbreaks for the public if after those statements the mayor of smederevo , jasna avramovic , did not get into the public and announced that there was : no poisoning epidemic in smederevo . she said that she would be forced to seek protection from court institutions over the media articles published dealing with the alleged poisoning epidemic in smederevo despite the official statement of the institute of public health in pozarevac . she claimed that the term poisoning epidemic did not exist and presented the doubt that media titles were politically motivated ! another event in serbia which caused the great attention of the public was the presence of aflatoxin in milk in 2013 . the minister of agriculture said in the course of the affair that the recent discovery of elevated aflatoxin concentration in milk stunned his ministry . he stated that harmonization of serbian standards with those in use in the eu was done without enough understanding for reality and their enforceability and due to that these rules have brought major changes the primary responsibility for food quality is on the producers , and the government takes on the role of a controller . the trade minister rasim ljaji also addressed the session to say that the financial damage from the aflatoxin crisis could cost serbia between 100 and 125 million euros . from the reactions of the competent authorities in those events , it is clear that serbian policy - makers are not familiar with the way of adequate risk communication . in serbian society the risk communication is far from favourable and lags behind the modern risk approaches ( 26 ) . in the last two years serbia recorded numerous events where state stakeholders stayed silent for a while , as in food outbreak in smederevo , food outbreak in six elementary belgrade schools and so on . the urgent action in the area of appropriate risk communication during the food outbreak , the improvement of surveillance , and established coordination between all stakeholders has to be an urgent future task for policy - makers . the only way to build trust between health care workers and the public is by delivering accurate and timely information , even if it might be scary . in serbia epidemiological investigations and public health surveillance include the ability to create , maintain , support , and strengthen surveillance and detection systems and epidemiological investigation processes , as well as to expand these systems and processes in response to the incidents of public health significance . the basic goals in these investigations are : to protect population ; to stop spread of disease and , of course , to protect the health personnel . one of the important actions in the usa is connected with work of the office of public health preparedness and response / centers for disease control and response ( cdc ) . the cdc applied systematic approach to develop the public health preparedness capabilities . in march 2011 , the cdc provided a guide according to which the state and local jurisdictions can be used to better organize their work , plan their priorities , and decide which capabilities they have the resources to build or sustain ( 27 ) . the capability of stakeholders in this action should consist of the ability to perform the following functions : conduct public health surveillance and detection ; conduct public health and epidemiological investigations ; recommend and analyze monitoring action , and improve public health surveillance and epidemiological investigation systems . when the foodborne outbreak happens , the stakeholders in serbia have to apply the scientific method of epidemiological investigation . in that process the epidemiologists use laboratory scientists , statisticians , physicians and other public health professionals to get to the root of health problems and outbreaks in a community . hence , in serbia the epidemiologists are not familiar with the broader application of joint investigations between epidemiologists and law enforcement in the investigation processes . in serbia despite their regular medical education the epidemiologist have a little chance to gain some additional knowledge and skills necessary for conducting epidemiological investigations . they have just a few trainings which were provided as a part of some international projects . disease detectives - epidemiologists have to conduct epidemiological investigations by combined use of diplomacy , logical thinking , problem - solving ability , quantitative skills , epidemiologic know - how , and judgment ( 28 ) . the first step in investigation is to quickly collect the accurate data . in other words , epidemiologists can not afford to conduct an investigation that is quick and dirty . they must conduct investigations that are quick and clean ( 29 ) . the authors of the study found that in serbia the epidemiologists faced with numerous objective and subjective obstacles in their investigation . serbia still does not have a reference laboratory which is able to conduct some specific analyses like in aflatoxin affair , or the detection of cyanobacterical toxins from algae in vrutci lake which caused the actual long - term interruption of water supply for 60,000 citizens in the town of uzice . the objective of the european commission project regarding dnrl in batajnica to make it fully operational in order to be in line with the eu best practice and standards is still without success . the project titled equipment and courier service supply and capacity building of serbian national referent laboratories directorate in food chain started by providing laboratories with the equipment during 2003 , but that equipment was turned over to the official laboratories in the field of food safety , veterinary and phytosanitary controls . serbia still does not have dnrl and that was the reason for seeking rikelt collaboration in netherlands ( 30 ) . in serbian society it is well known that many food outbreaks may go undetected because of health habits of the population . recent changes in the practice of the primary health care institutions are another reason for this behaviour of patients . without previously making an appointment with their chosen physician , the patients are not able to go seek a medical examination except in case of emergency . this is obligatory and as this regulation is seen by the patients as complicated and enforced by the health institutions without any agreement with the patients , they do not go to the physician and take some alternative medicines , or go to private practice which is not so strict in the process of reporting the disease which might be caused by foodborne outbreak . the most important task for the epidemiologists is to decide whether to investigate a possible outbreak . the decisions regarding where and how extensively to investigate a potential outbreak depend on a variety of factors . these usually include some factors related to the existing problem , some are related to the availability of staff and resources of a health department and some are related to external concerns . hence , public , political or legal concerns can also be a force behind the decision to conduct an investigation . in the case of aflatoxin in maize in serbia it was obvious that some oppositional political parties seized the moment to gain advantage over their opponents and accused the government parties for announcing inaccurate information about the level of contamination in milk . this was a reason to send the collected samples to an independent reference laboratory in wageningen the most important public health reasons for investigation of an outbreak are to help guide disease prevention and control strategies . the importance of outbreak investigation could be seen from the following actions ( 31 ) : stop the current outbreak from spreading ; prevent future similar outbreaks ; provide scientific explanation of the event ; provide knowledge for the understanding of the disease process ; react to and calm public and political concerns , and train epidemiologists . the recommendation to the future epidemiological investigations is that sometimes when the epidemiology does not fit the usual or natural patterns of transmission , the investigators should think about the intentional modes of transmission . in the usa after 2001 terrorism and onwards , bioterrorism is recognized as a new and increasing threat to the americans and this requires the different approaches in the protection of public health of the population . the new approach requires law enforcement , other public safety organizations , and public health agencies to work together ( 32 ) . to foster the improved understanding of the investigative goals and methodology , and strengthen interdisciplinary collaborative effectiveness in response to future attacks involving biological agents , in the spring of 2002 the public health law program of the us cdc in partnership with other agencies and organizations undertook the development of a module for the joint training of law enforcement and public health officials ( 33 ) . the field investigations of disease outbreaks is the element of public health which is recognized in the usa as one that will most resemble law enforcement investigations because of the types of information collected and the means by which they are collected . how this collaboration could be useful for any country is proven in a case in dallas , oregon . the investigators of an outbreak of salmonellosis were stumped when they were able to implicate salad bars in several local restaurants , but could not identify any common ingredients or distribution system . a year later , a member of a local cult admitted that the cult had intentionally contaminated the salads bars with salmonella organisms ( 34 ) . the next improvement in the epidemiological investigation in serbia could be engagement of private investigators or specific places in public health departments which use specific kind of epidemiological investigations , so called food sleuths . one of the famous cases was when the cdc s disease detective casey barton behravesh helped track the source of a 2010 outbreak of salmonella infections that sickened more than 270 people in more than 40 states . the resourceful use of unconventional data helped the cdc and its partners across the country quickly identify the source of the problem and stop the outbreak . in an investigation of a food outbreak , the health department may be able to allay population fears by documenting that the outbreak was the result of an inadvertent or naturally occurring exposure . the need for communicating with the public health and clinical community has long been acknowledged in serbia , but the need for communicating quickly and effectively with the elected officials and the public became obvious in 2013 during the affair with aflatoxin in milk and dairy products . the law on food safety stipulates the adoption of the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment , the causes of which are the food or feed and the occurrence of which could not be foreseen , prevented , eliminated or abated to the acceptable level . the programme has to be adopted by the government , and it governs precisely the following : the type of situation in which direct or indirect risk to human health caused by food or feed exists ; the measures that have to be implemented without delay once it is established that food or feed is posing a serious threat to humans or animals , either directly or indirectly through the environment ; the crisis management procedures which include the principle of transparency and communication ; the plan of exercises and simulations for crisis management purposes . pursuant to the programme , the minister of agriculture shall adopt , with the consent of the minister responsible for public health , a special crisis management plan depending on the types of risks and shall form a crisis team to implement the special plan , but in the affair of aflatoxin both the officials and the public recognized that this program was not established . the positive practice from the usa , where depending on the type of outbreak the number of involved agencies may be quite large , is not recognized in serbia . apart from the epidemiologists , it is not known in the public that anyone else was in charge to conduct epidemiological investigation . it would be useful if serbian policy - makers recognized that the staffs from different agencies have different perspectives , approaches , and priorities that must be useful in any kind of outbreak . for example , whereas the public health investigation may focus on identifying a pathogen , the source and mode of transmission , a criminal investigation is likely to focus on finding the perpetrator who has to be punished at the conclusion of the investigation . the criminal law in serbia has changed recently and the greatest change for the stakeholders in the future will be the new kind of prosecutors investigation . as for the opinion of experts , its implementation in practice is questionable because of a lot of uncertainties . among all goals which should be achieved in the process of an epidemiological investigation of foodborne outbreak , one is particularly important for the population and the interested parties : to maximize resources and facilitate communication and interaction among public health officials and law enforcement in serbia following the practice from the developed countries . serbia is a part of south - eastern europe ( see ) and today it is an eu candidate country . to facilitate the process of legal approximation , the serbian parliament adopted the national plan for the adoption of the acquis 2013 - 2016 ( npaa ) ( 16 ) therefore , acquis communautaire in chapter 12 - food safety , veterinary and phytosanitary policy has been transposed into serbian national legislation . competencies in the field of food safety have been divided between the ministry of agriculture , forestry and water management ( mafwm ) and the ministry of health ( mh ) . the similar organization is accepted in almost all countries in the region of west balkans following the recommendation of the european union ( eu ) , croatia , macedonia , montenegro etc . the ministry of internal and external trade and teleco - mmu - nication ( mott ) is responsible for the implementation of the law on standardization , and the market inspection department is responsible for inspecting food quality at the retail level . there are also a significant number of academic institutions departments at universities which undertake research contributing to the area of food safety . hence , the influential organizations are also the serbian chamber of commerce , and the national statistical office . mafwm is responsible for veterinary , phytosanitary and food safety policies ( the safety of food of animal origin , composite food , food of plant origin and feed ) . the mafwm supervises the legality of work through its four directorates : veterinary directorate ; plant protection directorate ; general inspectorate and directorate for national reference laboratories ( dnrl ) . the mafwm is central competent authority responsible for the organizations of official control and for ensuring efficient and effective coordination among all authorities and their directorates responsible for carrying out official controls of food . veterinary , phytosanitary and agricultural inspections are managed centrally but distributed territorially , so as to cover the entire territory of the rs . in the autonomous province of vojvodina ( apv ) , the tasks related to food safety that fall under the competency of the mh have been conferred to the secretary of the health of the province . the flow diagram presenting the serbian food safety system is given in fig.1 and it is useful for understanding the responsibilities of different sectors , inspection units and other stakeholders . it is clear that due to numerous organisational units the communication among stakeholders is not always simple and easy to achieve , especially in the case of emergency . competence of authorities in charge of food safety , veterinary and phytosanitary policies ( source : serbian government , 2011 ) the surveillance of foodborne diseases in the rs is a part of the communicable disease information system . the provisions for surveillance of foodborne diseases are based on the law on protection of population from communicable diseases ( 17 ) , under which the first contact physician is obliged to report an infectious disease or epidemic to the epidemiological service in charge . a national communication centre for surveillance is established , based on the european centre for disease prevention and control ( ecdc ) methodology , and capable to provide on - line communication in the case of pandemics in order to coordinate the national health care network and be in contact with the ecdc ( 18 ) . this part is done by the serbian national institute of public health ( sniph ) and through the network of regional public health institutes . pursuant to the open competition conducted by the general inspectorate , the authorities established a list of laboratories for laboratory tests in the field of food and feed safety and the implementation of monitoring programme . with entry into force of the rulebook on general and specific requirements for food hygiene in all stages of production , processing and circulation governing the microbiological criteria for foodstuffs , as of 1 june 2011 all food testing laboratories will apply test methods in accordance with the ec ( 19 ) . according to the food safety law as from 2009 food business operators should implement hazard analysis and critical control points ( haccp ) principles in all establishments involved in the production of animal and non animal food . serbia uses a lot of sources from international community for the purposes of food safety . under the 2003 community assistance for reconstruction , development and stability in the balkans programme , technical assistance was provided to two ministries to strengthen the protection of food safety . the health care system in serbia is characterized by a well - developed network of health institutions , with predominantly state - owned health care facilities . their work is financed from the budget of the government and the republic fund of health insurance ( rfhi ) . serbia allocated roughly the same percentage of its gdp to health care as the eu average ( 10.4% - serbia ; 9.5% eu average in 2010 ) . however , in terms of total money allocated , serbia allocates less than half of the eu average . due to the economic crisis , public health expenditure is decreasing while private expenditure for health increases ( 20 ) . therefore , the serbian health care system is facing serious problems due to political circumstances and financial constraints . democratic change brought expectations for a better future of the health system , and the first recovery signs were visible thanks to enormous number of international donations ( 21 ) . in serbia , the most important institution of public health is the national institute of public health ( sniph ) dr milan jovanovi - batut . the structural characteristics and the functioning of the sniph in serbia are regulated by a separate law on public health adopted in 2009 ( 22 ) . the sniph is financed from the governmental budget and by the republic fund of health insurance ( rhif ) . in the recently published report - european health consumer index ( ehci ) -serbian health system was listed as the last in the group of 35 eu countries ( 23 ) . the public recognized the health care system in serbia as the least efficient and the most corrupted and inefficient . therefore , the government and mh undertook many activities in order to improve the status of the health care system ( 24 ) . many countries are applying different measures in the area of protection of their population from foodborne diseases . the iranian ministry of health and medical education has developed a method for the disinfection of raw eaten fruits and vegetables ( 25 ) . serbia is developing the rapid alert system for food and feed ( rasff ) and in the last year the products from serbia have been the subject of notifications in the european rasff system , particularly related to fruit and vegetables and most recently ( 2013 ) related to norovirus contamination of frozen raspberries and aflatoxin contamination of maize . the products originating from serbia have been the subject of 48 notifications from 20082013 due to the data presented on the rasff portal database . in 2013 , the serbian public focused on food outbreak in smederevo , and aflatoxin affair in maize , and due to that in milk and other dairy products , etc . each of these events caused a great odium in the public due to the mutual accusations between the competent authorities . the first event happened in the town of smederevo during the famous tourist and business manifestation the institute of public health in pozarevac announced the epidemic of diarrhea and gastroenteritis because according to the smederevo general hospital , in two days medical advice was sought by about 310 patients , including 220 children and 90 adults . they undertook the necessary measures : epidemiological investigation , curing , health education work and continual cooperation with sanitary and veterinary inspections of podunavski county and the epidemic was recalled on 1 october . the institute stated that the source of disease was not recognized but they think there is no evidence of the connection between food poisoning and what was happening in smederevo . they also witnessed that during the epidemic they took a small number of laboratory samples and could not have any clue about the route of the foodborne outbreak . this event would not be different from other similar food outbreaks for the public if after those statements the mayor of smederevo , jasna avramovic , did not get into the public and announced that there was : no poisoning epidemic in smederevo . she said that she would be forced to seek protection from court institutions over the media articles published dealing with the alleged poisoning epidemic in smederevo despite the official statement of the institute of public health in pozarevac . she claimed that the term poisoning epidemic did not exist and presented the doubt that media titles were politically motivated ! another event in serbia which caused the great attention of the public was the presence of aflatoxin in milk in 2013 . the minister of agriculture said in the course of the affair that the recent discovery of elevated aflatoxin concentration in milk stunned his ministry . he stated that harmonization of serbian standards with those in use in the eu was done without enough understanding for reality and their enforceability and due to that these rules have brought major changes the primary responsibility for food quality is on the producers , and the government takes on the role of a controller . the trade minister rasim ljaji also addressed the session to say that the financial damage from the aflatoxin crisis could cost serbia between 100 and 125 million euros . from the reactions of the competent authorities in those events , it is clear that serbian policy - makers are not familiar with the way of adequate risk communication . in serbian society the risk communication is far from favourable and lags behind the modern risk approaches ( 26 ) . in the last two years serbia recorded numerous events where state stakeholders stayed silent for a while , as in food outbreak in smederevo , food outbreak in six elementary belgrade schools and so on . the urgent action in the area of appropriate risk communication during the food outbreak , the improvement of surveillance , and established coordination between all stakeholders has to be an urgent future task for policy - makers . the only way to build trust between health care workers and the public is by delivering accurate and timely information , even if it might be scary . epidemiological investigations and public health surveillance include the ability to create , maintain , support , and strengthen surveillance and detection systems and epidemiological investigation processes , as well as to expand these systems and processes in response to the incidents of public health significance . the basic goals in these investigations are : to protect population ; to stop spread of disease and , of course , to protect the health personnel . one of the important actions in the usa is connected with work of the office of public health preparedness and response / centers for disease control and response ( cdc ) . the cdc applied systematic approach to develop the public health preparedness capabilities . in march 2011 , the cdc provided a guide according to which the state and local jurisdictions can be used to better organize their work , plan their priorities , and decide which capabilities they have the resources to build or sustain ( 27 ) . the capability of stakeholders in this action should consist of the ability to perform the following functions : conduct public health surveillance and detection ; conduct public health and epidemiological investigations ; recommend and analyze monitoring action , and improve public health surveillance and epidemiological investigation systems . when the foodborne outbreak happens , the stakeholders in serbia have to apply the scientific method of epidemiological investigation . in that process the epidemiologists use laboratory scientists , statisticians , physicians and other public health professionals to get to the root of health problems and outbreaks in a community . hence , in serbia the epidemiologists are not familiar with the broader application of joint investigations between epidemiologists and law enforcement in the investigation processes . in serbia despite their regular medical education the epidemiologist have a little chance to gain some additional knowledge and skills necessary for conducting epidemiological investigations . they have just a few trainings which were provided as a part of some international projects . disease detectives - epidemiologists have to conduct epidemiological investigations by combined use of diplomacy , logical thinking , problem - solving ability , quantitative skills , epidemiologic know - how , and judgment ( 28 ) . the first step in investigation is to quickly collect the accurate data . in other words , epidemiologists can not afford to conduct an investigation that is quick and dirty . they must conduct investigations that are quick and clean ( 29 ) . the authors of the study found that in serbia the epidemiologists faced with numerous objective and subjective obstacles in their investigation . serbia still does not have a reference laboratory which is able to conduct some specific analyses like in aflatoxin affair , or the detection of cyanobacterical toxins from algae in vrutci lake which caused the actual long - term interruption of water supply for 60,000 citizens in the town of uzice . the objective of the european commission project regarding dnrl in batajnica to make it fully operational in order to be in line with the eu best practice and standards is still without success . the project titled equipment and courier service supply and capacity building of serbian national referent laboratories directorate in food chain started by providing laboratories with the equipment during 2003 , but that equipment was turned over to the official laboratories in the field of food safety , veterinary and phytosanitary controls . serbia still does not have dnrl and that was the reason for seeking rikelt collaboration in netherlands ( 30 ) . in serbian society it is well known that many food outbreaks may go undetected because of health habits of the population . recent changes in the practice of the primary health care institutions are another reason for this behaviour of patients . without previously making an appointment with their chosen physician , the patients are not able to go seek a medical examination except in case of emergency . this is obligatory and as this regulation is seen by the patients as complicated and enforced by the health institutions without any agreement with the patients , they do not go to the physician and take some alternative medicines , or go to private practice which is not so strict in the process of reporting the disease which might be caused by foodborne outbreak . the most important task for the epidemiologists is to decide whether to investigate a possible outbreak . the decisions regarding where and how extensively to investigate a potential outbreak depend on a variety of factors . these usually include some factors related to the existing problem , some are related to the availability of staff and resources of a health department and some are related to external concerns . hence , public , political or legal concerns can also be a force behind the decision to conduct an investigation . in the case of aflatoxin in maize in serbia it was obvious that some oppositional political parties seized the moment to gain advantage over their opponents and accused the government parties for announcing inaccurate information about the level of contamination in milk . this was a reason to send the collected samples to an independent reference laboratory in wageningen the most important public health reasons for investigation of an outbreak are to help guide disease prevention and control strategies . the importance of outbreak investigation could be seen from the following actions ( 31 ) : stop the current outbreak from spreading ; prevent future similar outbreaks ; provide scientific explanation of the event ; provide knowledge for the understanding of the disease process ; react to and calm public and political concerns , and train epidemiologists . the recommendation to the future epidemiological investigations is that sometimes when the epidemiology does not fit the usual or natural patterns of transmission , the investigators should think about the intentional modes of transmission . in the usa after 2001 terrorism and onwards , bioterrorism is recognized as a new and increasing threat to the americans and this requires the different approaches in the protection of public health of the population . the new approach requires law enforcement , other public safety organizations , and public health agencies to work together ( 32 ) . to foster the improved understanding of the investigative goals and methodology , and strengthen interdisciplinary collaborative effectiveness in response to future attacks involving biological agents , in the spring of 2002 the public health law program of the us cdc in partnership with other agencies and organizations undertook the development of a module for the joint training of law enforcement and public health officials ( 33 ) . the field investigations of disease outbreaks is the element of public health which is recognized in the usa as one that will most resemble law enforcement investigations because of the types of information collected and the means by which they are collected . how this collaboration could be useful for any country is proven in a case in dallas , oregon the investigators of an outbreak of salmonellosis were stumped when they were able to implicate salad bars in several local restaurants , but could not identify any common ingredients or distribution system . a year later , a member of a local cult admitted that the cult had intentionally contaminated the salads bars with salmonella organisms ( 34 ) . the next improvement in the epidemiological investigation in serbia could be engagement of private investigators or specific places in public health departments which use specific kind of epidemiological investigations , so called food sleuths . one of the famous cases was when the cdc s disease detective casey barton behravesh helped track the source of a 2010 outbreak of salmonella infections that sickened more than 270 people in more than 40 states . the resourceful use of unconventional data helped the cdc and its partners across the country quickly identify the source of the problem and stop the outbreak . in an investigation of a food outbreak , the health department may be able to allay population fears by documenting that the outbreak was the result of an inadvertent or naturally occurring exposure . the need for communicating with the public health and clinical community has long been acknowledged in serbia , but the need for communicating quickly and effectively with the elected officials and the public became obvious in 2013 during the affair with aflatoxin in milk and dairy products . the law on food safety stipulates the adoption of the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment , the causes of which are the food or feed and the occurrence of which could not be foreseen , prevented , eliminated or abated to the acceptable level . the programme has to be adopted by the government , and it governs precisely the following : the type of situation in which direct or indirect risk to human health caused by food or feed exists ; the measures that have to be implemented without delay once it is established that food or feed is posing a serious threat to humans or animals , either directly or indirectly through the environment ; the crisis management procedures which include the principle of transparency and communication ; the plan of exercises and simulations for crisis management purposes . pursuant to the programme , the minister of agriculture shall adopt , with the consent of the minister responsible for public health , a special crisis management plan depending on the types of risks and shall form a crisis team to implement the special plan , but in the affair of aflatoxin both the officials and the public recognized that this program was not established . the positive practice from the usa , where depending on the type of outbreak the number of involved agencies may be quite large , , it is not known in the public that anyone else was in charge to conduct epidemiological investigation . it would be useful if serbian policy - makers recognized that the staffs from different agencies have different perspectives , approaches , and priorities that must be useful in any kind of outbreak . for example , whereas the public health investigation may focus on identifying a pathogen , the source and mode of transmission , a criminal investigation is likely to focus on finding the perpetrator who has to be punished at the conclusion of the investigation . the criminal law in serbia has changed recently and the greatest change for the stakeholders in the future will be the new kind of prosecutors investigation . as for the opinion of experts , its implementation in practice is questionable because of a lot of uncertainties . among all goals which should be achieved in the process of an epidemiological investigation of foodborne outbreak , one is particularly important for the population and the interested parties : to maximize resources and facilitate communication and interaction among public health officials and law enforcement in serbia following the practice from the developed countries . the study results confirmed that the area of food safety and the current state - of - affairs of the health care system in serbia have a lot of room for improvement . decision- and policy - makers have to take urgent steps to develop the policies in which public health will be in the centre of decisions in food safety area . serbian government with competent authorities and in collaboration with the international community , mostly with the eu , works permanently on developing the new approaches in the area of food safety and accelerating the public health response to foodborne illnesses at the local , regional and national level . the actual legislation in the area of food and food safety area is not adequate and needs to be more precise in the area of obligations of stakeholders because of food safety challenges that persist in today s complex , dynamic and global food system ( 35 ) . after the examination of relevant documents from various sources , it is interesting to compare the similarities between serbia and some european countries in the area of food safety system improvement . first of all , there is a need to highlight the continuous need to change the system if it proves inefficient in any part . the usa had already done it , as well as the united kingdom ( uk ) . the food safety and hygiene ( england ) regulations came into effect on 31 december 2013 . these regulations revoke and re - enact the food hygiene ( england ) regulations 2006 ( and amendments ) and certain provisions of the general food regulations 2004 . the secretary of state has been designated for the purposes of that section in relation to measures relating to food ( including drink ) , including the primary production of food and measures in the veterinary and phytosanitary fields for the protection of public health . the chief changes will have to do with enforcement action taken against those who contravene the regulations , including the powers of courts ( 36 ) . serbia should take the same path , do the same and have power to punish violators of legal framework regarding the food safety area . another important issue similar to the current state regarding the number of referent laboratories and samples also came from the uk . leading food expert professor chris elliott has warned that the integrity of the food supply chain is being endangered by budget cuts . the warnings come after the number of public analyst laboratories has been reduced from 15 to 11 in the last three years while trading standards officers ( tso ) are facing a 40 percent cut in funding . the department for environment food and rural affairs ( defra ) claimed that the budget is spent in action to prevent food crime , including increasing unannounced inspections of meat cutting plants and boosting funding to 2 million to support local authorities food sampling programmes ( 37 ) . the project concerning food safety in less developed countries could not be performed without help of the international community . the currently ongoing project in the area of food safety in belarus which started in 2010 is supported with funds from the austrian ministry of finance . the main goal of the project is to increase the competitiveness of belarus food producers by improving their food safety practices ( 38 ) . in 2013 , georgia food safety improvement project was completed , which started in 2010 supported also with funds from the austrian ministry of finance and bp and its oil and gas partners . the project was designed to improve food safety practices among georgian food producers , build local food safety capacity , and harmonize national food safety legislation with the requirements of the european union ( 39 ) . there are still not enough such activities in serbia due to current state at borders regarding the improvement of import food safety . therefore , it is important to learn from the experiences of other countries which are presented in the articles from the united states , latin america , europe , and asia about a variety of regulatory approaches to food safety around the world ( 40 ) . the collaboration which in general enormously contributes to security in the entire food supply chain from farm to table in western balkans was performed through various specific projects with significant number of countries like slovenia , greece , bosnia and herzegovina , fyr macedonia , and serbia . despite all obstacles recognized in serbia , it is important to address that the european commission progress report 2012 reported little progress with regard to general food safety principles . hence , even though some progress is detected , there is still a need to move forward to a better operational level in practice to prevent and protect serbian population from foodborne diseases . serbia does not have the required human , material and financial resources , and in current macroeconomic conditions it is not able to execute the previous plans in the expected scope . therefore , the authors detected the main issues which have to be solved in the future . they are : the directorate for national reference laboratory ( dnrl ) has to be fully operational in terms of performing laboratory testing . it is unbelievable that even though it was established in 2009 , and presented expectations that it would be complete at the beginning of 2011 , serbia had to ask for the results from foreign laboratories in 2013 ; rapid communication and alert system for food and feed ( rasff ) still does not exist in serbia and it is not possible to share any information at either national or international level even though the rulebook on the establishment and organization of the rapid alert system for food and feed has been published ( 41 ) ; serbia imports a lot of goods but at some cross border points the trade control and expert system ( traces ) has not been established ; in almost all documents there are no any signs of coordination between health care communities and other authorities . therefore , there are no established official task forces for communication and information exchange on a regular basis for food safety ; the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment have to be established at the national level ; the external services are not included in the education program within agriculture community ; the risk analysis and the implementation of haccp which started in the past should be improved significantly in the future ; health care workers work in isolation in the course of an epidemiological investigation and do not collaborate with the law enforcement , and academic community has to be more proactive in the improvement of current courses and creation of studying programs which will follow the practice of foreign institutions . faster response to foodborne outbreaks in serbia will be provided only if all authorities start to work more effectively . budget shortfalls have to be overcome and financial means have to provide for the use of the best methods in public health laboratories to quickly identify , characterize , and improve integration of foodborne illness surveillance systems as well as to expand data sharing among the health professionals and other stakeholders from non health sectors . in this process , the law enforcement has to be recognized as an important factor in epidemiological investigations . in the future , serbia has to avoid the obvious interweaving of policy - making with inspection and epidemiological work . it is time to change a habit of bureaucratic work which delays response to outbreak and causes the permanent lack of coordination among different ministries , the institutes of public health and the local authorities . hence , the transparency and professionalism should be recognized as the first step which would build trust between the public and food safety and health system authorities . in the last few years serbian population started to seek answers to many questions due to the increasing evidence about food outbreaks presented in mass media . therefore , the protection against foodborne risks starts to be of paramount interest in the public health care system . serbian policy - makers have to follow the actions from the developed countries and work on the food safety modernization . the health care system is the first one to deal with the consequences of foodborne outbreaks . hence , in that difficult task it also has to apply different approaches in epidemiological investigations in some cases . shoe leather epidemiology and get out of their high tech , or less high tech laboratories asking people thoughtful questions and getting the answers what is going around and find the root of disease ( 42 ) . in epidemiological investigations , public health and law enforcements agencies could ensure that in cases of intentional food poisoning the perpetrators will be punished at the scientific conclusion of the investigation and respecting the legislation . similar intention has been recognized in germany during the e. coli crisis where the need was addressed to establish one central authority similar to a national investigative police agency that is capable to observe and act across state borders ( 43 ) . in the end , all authorities must have in mind what obolensky addressed in his study : human beings rely on food for sustenance and nutrition , and our health and well - being is dependent on the vitality of the food we consume . hence , improving the food safety system will increase the effectiveness of coordination and cooperation , communication and reporting between all organisational units and stakeholders , as well as communication with the public regarding food safety . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

abstractbackgroundfood safety issues are not a new issue in science , but due to the dynamic changes in the modern world it is as equally important as decades ago . the aim of the study was to address the efforts in the development of a comprehensive food safety system in serbia , and make specific recommendations regarding the improvement of epidemiological investigation capacity as a useful tool which contributes to improving the public health by joint efforts of epidemiologists and law enforcement.methodswe used the methodology appropriate for social sciences.resultsthe findings show the current state - of - affairs in the area of food safety and health care system and present some most important weaknesses which have to be overcome . policy makers need timely and reliable information so that they can make informed decisions to improve the population health in an ongoing process of seeking full membership in the european union.conclusionserbia has to apply significant changes in practice because the current state - of - affairs in the area of food safety and health care system is not so favourable due to numerous both objective and subjective factors . hence , the policy - makers must work on the development of epidemiological investigation capacities as a firm basis for greater efficiency and effectiveness . epidemiologists would not stay alone in their work . law enforcement as well as many other stakeholders should recognize their new role in the process of the development of epidemiological investigation capacity as a tool for the development of a comprehensive food safety system in serbia .

Introduction Methods Overview of Serbian food safety and health care system The importance of the process of epidemicological investigation in the development of food safety system in the Republic of Serbia Results and discussion Conclusion Ethical considerations

who works closely with the food and agriculture organization of the united nations ( fao ) , who for animal health ( oie ) , and other international organizations to address food safety issues along the entire food chain . in the european region there are a lot of documents that outline the addressed policies about food supplies , food safety and nutrition , and also about mobilization resources for the activities on poverty and health ( 8 , 9 ) . the republic of serbia ( rs ) has experienced important changes on its way to reach full membership in the european union . despite numerous efforts to provide adequate level of food safety the latest data have confirmed that legal framework is not the firm base for prevention of violations of current regulations in the area of food safety . considering the above mentioned , the authors decided to analyze in this study how it is possible to improve the food safety system in serbia and avoid suffering of population as well as the health system obviously overwhelmed by numerous problems . the purpose of this study is to address a current state in the food safety area in the republic of serbia and its inseparable connection with the public health . the methodology used in the study is the most appropriate for the social science . the second chapter is devoted to the organization of food safety system and health care in serbia regarding the european regulations . the last chapter presents the authors recommendation to the policy makers about the development of epidemiological investigation capacities as a tool for providing better food safety in serbia . among many other needs of innovation , there is one in particular to be highlighted and that is a change in conducting the epidemiological investigation by join efforts of both public health workforce and law enforcement in the food safety area . the main objective of this study is to consider current state in the food safety system in serbia . the additional objective is to present the need for improvement of epidemiological investigation in the food outbreak , which could be useful in the improvement of the system performances and these facts can be used to punish the violators . the official publications of the relevant authorities in serbia and from the european union , and broader international community were examined . this research is related to the events and changes in the area of health , food safety and in social sphere which occurred in serbia during the period 2008 - 2013 . to facilitate the process of legal approximation , the serbian parliament adopted the national plan for the adoption of the acquis 2013 - 2016 ( npaa ) ( 16 ) therefore , acquis communautaire in chapter 12 - food safety , veterinary and phytosanitary policy has been transposed into serbian national legislation . there are also a significant number of academic institutions departments at universities which undertake research contributing to the area of food safety . in the autonomous province of vojvodina ( apv ) , the tasks related to food safety that fall under the competency of the mh have been conferred to the secretary of the health of the province . the flow diagram presenting the serbian food safety system is given in fig.1 and it is useful for understanding the responsibilities of different sectors , inspection units and other stakeholders . it is clear that due to numerous organisational units the communication among stakeholders is not always simple and easy to achieve , especially in the case of emergency . a national communication centre for surveillance is established , based on the european centre for disease prevention and control ( ecdc ) methodology , and capable to provide on - line communication in the case of pandemics in order to coordinate the national health care network and be in contact with the ecdc ( 18 ) . pursuant to the open competition conducted by the general inspectorate , the authorities established a list of laboratories for laboratory tests in the field of food and feed safety and the implementation of monitoring programme . the health care system in serbia is characterized by a well - developed network of health institutions , with predominantly state - owned health care facilities . therefore , the serbian health care system is facing serious problems due to political circumstances and financial constraints . in serbia , the most important institution of public health is the national institute of public health ( sniph ) dr milan jovanovi - batut . the public recognized the health care system in serbia as the least efficient and the most corrupted and inefficient . therefore , the government and mh undertook many activities in order to improve the status of the health care system ( 24 ) . he stated that harmonization of serbian standards with those in use in the eu was done without enough understanding for reality and their enforceability and due to that these rules have brought major changes the primary responsibility for food quality is on the producers , and the government takes on the role of a controller . from the reactions of the competent authorities in those events , it is clear that serbian policy - makers are not familiar with the way of adequate risk communication . the urgent action in the area of appropriate risk communication during the food outbreak , the improvement of surveillance , and established coordination between all stakeholders has to be an urgent future task for policy - makers . in serbia epidemiological investigations and public health surveillance include the ability to create , maintain , support , and strengthen surveillance and detection systems and epidemiological investigation processes , as well as to expand these systems and processes in response to the incidents of public health significance . when the foodborne outbreak happens , the stakeholders in serbia have to apply the scientific method of epidemiological investigation . hence , in serbia the epidemiologists are not familiar with the broader application of joint investigations between epidemiologists and law enforcement in the investigation processes . the authors of the study found that in serbia the epidemiologists faced with numerous objective and subjective obstacles in their investigation . the project titled equipment and courier service supply and capacity building of serbian national referent laboratories directorate in food chain started by providing laboratories with the equipment during 2003 , but that equipment was turned over to the official laboratories in the field of food safety , veterinary and phytosanitary controls . this is obligatory and as this regulation is seen by the patients as complicated and enforced by the health institutions without any agreement with the patients , they do not go to the physician and take some alternative medicines , or go to private practice which is not so strict in the process of reporting the disease which might be caused by foodborne outbreak . in the usa after 2001 terrorism and onwards , bioterrorism is recognized as a new and increasing threat to the americans and this requires the different approaches in the protection of public health of the population . to foster the improved understanding of the investigative goals and methodology , and strengthen interdisciplinary collaborative effectiveness in response to future attacks involving biological agents , in the spring of 2002 the public health law program of the us cdc in partnership with other agencies and organizations undertook the development of a module for the joint training of law enforcement and public health officials ( 33 ) . the next improvement in the epidemiological investigation in serbia could be engagement of private investigators or specific places in public health departments which use specific kind of epidemiological investigations , so called food sleuths . the need for communicating with the public health and clinical community has long been acknowledged in serbia , but the need for communicating quickly and effectively with the elected officials and the public became obvious in 2013 during the affair with aflatoxin in milk and dairy products . the law on food safety stipulates the adoption of the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment , the causes of which are the food or feed and the occurrence of which could not be foreseen , prevented , eliminated or abated to the acceptable level . the programme has to be adopted by the government , and it governs precisely the following : the type of situation in which direct or indirect risk to human health caused by food or feed exists ; the measures that have to be implemented without delay once it is established that food or feed is posing a serious threat to humans or animals , either directly or indirectly through the environment ; the crisis management procedures which include the principle of transparency and communication ; the plan of exercises and simulations for crisis management purposes . pursuant to the programme , the minister of agriculture shall adopt , with the consent of the minister responsible for public health , a special crisis management plan depending on the types of risks and shall form a crisis team to implement the special plan , but in the affair of aflatoxin both the officials and the public recognized that this program was not established . apart from the epidemiologists , it is not known in the public that anyone else was in charge to conduct epidemiological investigation . for example , whereas the public health investigation may focus on identifying a pathogen , the source and mode of transmission , a criminal investigation is likely to focus on finding the perpetrator who has to be punished at the conclusion of the investigation . among all goals which should be achieved in the process of an epidemiological investigation of foodborne outbreak , one is particularly important for the population and the interested parties : to maximize resources and facilitate communication and interaction among public health officials and law enforcement in serbia following the practice from the developed countries . to facilitate the process of legal approximation , the serbian parliament adopted the national plan for the adoption of the acquis 2013 - 2016 ( npaa ) ( 16 ) therefore , acquis communautaire in chapter 12 - food safety , veterinary and phytosanitary policy has been transposed into serbian national legislation . there are also a significant number of academic institutions departments at universities which undertake research contributing to the area of food safety . in the autonomous province of vojvodina ( apv ) , the tasks related to food safety that fall under the competency of the mh have been conferred to the secretary of the health of the province . the flow diagram presenting the serbian food safety system is given in fig.1 and it is useful for understanding the responsibilities of different sectors , inspection units and other stakeholders . it is clear that due to numerous organisational units the communication among stakeholders is not always simple and easy to achieve , especially in the case of emergency . a national communication centre for surveillance is established , based on the european centre for disease prevention and control ( ecdc ) methodology , and capable to provide on - line communication in the case of pandemics in order to coordinate the national health care network and be in contact with the ecdc ( 18 ) . pursuant to the open competition conducted by the general inspectorate , the authorities established a list of laboratories for laboratory tests in the field of food and feed safety and the implementation of monitoring programme . the health care system in serbia is characterized by a well - developed network of health institutions , with predominantly state - owned health care facilities . therefore , the serbian health care system is facing serious problems due to political circumstances and financial constraints . in serbia , the most important institution of public health is the national institute of public health ( sniph ) dr milan jovanovi - batut . the public recognized the health care system in serbia as the least efficient and the most corrupted and inefficient . therefore , the government and mh undertook many activities in order to improve the status of the health care system ( 24 ) . he stated that harmonization of serbian standards with those in use in the eu was done without enough understanding for reality and their enforceability and due to that these rules have brought major changes the primary responsibility for food quality is on the producers , and the government takes on the role of a controller . from the reactions of the competent authorities in those events , it is clear that serbian policy - makers are not familiar with the way of adequate risk communication . the urgent action in the area of appropriate risk communication during the food outbreak , the improvement of surveillance , and established coordination between all stakeholders has to be an urgent future task for policy - makers . epidemiological investigations and public health surveillance include the ability to create , maintain , support , and strengthen surveillance and detection systems and epidemiological investigation processes , as well as to expand these systems and processes in response to the incidents of public health significance . when the foodborne outbreak happens , the stakeholders in serbia have to apply the scientific method of epidemiological investigation . hence , in serbia the epidemiologists are not familiar with the broader application of joint investigations between epidemiologists and law enforcement in the investigation processes . the authors of the study found that in serbia the epidemiologists faced with numerous objective and subjective obstacles in their investigation . the project titled equipment and courier service supply and capacity building of serbian national referent laboratories directorate in food chain started by providing laboratories with the equipment during 2003 , but that equipment was turned over to the official laboratories in the field of food safety , veterinary and phytosanitary controls . this is obligatory and as this regulation is seen by the patients as complicated and enforced by the health institutions without any agreement with the patients , they do not go to the physician and take some alternative medicines , or go to private practice which is not so strict in the process of reporting the disease which might be caused by foodborne outbreak . in the usa after 2001 terrorism and onwards , bioterrorism is recognized as a new and increasing threat to the americans and this requires the different approaches in the protection of public health of the population . to foster the improved understanding of the investigative goals and methodology , and strengthen interdisciplinary collaborative effectiveness in response to future attacks involving biological agents , in the spring of 2002 the public health law program of the us cdc in partnership with other agencies and organizations undertook the development of a module for the joint training of law enforcement and public health officials ( 33 ) . the next improvement in the epidemiological investigation in serbia could be engagement of private investigators or specific places in public health departments which use specific kind of epidemiological investigations , so called food sleuths . the need for communicating with the public health and clinical community has long been acknowledged in serbia , but the need for communicating quickly and effectively with the elected officials and the public became obvious in 2013 during the affair with aflatoxin in milk and dairy products . the law on food safety stipulates the adoption of the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment , the causes of which are the food or feed and the occurrence of which could not be foreseen , prevented , eliminated or abated to the acceptable level . the programme has to be adopted by the government , and it governs precisely the following : the type of situation in which direct or indirect risk to human health caused by food or feed exists ; the measures that have to be implemented without delay once it is established that food or feed is posing a serious threat to humans or animals , either directly or indirectly through the environment ; the crisis management procedures which include the principle of transparency and communication ; the plan of exercises and simulations for crisis management purposes . pursuant to the programme , the minister of agriculture shall adopt , with the consent of the minister responsible for public health , a special crisis management plan depending on the types of risks and shall form a crisis team to implement the special plan , but in the affair of aflatoxin both the officials and the public recognized that this program was not established . the positive practice from the usa , where depending on the type of outbreak the number of involved agencies may be quite large , , it is not known in the public that anyone else was in charge to conduct epidemiological investigation . for example , whereas the public health investigation may focus on identifying a pathogen , the source and mode of transmission , a criminal investigation is likely to focus on finding the perpetrator who has to be punished at the conclusion of the investigation . among all goals which should be achieved in the process of an epidemiological investigation of foodborne outbreak , one is particularly important for the population and the interested parties : to maximize resources and facilitate communication and interaction among public health officials and law enforcement in serbia following the practice from the developed countries . the study results confirmed that the area of food safety and the current state - of - affairs of the health care system in serbia have a lot of room for improvement . decision- and policy - makers have to take urgent steps to develop the policies in which public health will be in the centre of decisions in food safety area . serbian government with competent authorities and in collaboration with the international community , mostly with the eu , works permanently on developing the new approaches in the area of food safety and accelerating the public health response to foodborne illnesses at the local , regional and national level . the actual legislation in the area of food and food safety area is not adequate and needs to be more precise in the area of obligations of stakeholders because of food safety challenges that persist in today s complex , dynamic and global food system ( 35 ) . after the examination of relevant documents from various sources , it is interesting to compare the similarities between serbia and some european countries in the area of food safety system improvement . the currently ongoing project in the area of food safety in belarus which started in 2010 is supported with funds from the austrian ministry of finance . the project was designed to improve food safety practices among georgian food producers , build local food safety capacity , and harmonize national food safety legislation with the requirements of the european union ( 39 ) . there are still not enough such activities in serbia due to current state at borders regarding the improvement of import food safety . despite all obstacles recognized in serbia , it is important to address that the european commission progress report 2012 reported little progress with regard to general food safety principles . therefore , the authors detected the main issues which have to be solved in the future . it is unbelievable that even though it was established in 2009 , and presented expectations that it would be complete at the beginning of 2011 , serbia had to ask for the results from foreign laboratories in 2013 ; rapid communication and alert system for food and feed ( rasff ) still does not exist in serbia and it is not possible to share any information at either national or international level even though the rulebook on the establishment and organization of the rapid alert system for food and feed has been published ( 41 ) ; serbia imports a lot of goods but at some cross border points the trade control and expert system ( traces ) has not been established ; in almost all documents there are no any signs of coordination between health care communities and other authorities . therefore , there are no established official task forces for communication and information exchange on a regular basis for food safety ; the crisis management programme and crisis management plan in the field of food and feed safety in the event of direct or indirect risks to human health , animal health or the environment have to be established at the national level ; the external services are not included in the education program within agriculture community ; the risk analysis and the implementation of haccp which started in the past should be improved significantly in the future ; health care workers work in isolation in the course of an epidemiological investigation and do not collaborate with the law enforcement , and academic community has to be more proactive in the improvement of current courses and creation of studying programs which will follow the practice of foreign institutions . budget shortfalls have to be overcome and financial means have to provide for the use of the best methods in public health laboratories to quickly identify , characterize , and improve integration of foodborne illness surveillance systems as well as to expand data sharing among the health professionals and other stakeholders from non health sectors . hence , the transparency and professionalism should be recognized as the first step which would build trust between the public and food safety and health system authorities . therefore , the protection against foodborne risks starts to be of paramount interest in the public health care system . serbian policy - makers have to follow the actions from the developed countries and work on the food safety modernization . hence , improving the food safety system will increase the effectiveness of coordination and cooperation , communication and reporting between all organisational units and stakeholders , as well as communication with the public regarding food safety .

fungal infection has rarely been reported in patients with primary biliary cirrhosis ( pbc ) ; however it may occur following live transplant ( lt ) in patients with pbc , predisposing factors for the development of fungal infection following lt include diabetes mellitus , cholestasis , hypertransfusion , acute rejection , treatment with high - dose of steroids and immunosuppressive agents , renal failure and bacterial infection ( 1 ) . the present study aimed to analyze the imaging , clinical and pathological features of fungal infection involvement in pbc by retrospectively analyzing and reviewing the features of two patients with fungal infection involvement in pbc who were admitted to our department . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . , tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital . physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative . laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 ) routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl . autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 ) g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 ) u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u / u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) . magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig . following admission to our hospital , the patient suffered from a cough producing white sputum . lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded . moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration . the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig . the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure . a 49-year - old female presented with intermittent fever and abdominal distension for more than a year . the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) . fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension . ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever . one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease , symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected . the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia . the patient suffered from tuberculosis for 12 years , which was subsequently been cured . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs . laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml . g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl . routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative . umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative . schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig . 4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible . abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum . she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . , basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . , hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) . following treatment , the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . , shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . , tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital . physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative . laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 ) routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl . autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 ) g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 ) u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u / u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) . magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig . following admission to our hospital , the patient suffered from a cough producing white sputum . lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded . moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration . the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig . the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure . a 49-year - old female presented with intermittent fever and abdominal distension for more than a year . the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) . fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension . ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever . one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease , symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected . the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia . the patient suffered from tuberculosis for 12 years , which was subsequently been cured . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs . laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml . g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl . routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative . umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative . schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig . 4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible . abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum . she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . , basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . , hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) . following treatment , the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . , shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . , tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital . physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative . laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 ) routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl . autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 ) g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 ) u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u / u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) . magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig . following admission to our hospital , the patient suffered from a cough producing white sputum . lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded . moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration . the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig . the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure . a 49-year - old female presented with intermittent fever and abdominal distension for more than a year . the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) . fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension . ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . anti - smooth muscle antibody and ama were positive . taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever . one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease , symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected . the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs . laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml . g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl . routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative . g / l ; tbil , 33.9 umol / l ; dbil , 19.4 umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative . schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig . 4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible . abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum . she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . , basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . , hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) . following treatment , the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . , shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition . this condition predominantly occurs in middle - aged women and is characterized by progressive bile duct destruction in the liver , inflammation and fibrosis in the portal area and around the periportal , and eventual portal hypertension , liver cirrhosis and liver failure ( 2 ) . with advances in diagnostic techniques and knowledge of the disease in recent years , the incidence rates of this disease have increased in china ( 3 ) . previous studies have confirmed that pbc and other autoimmune diseases , such as sjogren 's syndrome and rheumatoid arthritis , have a common immunopathologic basis ( 4,5 ) . there is currently a lack of satisfactory therapies for the disease , as symptomatic treatment remains the main therapy option . ursodeoxycholic acid has been successful in some patients for the relief of symptoms and has been shown to improve the indicators of liver function , delay disease progression and improve quality of life ( 6 ) . if the patient has an additional connective tissue diseases , the patient is required to take hormones and immunosuppressive therapy simultaneously . long - term use of hormones and immunosuppressive agents may increase the patient 's risk of developing a variety of concurrent infections , particularly opportunistic infections ( 7,8 ) . in the present study , two cases of fungal infection involvement in pbc , which was diagnosed in line with american association for the study of liver disease 's pbc diagnosis recommendations published in 2000 ( 9 ) . in case 1 , the patient suffered from subsequent respiratory and cardiac failure ; in case 2 , the patient 's condition was complicated by sjogren 's syndrome . both of the patients were receiving hormonal and immunosuppressive therapy due to pbc prior to the development of concurrent p. carinii and mucormycosis infection . individuals of any age can be infected with pcp , and people with normal immune function do not typically exhibit symptoms after infection . however , when the host 's immune system is weakened , pneumocystis bacteria begin to multiply , and spread in the lungs , resulting in interstitial plasma cell pneumonia . patients with connective tissue disease are at an increased risk of infection with pcp as they receive large doses of hormonal and immunosuppressive agents for long periods of time ( 10 ) . in recent years , with the extensive use of biological agents , the incidence of fungal infections has significantly increased ( 1114 ) . in japan , the rate of rheumatoid arthritis ( ra ) involvement in pcp infection after applying infliximab treatment is 0.4% , at an average of 8.5 weeks after the application of infliximab ( 15 ) . rituximab monoclonal antibody treatment of ra and wegener 's granulomatosis ( wg ) has also been demonstrated to increase involvement in pcp ( 16 ) . numerous clinical studies have reported systemic lupus erythematosus and wg involvement pcp infection ( 1618 ) . reports of wg involvement in pcp infection are most common . in a meta - analysis of 11,905 cases of patients with connective tissue disease , 12% of the 578 cases of wg were complicated by pcp infection , whereas dermatomyositis / polymyositis involvement in pcp infection was only 6% , sle was 5% and ra was 1% ( 19 ) . connective tissue disease patients ' infection with fungi and pcp is not only related to immune disorders of connective tissue disease but is also associated with the long - term use of hormonal and immunosuppressive therapy ( 20 ) . however , it is difficult to elucidate which factor specifically causes the disease . in the present study , the two cases exhibited leukopenia upon admission to our hospital , as leukopenia may increase the chance of opportunistic infection . connective tissue disease involvement in pcp infection is related to treatment with cyclophosphamide , methotrexate , hormones , azathioprine , cyclosporine and other biological agents , such as anti - tnf inhibitors and anti - cd20 inhibitors ( 21,22 ) . hormone therapy is the uppermost risk factor ; > 90% of patients who present with fungal infection involvement pcp have received long - term hormone therapy ( 23 ) . if elderly patients , patients with nutritional deficiency or patients with low - lymphocytes lipoproteinaemia receive > 16 mg / d equivalent hormone ( > 2 months ) or > 20 mg / d ( > 1-month ) hormone therapy , the risk of pcp infection markedly increases ( 24 ) . cyclophosphamide treatment involvement in pcp infection may be related to significant inhibition of the lymphocyte count ; therefore , patients regularly treated with cyclophosphamide should consider monitoring their cd4 lymphocyte count . to the best of our knowledge , there are no previous reports of pbc involvement in renal mucormycosis infection . in a clinical setting , there have been a small number of reports about liver involvement in mucormycosis infection after transplantation ; the majority involve lung mucormycosis infection and reports of renal mucormycosis infection are rare ( 25 ) . patients with the disease are predominantly complicated by diabetic acidosis , malnutrition , severe burns , trauma , leukemia , lymphoma , acquired immune deficiency syndrome or other serious wasting diseases , or have received long - term immunosuppressive agents , cytotoxic drugs or adrenocortical hormone ( 27 ) . most of the mucormycosis diseases are dangerous and the mortality rates are high ( 28 ) . the main feature of mucormycosis is sexual propagation producing zygosperm and asexual reproduction forming sporangia . features include : i ) hyphae are relatively thick , unseparated or rarely separated ; ii ) the mycelium wall is relatively thick , the side shoot is at right angle with the base shoot ; and iii ) tend to violate the vessel wall and lumen , thus forming a vessel clot that leads to ischemia , hemorrhage infarction and necrotizing inflammation of adjacent tissues ( 29 ) . the clinical manifestations of mucor infections are cerebral , lung , gastrointestinal , cutaneous and systemically - spreading infections . the symptoms of patients with acute disease progress quickly and patients often succumb to the disease within several days or several weeks ( 30 ) . chronic infection can be manifested as simple granuloma or pyogenic inflammation and granulomatous mixed inflammation ( 31,32 ) . clinical diagnosis is often based on clinical symptoms , susceptible factors , mycological examination and pathological biopsy , of which , locating hypha in pathological tissue is of most diagnostic significance . its initial performance is unilateral or bilateral bronchial pneumonia integrating into a large consolidation rapidly and often forming porosis . single or multiple nodules can also be detected . if the pulmonary embolism is relatively large , the wedge - shaped shadow close to the pleura at the bottom can be seen , which has diagnostic significance . high - resolution ct can detect symptoms that can not be seen on an x - ray , such as lumen blocking , vignette and pulmonary artery pseudoaneurysm caused by mucormycosis within the bronchus . mucormycosis progresses rapidly , therefore , early diagnosis and effective treatment are of decisive significance for the prognosis of patients with mucormycosis . renal involvement of systemically disseminated mucormycosis is rare , and simple renal mucormycosis is scarcer . case 2 received hormonal and immunosuppressive therapy due to sjogren 's syndrome involvement in pbc . following therapy , the patient was infected with renal mucormycosis . despite active amphotericin b anti - infective therapy , the present study provided a retrospective analysis of two cases of pbc involvement in opportunistic fungal infections . the patients in these cases were infected with fungi after taking hormones and immunosuppressive agents for a long period of time . this suggests that in patients with rheumatic autoimmune diseases , who receive long - term hormonal and immunosuppressive therapy , the probability of opportunistic pathogen infections increases . when confronted with an infection whose response to antibiotic therapy is not ideal , a sputum smear and tests for fungi should be conducted . when necessary , an early histopathological biopsy should be performed to make time for the treatment of the patient . at the same time , actively seeking pathological diagnosis will improve our understanding of these difficult diseases .

the present study aimed to analyze the imaging , clinical and pathological features of fungal infection involvement in primary biliary cirrhosis ( pbc ) by retrospectively analyzing and reviewing the features of two patients with fungal infection involvement in pbc . both patients were female . one patient had a confirmed diagnosis of pbc . the other patient had confirmed sjogren syndrome and pbc . the two cases of pbc were infected with fungal infection after treatment with hormonal and immunosuppressive agents . rcr of sputum confirmed pneumocystis spp . infection in the patient with pbc alone . the mucormycosis infection was confirmed in the other patient after pathological examination of a renal biopsy . the state of the illnesses progressed quickly and both patients ultimately succumbed to their conditions . the patient prognosis of fungal infection involvement pbc is poor . patients treated with long - term hormone and immunosuppressive agents should be monitored .

Introduction Case report None Case 1 Case 2 Discussion

fungal infection has rarely been reported in patients with primary biliary cirrhosis ( pbc ) ; however it may occur following live transplant ( lt ) in patients with pbc , predisposing factors for the development of fungal infection following lt include diabetes mellitus , cholestasis , hypertransfusion , acute rejection , treatment with high - dose of steroids and immunosuppressive agents , renal failure and bacterial infection ( 1 ) . the present study aimed to analyze the imaging , clinical and pathological features of fungal infection involvement in pbc by retrospectively analyzing and reviewing the features of two patients with fungal infection involvement in pbc who were admitted to our department . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig . ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum . she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . due to the severity of the illness , the patient succumbed to the condition . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital . magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig . following admission to our hospital , the patient suffered from a cough producing white sputum . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . during the course of the disease , symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs . schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . due to the severity of the illness , the patient succumbed to the condition . the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 . liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . , hurley , uk ) , the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved . a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) . the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) . the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following admission to our hospital , the patient suffered from a cough producing white sputum . , serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp . the patient was treated with caspofungin acetate ( 70 mg ; i.v . the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig . the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) . the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . , the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon . the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs . plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear . she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . following treatment , the patient 's body temperature dropped to within normal levels . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs . flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot . the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation . the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed . to identify the nature of the disease , the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . due to the severity of the illness , the patient succumbed to the condition . this condition predominantly occurs in middle - aged women and is characterized by progressive bile duct destruction in the liver , inflammation and fibrosis in the portal area and around the periportal , and eventual portal hypertension , liver cirrhosis and liver failure ( 2 ) . previous studies have confirmed that pbc and other autoimmune diseases , such as sjogren 's syndrome and rheumatoid arthritis , have a common immunopathologic basis ( 4,5 ) . if the patient has an additional connective tissue diseases , the patient is required to take hormones and immunosuppressive therapy simultaneously . long - term use of hormones and immunosuppressive agents may increase the patient 's risk of developing a variety of concurrent infections , particularly opportunistic infections ( 7,8 ) . in the present study , two cases of fungal infection involvement in pbc , which was diagnosed in line with american association for the study of liver disease 's pbc diagnosis recommendations published in 2000 ( 9 ) . in case 1 , the patient suffered from subsequent respiratory and cardiac failure ; in case 2 , the patient 's condition was complicated by sjogren 's syndrome . both of the patients were receiving hormonal and immunosuppressive therapy due to pbc prior to the development of concurrent p. carinii and mucormycosis infection . however , when the host 's immune system is weakened , pneumocystis bacteria begin to multiply , and spread in the lungs , resulting in interstitial plasma cell pneumonia . patients with connective tissue disease are at an increased risk of infection with pcp as they receive large doses of hormonal and immunosuppressive agents for long periods of time ( 10 ) . in recent years , with the extensive use of biological agents , the incidence of fungal infections has significantly increased ( 1114 ) . in japan , the rate of rheumatoid arthritis ( ra ) involvement in pcp infection after applying infliximab treatment is 0.4% , at an average of 8.5 weeks after the application of infliximab ( 15 ) . rituximab monoclonal antibody treatment of ra and wegener 's granulomatosis ( wg ) has also been demonstrated to increase involvement in pcp ( 16 ) . reports of wg involvement in pcp infection are most common . in a meta - analysis of 11,905 cases of patients with connective tissue disease , 12% of the 578 cases of wg were complicated by pcp infection , whereas dermatomyositis / polymyositis involvement in pcp infection was only 6% , sle was 5% and ra was 1% ( 19 ) . connective tissue disease patients ' infection with fungi and pcp is not only related to immune disorders of connective tissue disease but is also associated with the long - term use of hormonal and immunosuppressive therapy ( 20 ) . in the present study , the two cases exhibited leukopenia upon admission to our hospital , as leukopenia may increase the chance of opportunistic infection . connective tissue disease involvement in pcp infection is related to treatment with cyclophosphamide , methotrexate , hormones , azathioprine , cyclosporine and other biological agents , such as anti - tnf inhibitors and anti - cd20 inhibitors ( 21,22 ) . hormone therapy is the uppermost risk factor ; > 90% of patients who present with fungal infection involvement pcp have received long - term hormone therapy ( 23 ) . if elderly patients , patients with nutritional deficiency or patients with low - lymphocytes lipoproteinaemia receive > 16 mg / d equivalent hormone ( > 2 months ) or > 20 mg / d ( > 1-month ) hormone therapy , the risk of pcp infection markedly increases ( 24 ) . cyclophosphamide treatment involvement in pcp infection may be related to significant inhibition of the lymphocyte count ; therefore , patients regularly treated with cyclophosphamide should consider monitoring their cd4 lymphocyte count . to the best of our knowledge , there are no previous reports of pbc involvement in renal mucormycosis infection . in a clinical setting , there have been a small number of reports about liver involvement in mucormycosis infection after transplantation ; the majority involve lung mucormycosis infection and reports of renal mucormycosis infection are rare ( 25 ) . patients with the disease are predominantly complicated by diabetic acidosis , malnutrition , severe burns , trauma , leukemia , lymphoma , acquired immune deficiency syndrome or other serious wasting diseases , or have received long - term immunosuppressive agents , cytotoxic drugs or adrenocortical hormone ( 27 ) . most of the mucormycosis diseases are dangerous and the mortality rates are high ( 28 ) . the symptoms of patients with acute disease progress quickly and patients often succumb to the disease within several days or several weeks ( 30 ) . mucormycosis progresses rapidly , therefore , early diagnosis and effective treatment are of decisive significance for the prognosis of patients with mucormycosis . case 2 received hormonal and immunosuppressive therapy due to sjogren 's syndrome involvement in pbc . following therapy , the patient was infected with renal mucormycosis . despite active amphotericin b anti - infective therapy , the present study provided a retrospective analysis of two cases of pbc involvement in opportunistic fungal infections . the patients in these cases were infected with fungi after taking hormones and immunosuppressive agents for a long period of time . this suggests that in patients with rheumatic autoimmune diseases , who receive long - term hormonal and immunosuppressive therapy , the probability of opportunistic pathogen infections increases . when confronted with an infection whose response to antibiotic therapy is not ideal , a sputum smear and tests for fungi should be conducted . when necessary , an early histopathological biopsy should be performed to make time for the treatment of the patient .

cocaine dependence is associated with increased impulsivity ( chamberlain and sahakian , 2007 ; moeller et al . , 2001a ) in humans ( colzato et al . , 2007 , 2006b ; verdejo - garcia et al . , 2007 ) and animals ( anastasio et al . , 2011 ; anker et al . , 2009 ; paine et al . , 2003 ; paine and olmstead , 2004 ; winstanley et al . , impulsivity may serve as a premorbid trait that confers vulnerability to cocaine dependence ( buckholtz et al . , 2010 ; cunningham and anastasio , 2014 ; verdejo - garcia et al . , 2008 ; winstanley et al . , 2010 ) . in addition , cocaine dependent ( cd ) subjects with higher baseline impulsivity predict reduced retention in outpatient treatment trials for cocaine dependence than cd subjects with lower baseline impulsivity ( moeller et al . , 2001b ) . both cocaine dependence and impulsive behavior are under the regulatory control of cortico - striatal networks ( aron , 2011 ; cunningham and anastasio , 2014 ; dalley et al . , 2011 ; ersche et al . , 2011 ; fineberg et al . , 2010 ; ghahremani et al . volkow et al . , 2011 ; winstanley , 2007 ) with the theories of addiction ( bickel et al . , 2007 ) positing that impulsivity and maladaptive drug - taking result from insufficient communication between frontocortical behavioral control centers and subcortical ( striatal ) incentive - motivational circuitry . however , there is no direct evidence for this putative disruption of directional information flow in cortico - striatal networks in humans , either in cocaine use disorder research or impulsivity research . response inhibition ( ability to withhold a prepotent response ) is one main measure of impulsivity ( moeller et al . most neuroimaging analyses of response inhibition have used either a go / nogo task or a stop - signal task ( colzato et al . , 2007 ; fillmore et al . , 2002 ; fillmore and rush , 2002 ; meta - analyses ( e.g. , buchsbaum et al . , 2005 ; simmonds et al . , 2008 ; swick et al . , 2011 ) of go / nogo neuroimaging studies have shown activation of frontal , subcortical , parietal , and insular regions with right hemispheric dominance during response inhibition under the nogo condition . it has been hypothesized that the dorsolateral prefrontal cortex ( dlpfc ) , ventrolateral prefrontal cortex ( vlpfc ) , and pre - supplementary motor area are particularly important for response inhibition during nogo conditions ( chikazoe , 2010 ) . the go / nogo task has revealed altered patterns of cortical recruitment under acute demands to curtail a prepotent response in subjects with cocaine dependence . for example , kaufman et al . ( 2003 ) conducted a functional magnetic resonance imaging ( fmri ) study with a go / nogo task and found poorer behavioral performance and lower activation in the cingulate , pre - supplementary motor cortex , and insula during response inhibition in active cocaine users compared to cocaine - naive controls . in another fmri study using a ( 2012 ) found that although there was no group difference in behavioral performance , cocaine users with short - term abstinence had greater inhibition - elicited activation than controls in the right middle frontal gyrus ( mfg ) , right precentral gyrus , right superior frontal gyrus , and right middle temporal region . in addition , cocaine users with long - term abstinence had greater activation than controls in the right inferior frontal gyrus ( ifg ) , right mfg , right precentral gyrus , left superior temporal gyrus , and cerebellar tonsils . however , traditional regional activation fmri studies have been unable to answer questions about effective neuronal connectivity and directional relationships among functionally - related brain regions , i.e. , whether a particular neuronal region ( region 1 ) directionally influences another region ( region 2 ) , whether region 2 directionally influences region 1 , or whether the regions reciprocally influence each other . in the present study , 2003 ; li et al . , 2011 ) to test whether cd subjects have altered directional neuronal connectivity underlying their inhibitory behavioral control . we measured response inhibition using the go / nogo task ( lane et al . , 2007 ) , in which the subject was instructed to respond ( go ) when a target stimulus was presented and to withhold responding ( nogo ) when a non - target stimulus was presented . unique from other analytic techniques , effective ( directional ) connectivity in dcm is modeled at the neuronal level rather than the observed blood oxygen level dependent ( bold ) signal level ( friston et al . , 2003 ) . this is important for fmri studies of individuals with substance use disorders because it is known that the bold signal could be confounded by disruption from disease ( i.e. , alzheimer 's ) or drug effects on neurovascular coupling and/or hemodynamic responses ( iannetti and wise , 2007 ) . this is attractive because sometimes disease - related impaired cognitive functions can only be observed during special experimental conditions . ( 2007 ) used a go / nogo task with two - level nogo difficulty ( easy and hard , in terms of similarity between targets and non - targets ) , and found that cd subjects showed poorer behavioral performance than controls only during hard nogo trials rather than easy nogo trials . the dcm analysis in this study was conducted on fmri data acquired from 13 cd subjects and 10 normal healthy cocaine naive controls while they performed a go / nogo task as used in lane et al . based on the hypothesis that cocaine use disorder and inhibitory behavior are regulated through top - down control of the prefrontal cortex reflective system over an amygdala striatum impulsive system ( aron , 2011 ; bechara , 2005 ; cunningham and anastasio , 2014 ; dalley et al . , 2011 ; ersche et al . , 2011 ; fineberg et al . , 2010 ; ghahremani et al . , 2012 ; , we hypothesized that the effective connectivity from prefrontal regions to sub - cortical regions would be altered in cd subjects compared to controls during successful response inhibition . the study was officially approved by the committee for the protection of human subjects ( cphs ) in university of texas health science center , houston , tx and university of texas medical branch , galveston , tx , and was performed in accordance with the code of ethics of the world medical association ( declaration of helsinki ) . the subjects included in this study were from two separate projects that assessed the acute effects of medication versus placebo on brain activation and brain connectivity . four subjects participated in both projects . among the 23 subjects included in the final analyses , 15 subjects ( five cd subjects and 10 controls ) were from the first project , and eight subjects ( all cd subjects ) were from the second project . all subjects were screened using the structured clinical interview for dsm - iv ( scid ) ( first et al . , 1996 ) . all subjects underwent physical examination and medical history . the addiction severity index ( mclellan et al . , 1992 ) each subject 's urine was screened for tetrahydrocannabinol , opiates , cocaine , amphetamines , and benzodiazepines ( syva company , deerfield , il ) , and each subject was screened for alcohol with an intoximeter alco - sensor iii breathalyzer ( intoximeters , inc . , st . subject inclusion criteria were : ( 1 ) 1855 years old ; ( 2 ) right - handed ; ( 3 ) free of alcohol at the time of mri scanning ; ( 4 ) cd subjects met diagnostic and statistical manual fourth edition ( american psychiatric association , 2000 ) criteria for current cocaine dependence as determined by structured clinical interview for dsm - iv ( scid ) ( first et al . , 1996 ) , and ( 5 ) normal control subjects had no current or lifetime history of any dsm - iv substance use or psychiatric disorder . exclusion criteria were : ( 1 ) cd subjects who met current or past dsm - iv axis i disorder other than substance abuse or substance dependence ; ( 2 ) medical disorders or taking medication that may affect the central nervous system ; ( 3 ) claustrophobia experienced during mri simulator sessions ; ( 4 ) any definite or suspected clinically significant abnormalities of the brain on fluid - attenuated inversion recovery ( flair ) mri scans , as read prior to data analysis by a board - certified radiologist ; ( 5 ) positive urine drug screen for control subjects ; and ( 6 ) positive pregnancy test result . in addition to the 10 completed control subjects analyzed in this report , seven other control subjects were excluded for the following reasons : taking medications that may affect the central nervous system ( one subject ) ; behavioral performance ( percentage of correct responses < 50% ) ( two subjects ) ; and unmatched age ( younger than 23 years old ) ( four subjects ) . in addition to the 13 completed cd subjects , 13 additional cd subjects were excluded for the following reasons : behavioral performance ( percentage of correct response < 50% ) ( one subject ) ; excessive head motion during the fmri scan ( two subjects ) ; clinically significant abnormal results on flair scan of the brain ( four subjects ) ; subject 's request to terminate the scanning ( three subjects ) ; and current alcohol dependence ( three subjects ) . see fig . 1 for the flow chart showing subject inclusion / exclusion . based on the inclusion and exclusion criteria , 13 cd subjects ( cd group ) and 10 controls ( control group ) none of the control subjects had a dsm - iv diagnosis of any present or past drug abuse or dependence . the cd group was composed of one female and 12 males ; their mean age was 37.4 5.3 years ( mean standard deviation ) , ranging from 27.5 to 44.1 years . the control group had three females and seven males ; the mean age was 35.2 7.3 years , ranging from 23.3 to 43.6 years . the educational duration of cd group was 11.8 2.0 years , ranging from 7 to 15 years ; and the educational duration of the control group was 13.8 2.0 years , ranging from 11 to 17 years . fisher 's exact test revealed that there was no significant difference in proportion of female and male subjects between groups ( p = 0.281 , two tail ) . there was no significant group difference in age ( t = 0.837 , degree of freedom [ df ] = 21 , p = 0.412 ) . the cd group had significantly lower educational durations than the control group ( t = 2.220 , df = 21 , p = 0.038 ) , with a 2-year difference in means . a rapid - presentation event - related go / nogo task ( lane et al . , 2014b ) was used for analyses of response inhibition during fmri . for all subjects , there were two go / nogo fmri runs . the go / nogo task has been described in detail elsewhere ( lane et al . , 2007 ; ma et al . , 2014b ) . in brief , during each fmri run , 208 visual stimuli ( including go , easy nogo , or hard nogo , please see below ) were sequentially presented in random order . the neighboring stimuli in time were separated by a blank screen lasting 1900 ms , 2100 ms , or 2300 ms ( jittered randomly ) . each of the stimuli consisted of line segments enclosed within two boxes that were presented simultaneously side by side on the same screen . each subject was instructed to discriminate the direction of the lines by pressing a button using their right index finger when both boxes showed parallel diagonal lines in the same direction in both boxes ( go trial ) . each subject was instructed not to press the button when both boxes showed horizontal lines ( easy nogo trial ) , or when one box contained diagonal lines that were in the opposite direction of the diagonal lines in the other box ( hard nogo trial ) . for go trials , a key press , completed greater than 100 ms and less than 600 ms after the stimulus , was defined as a correct response . for nogo trials , a key press , completed within 600 ms after the stimulus , was defined as an incorrect response . each fmri run duration was 10 min 40 s , including 156 go trials ( 75% ) , 26 easy nogo trials ( 12.5% ) , and 26 hard nogo trials ( 12.5% ) . each subject completed a practice go / nogo test during a mock fmri session in order to stabilize performance and provide familiarity with the task prior to actual mri scanning . the discrimination accuracy measure ( d ) ( forman et al . , 2004 ; gescheider , 1985 ; lane et al . , 2007 ) was used to measure behavioral performance on the go / nogo task in the scanner . mri data were acquired on a philips 3.0 t intera system ( philips medical systems , best , netherlands ) with an eight - channel receive head coil . single shot spin - echo echoplanar imaging ( epi ) was used for acquiring fmri data . the spin - echo epi sequence eliminates signal losses caused by through - slice dephasing in medial orbitofrontal cortex ( kruger et al . , 2001 ) and is sensitive ( norris et al . , 2002 ) to blood oxygen level dependent ( bold ) signal in fmri . the fmri acquisition parameters were as follows : sense acceleration factor 2.0 , repetition time 2500 ms , echo time 75 ms , flip angle 90 , field - of - view 240 240 mm , in - plane resolution 3.75 3.75 mm , 25 axial slices , slice thickness 3.75 mm , interslice gap 1.25 mm , 256 repetitions per run after 10 dummy acquisitions . a t1-weighted 3-dimensional spoiled gradient recalled ( spgr ) anatomical scan ( in - plane resolution 0.94 0.94 mm , slice thickness 1 mm ) was acquired for co - registration with the fmri images . a fluid attenuated inversion recovery ( flair ) scan and t2-weighted spin - echo scan were acquired , and were read by a board - certified radiologist in order to rule - out incidental brain abnormalities . although two fmri runs were acquired for each subject , some subjects only had one usable fmri run . thus we decided to use only one fmri run for each subject in order to avoid potential bias effects . the first run was used if a subject had two usable fmri runs . during each fmri run , individual images in which the fmri signal exceeded plus or minus four standard deviations from the mean for the run were considered to be outliers and were replaced by the mean of the two nearest neighbors using the analysis of functional neuroimages ( afni ) ( cox , 1996 ) software command 3ddespike ( http://afni.nimh.nih.gov/afni/ ) . all remaining preprocessing used statistical parametric mapping 8 ( spm8 ) software ( http://www.fil.ion.ucl.ac.uk/spm/ ) implemented in matlab r2007b ( mathworks inc . , sherborn , ma , usa ) . then , the fmri series was realigned to the first image to correct for head motion . runs with head motion greater than 1 voxel ( 3.75 mm translation on any axis ) or rotation greater than 3.75 were excluded from the analysis . as an optional feature of spm software , the head motion parameters can be regressed out in the spm level 1 general linear model analysis , and if so , the motion parameters can be then be regressed out from the effects of interest when generating the roi time series for the dcm analysis . however , many studies do not enter head motion parameters as regressors because regressing out task - related head motion may eliminate much of the true signal when the movement parameters are related to the experimental task design ( ashburner and friston , 2007 ) . for this reason , we do not routinely include head motion parameters as regressors in our fmri studies that involve activation tasks , including the present study . however , fmri series with severe head movement were excluded from this study , and motion correction was conducted with the spm8 realignment module for all included fmri series . as noted previously , for all subjects , the first run without artifacts and without excessive motion was included in the analysis . the anatomical image was coregistered to the fmri images and spatially transformed to montreal neurological institute ( mni ) standard atlas coordinates using the spm8 normalise module with the spm8 t1 mni template image . after that , the fmri images were resliced to 2 mm isotropic resolution and spatially smoothed with a gaussian filter of 8 mm isotropic full width at half maximum . the univariate statistical analyses of the fmri data were conducted using spm8 . after specifying the design matrix , the parameters for the effects of different conditions were estimated at the first level for all subjects as an event related design according to the general linear model at each voxel , using stick functions modeling the onsets of correct nogo trials convolved with the spm8 canonical hemodynamic response function as a basis function . standard spm8 basis functions for temporal and dispersion derivatives were also included in the model . incorrect trials were entered as a separate covariate of no interest so that the remaining implicit baseline consisted only of the correct go trials . a 1/128 hz high - pass temporal filter was applied . one contrast image was constructed for all subjects for each of the following contrasts of parameter estimates : ( 1 ) correct easy nogo minus correct go ; ( 2 ) correct hard nogo minus correct go ; and ( 3 ) correct hard nogo minus correct easy nogo . in the remainder of this paper , for brevity the word correct will be omitted , but the nogo and go conditions will be understood to consist of correct responses only . while incorrect responses merit theoretic and clinical interest , they were too few to allow valid activation analyses . in order to determine differences in bold activation between groups , a spm8 second level random effects ( holmes and friston , 1998 ) statistical analysis was conducted voxel - wise throughout the whole brain for each of the contrast images listed in the above paragraph . for each contrast , the spm8 second - level two - sample t - test with the default non - sphericity correction for unequal variance between groups was used . according to the practical steps in a typical dcm analysis suggested by seghier et al . ( 2010 ) , random effects group analysis can be used to determine the dcm nodes . 's recommendation and previous dcm studies ( e.g. , bitan et al . , 2005 ; deserno et al . , 2012 ; dima et al . , 2009 ; diquattro and geng , 2011 ; wang et al . , 2011 ) , we used random effects group analysis to determine the regions that significantly activated across both groups in this study , after excluding the brain regions showing group differences . a separate spm8 second level ( random effects ) statistical analysis was conducted voxel - wise throughout the whole brain for each of the contrast images listed above . for each contrast , the spm8 second - level one - sample t - test with the default settings was used to determine bold activations significantly different from zero . for all spm second level group analyses , statistical significance was defined as family - wise error ( fwe ) corrected cluster probability ( p ) less than 0.05 ( two tail ) . uncorrected cluster p less than 0.05 ( two tail ) was used as the threshold for the brain activations used for dcm regions of interest selection . approximate anatomical labels for regions of activation were determined using the anatomical automatic labeling ( aal ) toolbox ( tzourio - mazoyer et al . fmri based dcm is a biophysical model of the underlying neuronal connectivity and of how the neuronal connectivity generates the observed bold signal ( friston et al . , 2003 ) . dcm has been described elsewhere ( friston et al . , 2003 ; ma et al . , 2012 , 2014a ; 2014b ) . in brief , the mathematical model of the underlying neuronal connectivity among an a priori selected set of brain regions ( or dcm nodes ) is a system of bilinear differential state equations with coefficients specified by three matrices ( a matrix , b matrix and c matrix ) ( friston et al . , 2003 ) . in this model , experimental conditions ( e.g. , go , easy nogo , or hard nogo ) can serve as inputs to the model as either driving inputs , or modulatory inputs , or both . the dcm analysis determines which particular nodes in the model exhibit effective ( directional ) connectivity with other specific nodes in the model , which nodes receive driving inputs from experimental conditions into the model , and which specific connectivities between nodes in the model are modulated during experimental conditions . a node in the model that receives driving inputs , as quantified by the c matrix parameters , is the brain region among the nodes in the model which first experiences a change in neuronal activity associated with experimental conditions . the node that receives the driving input then influences ( drives ) the connectivity to other nodes in the model . the endogenous ( or fixed ) connectivity in dcm is quantified by the a matrix parameters , which measure the effective connectivity strengths ( in units of hz ) between nodes , regardless of the moment - to - moment switching on and off of inputs . these modulation effects are quantified by the b matrix parameters as increased or decreased connectivity strength compared to the endogenous connectivity at different times in the experiment that are related to the timing of changes in the particular experimental conditions . nonlinear connectivity effects that are gated by other regions in the system can be modeled by another matrix ( d matrix ) ( stephan et al . , 2007 ) . in the present study , the nonlinear option was not applied , and thus the term modulation effects in the present paper denotes bilinear modulation effects , where bilinear refers to the mathematical form of the equations determining the b matrix parameters . in addition , the stochastic option for dcm was used in which random fluctuations were modeled as inputs to the system as well as the driving inputs due to experimental conditions ( daunizeau et al . , 2009 , 2012a , 2012b , 2013 ; li et al . , 2011 ) . the random fluctuations in physiological noise may contribute to the system connectivity input ( li et al . , 2011 ) due to stochastic fluctuations in neuronal and vascular responses ( kruger and glover , 2001 ; li et al . , 2011 ) . ( 2011 ) have shown that stochastic dcm can improve parameter estimation over deterministic dcm . ( 2012 ) have validated stochastic dcm and shown that stochastic dcm is superior to deterministic dcm in terms of both model structure inference and model parameter inference . following the procedures in ma et al . ( 2012 , 2014a ) , the regions ( nodes ) for the dcm analysis in the present study were chosen based on simultaneously meeting the following three criteria : ( 1 ) the region must show activation that is at least significant at the uncorrected cluster level in the present univariate spm second - level analysis ; ( 2 ) the region must also show activation ( nogo vs. go or nogo vs. baseline ) in previous fmri studies using go / nogo tasks ( e.g. , meta studies , buchsbaum et al . , 2005 ; simmonds et al . , 2008 ; swick et al . , 2011 ) , and ( 3 ) the region must also be regarded to be involved in inhibitory behavior in the previous literature ( e.g. , bechara , 2005 ; chikazoe , 2010 ; heatherton and wagner , 2011 ; volkow et al . , 2011 ) . thus , the following seven nodes were used for the dcm analyses in the present study : ( 1 ) left ( l ) dorsolateral prefrontal cortex ( dlpfc ) ; ( 2 ) right ( r ) dlpfc ; ( 3 ) l anterior cingulate cortex ( acc ) ; ( 4 ) r acc ; ( 5 ) r ventrolateral prefrontal cortex ( vlpfc ) ; ( 6 ) l caudate ( cau ) ; and ( 7 ) r hippocampus ( hipp ) . because of uncertainty about the exact gross anatomical boundaries in humans of the dlpfc ( fuster , 2008 ) , the dlpfc in the present paper was defined by the middle frontal gyrus , which comprises a major portion of the dlpfc in humans ( fuster , 2008 ) . the vlpfc in the present paper was defined by inferior frontal gyrus , on which the major part of the vlpfc of the human brain lies ( petrides , 2005 ) . ( 2012 , 2014a ; 2014b ) to construct the volumes of interest ( vois ) . the atlas - derived binary masks corresponding to the aforementioned seven nodes were obtained from the anatomical automatic labeling ( aal ) atlas ( tzourio - mazoyer et al . , 2002 ) which was implemented in the wfu ( wake forest university ) pickatlas spm toolbox ( maldjian et al . , 2003 ; maldjian et al . , 2004 ) . the binary mask of vlpfc was defined as the set - theoretic union of the atlas - based binary masks of inferior frontal gyrus ( pars opercularis , pars triangularis , and pars orbitalis ) . each voi was obtained by the set - theoretic intersection of the atlas - based binary masks and the activation clusters ( at least significant at uncorrected cluster level ) that were determined by the second - level random effects univariate spm analysis . the standard spm procedure in which nogo and go conditions were explicitly modeled was conducted by using the principal eigenvariate of each voi as a summary of the functional activity time - series in that voi ( ma et al . , 2014a ) , and each principal eigenvariate time series was also adjusted for the f - contrast of effects of interest ( stephan et al . , 2010 ) . the number of voxels , volume , and center of mass of the seven vois used as nodes for the dcm analysis are shown in table 1 . these vois did not overlap with the regions showing significant group difference ( see the results section ) . dcm structure inference , as applied to task - fmri experiments such as this study , searches for a model of the underlying neuronal connectivity among an a priori selected set of brain regions , in which the combined presence of some endogenous connectivities ( and/or modulation / driving input effects ) and the absence of some other endogenous connectivities ( and/or modulation / driving input effects ) best explain the observed fmri data . in this study , dcm structure inference was conducted using dcm network discovery ( dnd ) ( friston et al . the rationale for us to conduct dcm structure inference using dnd has been described elsewhere ( ma et al . , 2014b ) . dnd was conducted using the post - hoc optimization ( spm_dcm_post_hoc routine ) as implemented in the spm12b software . before the dnd analysis was conducted , an initial single full model ( friston et al . , 2011 ) was specified for all subjects . the term full is used here in the sense that ( 1 ) each of the three experimental conditions ( i.e. , go , easy nogo , and hard nogo conditions ) can be a driving input and a modulatory input ; ( 2 ) each of the putative driving inputs entered all of the seven nodes ; ( 3 ) each node was putatively interconnected to all other nodes , and ( 4 ) each of the modulatory inputs putatively modulated all of the 42 interconnectivities between nodes . only stimuli corresponding to correct responses were included in the dcm analysis because the incorrect responses were very few and sporadic for all included subjects . the full models were inverted ( estimated ) for all subjects . for each group , group level post - hoc optimization was conducted by selecting all inverted full models ( one per subject ) . the group level optimal sparse model was found at the group level , using bayesian parameter averaging ( bpa ) , which is integrated in the spm_dcm_post_hoc routine . student 's t - test and fisher 's exact test were used to assess group differences on continuous and categorical demographic variables , respectively . linear mixed models analysis , as implemented in ibm spss version 22 ( chicago , il ) for windows ( microsoft corp . , redmond , wa ) , was used to analyze the main effects of the two factors and their interaction effects on the behavioral performance . the between - subjects factor in this analysis was group ( cd and control groups ) , and the within - subjects factor was levels of nogo difficulty ( easy and hard ) . if main effects or interactions were statistically significant , then post - hoc analyses were conducted with the bonferroni correction for multiple comparisons . the mean and standard deviation of the discrimination accuracy measure ( d ) and percentage of correct response in each group during easy nogo and hard nogo are shown in table 2 . the spss linear mixed model analysis revealed significant main effects of difficulty level ( hard or easy nogo ) ( f = 18.810 ; df = 1 , 35.13 ; p < 0.001 ) . the main effects of group ( f = 0.014 ; df = 1,35.13 ; p = 0.905 ) and the interaction of group difficulty ( f = 0.111 ; df = 1,35.13 ; p post - hoc comparisons showed that d during easy nogo was significantly greater than during hard nogo for both groups ( cds and controls ) ( p < 0.001 ) , suggesting that the behavioral performance during hard nogo trials was less accurate than that during easy nogo trials . the spm8 univariate second level glm analysis of the fmri data revealed a statistically significant cluster ( p = 0.038 , two tail , fwe - corrected ) showing a group difference in activation ( cd group less than control group ) for the easy contrast . 2 and table 3 ) was found in portions of r middle frontal gyrus ( g ) , r precentral g , r middle cingulate cortex , r superior frontal g , and r paracentral lobule . no significant cluster was found for the reverse direction of comparison ( control group less than cd group ) for the easy contrast . no significant cluster was found for the other contrasts ( hard and hard - easy contrasts ) . spm8 second - level random - effects one - sample t - test analysis across both groups combined revealed several clusters for easy , hard , and hard easy activations with the cluster level p less than 0.05 ( uncorrected , two tail ) in portions of frontal , subcortical , and other brain regions . post - hoc optimization found a group - level optimum sparse model structure for each subject group . for the cd group , r hipp , l acc , and r vlpfc were reliable ( posterior probability > 0.9999 ) driving input locations for all three driving inputs ( go , easy nogo , and hard nogo ) . in addition , l caudate was a reliable driving input location for easy nogo and go inputs . furthermore , r acc was a reliable driving input location for the go input . for the control group , r hipp , l acc , l caudate , and r dlpfc were reliable driving input locations for all three driving inputs ( go , easy nogo , and hard nogo ) . the posterior mean strength of each driving input effect is shown in supplementary table 1 . the group level sparse structure regarding the endogenous connectivities is shown in supplementary table 2 , which also shows the posterior mean strength of each endogenous connectivity . three connectivities ( r dlpfc to r acc , r acc to r dlpfc , and r acc to l dlpfc ) among the 42 connectivities were switched off ( posterior probability = 0 ) by the post - hoc optimization for the cd group . two connectivities ( r vlpfc to r acc , and r acc to r vlpfc ) among the 42 connectivities were switched off ( posterior probability = 0 ) by the post - hoc optimization for the control group . the group level sparse structure regarding the modulation effects is shown in supplementary table 3 . 3 , for both cd group ( left panel ) and control group ( right panel ) . the mean strength of each endogenous connectivity modulated during nogo conditions is also shown in fig . 3 . for the cd group , only one ( l acc to l caudate ) of the 42 connectivity was reliably ( posterior probability > 0.9999 ) modulated during nogo conditions , and this connectivity was modulated during both easy ( modulation effect = 0.0584 hz ) and hard ( modulation effect = 0.0822 hz ) nogo conditions . for the control group , three of the 42 connectivity were reliably ( posterior probability > one connectivity ( l acc to l caudate ) was only modulated during the easy nogo condition ( modulation effect = 0.0229 hz ) . the other two connectivity , i.e. , r dlpfc to l caudate , and r vlpfc to l caudate , were only modulated during the hard nogo condition , with modulation effect = 0.2016 hz for the dlpfc caudate connectivity , and modulation effect = 0.2418 hz for the vlpfc caudate connectivity . a post - hoc analysis ( using student 's t - test ) was conducted to determine whether the two groups were significantly different in the modulation effects exerted by the nogo conditions . a t - test was conducted based on the posterior means and posterior standard deviations obtained from the two groups . there was no group difference ( uncorrected p = 0.3640 , 2-tail ) in the modulation effects exerted by the easy nogo condition ( on the connectivity from l acc to l caudate ) . all the modulation effects exerted by the hard nogo condition were significantly different between the groups ( p < 0.0008 , bonferroni corrected ) . these modulation effects were on the connectivities from l acc to l caudate ( cd group : 0.0822 hz ; control group : 0 hz ) , from r dlpfc to l caudate ( cd group : 0 hz ; control group : 0.2016 hz ) , and from r vlpfc to l caudate ( cd group : 0 hz ; control group : 0.2418 hz ) . the present study provides evidence that cortico - striatal circuits activated in cd subjects during inhibition of a prepotent response are distinct from those employed by normal healthy controls responding under the same task with similar performance . when the task demands were high ( hard nogo trials ) , cd subjects demonstrated acc connectivity to the caudate during successful response inhibition instead of the control subjects ' dlpfc or vlpfc connectivity to the caudate which was also during successful response inhibition . these data support the use of dcm to measure effective connectivity specific to certain experimental conditions . in the dcm analysis , a single optimum model with reliable driving input effects and modulatory effects endogenous connectivities , modulatory effects , and driving input effects with high posterior probability were retained , and those with low posterior probability were eliminated by the network discovery analysis . a modulation effect measures increased or decreased effective connectivity strength relative to the endogenous connectivity at different times in the experiment that are related to the timing of changes in a particular experimental condition . subjects in both groups performed the go / nogo tasks similarly well and demonstrated several commonalities during the nogo conditions . specifically , only prefrontal caudate connectivity was modulated during the nogo condition for both groups . this is consistent with the theories of top - down regulation ( bechara , 2005 ; heatherton and wagner , 2011 ; nol et al . 2011 ) and the involvement of the caudate ( aron et al . , 2003 ) in response inhibition . furthermore , the easy nogo condition modulated only l acc to l caudate effective connectivity . these modulation effects were not significantly different between the two groups and support a previous study that reported similar inhibitory behavioral performance in cd and control groups during the easy nogo condition ( lane et al . , 2007 ) . interestingly , control and cd subjects also achieved similar successful response inhibition during the hard nogo condition . this is in contrast to the poorer performance by cd subjects during hard nogo trials reported previously ( lane et al . , 2007 ) . we have used dcm to demonstrate differential modulation of effective cortico - striatal networks during hard nogo condition for cd vs. control subjects . specifically , performance during the hard nogo condition was associated with modulation of the effective connectivity of the r vlpfc to l caudate and r dlpfc to l caudate in control subjects only , and the l acc to l caudate in cd subjects only . the dcm analyses showed negative modulation of the effective connectivity from r vlpfc to l caudate during the hard nogo condition in control subjects . this supports cumulative evidence suggesting that the r vlpfc functions as a brake during inhibitory responding ( aron et al . , 2014 ) . as previously noted , response inhibition may be driven by reduced striatal activity and subsequent medial globus pallidus disinhibition and thalamocortical suppression via the direct pathway of the basal ganglia ( aron et al . , 2003 ; beiser et al . , 1997 ) . thus , our results may reflect that normal healthy subjects achieve successful response inhibition during hard nogo trials due to effective brake the dcm analyses further showed positive modulation of the effective connectivity from r dlpfc to l caudate during the hard nogo condition in control subjects . this result may reflect that healthy subjects achieve executive control through dlpfc activation during the hard nogo condition . while the effective connectivity from the r dlpfc to l caudate was modulated during the hard nogo condition , this pathway was not modulated during the easy nogo condition . this finding is similar to a previous study showing significant dlpfc activation during a complex , but not a simple , nogo task ( mostofsky et al . , 2003 ) . the results are also consistent with a previous study that demonstrated right hemisphere lateralization during executive control in healthy subjects ( tranel et al . , 2005 ) . further , the modulation of two inter - hemisphere prefrontal striatal connectivities ( r dlpfc to l caudate and r vlpfc to l caudate ) during the hard nogo condition in the control group is consistent with the hypothesis that dlpfc and vlpfc are essential for go / nogo response inhibition tasks and that the right pfc dominates the left pfc during response inhibition in healthy subjects ( chikazoe , 2010 ) . the present study demonstrates the unique finding that the hard nogo condition modulates different prefrontal particularly , the effective connectivity from l acc to l caudate was negatively modulated during the hard nogo condition in the cd group but unaffected in the control group . the acc is a critical region for both behavioral monitoring ( botvinick et al . , 1999 ; macdonald et al . , 2000 ) , an important aspect of executive control ( garavan and hester , 2007 ) , and emotional response inhibition ( albert et al . , thus , instead of employing the brake functionality of the vlpfc and executive control functionality of the dlpfc as in control subjects , the cd subjects may complete the task through the behavior monitoring functionality of the acc . alternatively , the difficulty level of the hard nogo trials may arouse frustration and consequently activate emotion - responsive neural nodes ( e.g. , acc ; albert et al . , 2012 ) during response inhibition , particularly as observed in the cd subjects . the results also may suggest that cd subjects employ an intact intra - hemisphere connectivity ( l acc to l caudate ) to compensate for an altered inter - hemisphere connectivity ( r vlpfc to l caudate ) to achieve successful response inhibition . while the key connectivity underlying response inhibition in cd subjects ( l acc to l caudate ) seems inconsistent with a previous study ( kaufman et al . , 2003 ) that found the acc to be hypoactive in cocaine users during a go / nogo task , it is important to consider that the current study showed no group differences in success during the nogo condition as opposed to the study that demonstrated acc hypoactivity concurrent with impairments in behavioral performance in cd subjects ( kaufman et al . , 2003 ) . nonetheless , the present study shows group differences in modulation of prefrontal striatal effective connectivity during hard nogo condition consistent with other studies ( e.g. , hanlon et al . , 2011 ; , 2014a ) and theories ( e.g. , volkow et al . , 2011 ) in cd subjects . the groupwise difference in brain signaling that underlies overtly similar task performance ( here discriminability ) is in keeping with perhaps a dominant finding in the task fmri of addiction ( connolly et al . , 2012 ; ma et al . , 2014a ; tomasi et al . , 2007 ; wilkinson and halligan , 2004 ) . we contend that these altered brain signatures may be indicative of reduced effective function in real world settings that may be infused with emotional context or may otherwise lack the vigilance - inducing conditions of observed behavior in a novel laboratory setting . the interpretive advantage of normative performance is that differences are not likely due to any differences in experience , perception of task errors , or frustration . normal healthy controls exhibited the ability to adapt dynamic neuronal connectivity dependent upon the difficulty level of the response inhibition task , while cd subjects demonstrated the same intra - hemispheric ( l acc to l caudate ) connectivity regardless of the difficulty level of the response inhibition task . this pattern of neuronal connectivity could directly result from chronic cocaine use associated with alterations in functional connectivity ( albein - urios et al . , 2013 , 2014 ; bednarski et al . , 2011 ; cisler et al . , 2013 ; gu et al . , 2010 ; hanlon et al . , 2011 ; lu et al . , 2014 ; , 2013 ; murnane et al . , 2015 ; velez - hernandez et al . , 2014 ; verdejo - garcia et al . , 2014 ; wilcox et al . , 2011 ; wisner et al . , 2013 ; worhunsky et al . , 2013 ; zhang et al . , 2014 ) and dysregulation within key brain regions ( e.g. , prefrontal cortex ) involved in cognitive processing ( fuster , 1997 ) . these alterations may be attributable to a combination of perfusion deficits ( holman et al . , 1991 , 1993 ; levin et al . 1991 ) , altered gray / white matter structure ( barros - loscertales et al . , 2011 ; ma et al . , 2009 ; moeller et al . , 2005 ) , and/or changes in metabolic activity ( volkow et al . , 1991 ) . furthermore , chronic cocaine use is characterized by a multitude of alterations in neurotransmitter function , particularly in the dopamine ( da ) , serotonin ( 5-ht ) , and glutamate systems that may occur consequent to or independent of the aforementioned changes ( cunningham and anastasio , 2014 ; volkow et al . , 2011 ) . monoaminergic da and 5-ht neurons projecting from the mid - brain regions densely innervate the cortical and subcortical systems ( kosofsky and molliver , 1987 ; vertes and linley , 2008 ) . both neurotransmitters interact profoundly at strategically localized receptor proteins ( e.g. , 5-ht2a receptor ( 5-ht2ar ) , 5-ht2cr , da d1 and d2 receptors ) within the complex cortico - striatal circuits , including those localized to the microcircuitry of the frontal cortex and dorsal striatum ( for review , howell and cunningham , 2015 ) . particularly , the 5-ht2ar and 5-ht2cr abundantly localize to pyramidal and gabaergic neurons within the frontal and cingulate cortex ( cornea - hebert et al . , 1999 ; liu et al . , 2007 ; pompeiano et al . , 1994 ; santana et al . , 2004 ) , dopaminergic and gabaergic neurons of the caudate putamen ( eberle - wang et al . , 1997 ; lopez - gimenez et al . , 1997 ) , and ascending dopaminergic mesolimbic neurons innervating the cortico - striatal networks ( bubar and cunningham , 2007 ; doherty and pickel , 2000 ; nocjar et al . serotonin exerts tonic and phasic neuromodulatory control over both da and glutamate neurotransmission through these receptors in the mesocortical and nigrostriatal pathways ( for reviews , alex and pehek , 2007 ; howell and cunningham , 2015 ) that govern cognitive / executive processes and motor / inhibitory response behaviors ( carli and invernizzi , 2014 ; cunningham and anastasio , 2014 ) . while there is an extensive body of literature supporting the role of these receptors in cocaine - related behavioral alterations ( cunningham and anastasio , 2014 ; bubar and cunningham , 2008 ) , it is unknown whether the altered top - down control in the cd subjects may be mediated , in part , by compromised 5-ht system interactions with da and glutamate . ( 1 ) it is possible that other neural interconnectivities are similarly important for inhibitory control but were not identified because the connecting regions were not included as nodes for the dcm analysis . one reason for the exclusion of potential nodes was the lack of sufficient statistical power on fmri activation due to the small sample size in this study . future studies with more subjects will be helpful in providing greater insight into the altered neuronal effective connectivity underlying inhibitory control in cd subjects . ( 2 ) all subjects in the present study received a placebo capsule as part of two larger studies in which they were enrolled . although unlikely , this may have contributed to unknown sources of variability in both the behavioral and fmri data . ( 3 ) while we have shown modulation of effective connectivity during the nogo conditions , the present study was unable to determine which brain regions mechanistically caused these modulation effects . this question could be answered in future studies through the utilization of non - linear dcm ( stephan et al . , 2007 ) . ( 4 ) although the go / nogo paradigm has been frequently used to investigate response inhibition mechanisms , regional brain activation elicited by go / nogo tasks may not be directly related to response inhibition ( criaud and boulinguez , 2013 ) . to avoid the emergence of trivial strategies ( for example , repeated responses to stimuli resulting in 75% correct performance ) , we motivated subjects by setting reward - values for nogo trials three times that of go trials ( either in terms of gain or loss ) , thereby balancing the relative value of go and nogo trials . therefore , the connectivity observed in the present study may reflect the engagement of other cognitive processes ( e.g. , attention , reward , and/or motivation ) in addition to response inhibition , which could confound our interpretations . ( 5 ) the mean education of control subjects was higher ( by about 2 years ) than that of cd subjects . although differences in education level could theoretically affect the study , it is unlikely since behavioral performance was the same in both groups . ( 6 ) these findings were from a small sample size ( 23 total subjects ) . thus , one should be cautious when generalizing these findings or interpreting effect sizes ( button et al . , 2013 ) . in summary , the control and cd subjects had similar levels of performance on the go / nogo task . given the nodes in our network model , the dcm network discovery analysis revealed that prefrontal striatal connectivities were modulated during the nogo conditions for both groups , consistent with the theory that successful inhibition is related to top - down control by a prefrontal , reflective system over a subcortical , impulsive system . while the effective connectivity from l acc to l caudate was similarly modulated during the easy nogo condition for both groups , differences in connectivity were observed during hard nogo trials between groups . in the control group , the effective connectivity from r vlpfc to l caudate was negatively modulated while the r dlpfc to l caudate was positively modulated during the hard nogo condition ; there were no modulation effects on these two connectivities during the hard nogo condition in the cd group . in the cd group , the effective connectivity from l acc to l caudate was negatively modulated during the hard nogo condition ; there was no modulation effect on this network during the hard nogo condition in the control group . these results indicate that cd subjects use different patterns of connectivity to achieve behavioral performance similar to control subjects during hard nogo trials .

cocaine dependence is associated with increased impulsivity in humans . both cocaine dependence and impulsive behavior are under the regulatory control of cortico - striatal networks . one behavioral laboratory measure of impulsivity is response inhibition ( ability to withhold a prepotent response ) in which altered patterns of regional brain activation during executive tasks in service of normal performance are frequently found in cocaine dependent ( cd ) subjects studied with functional magnetic resonance imaging ( fmri ) . however , little is known about aberrations in specific directional neuronal connectivity in cd subjects . the present study employed fmri - based dynamic causal modeling ( dcm ) to study the effective ( directional ) neuronal connectivity associated with response inhibition in cd subjects , elicited under performance of a go / nogo task with two levels of nogo difficulty ( easy and hard ) . the performance on the go / nogo task was not significantly different between cd subjects and controls . the dcm analysis revealed that prefrontal striatal connectivity was modulated ( influenced ) during the nogo conditions for both groups . the effective connectivity from left ( l ) anterior cingulate cortex ( acc ) to l caudate was similarly modulated during the easy nogo condition for both groups . during the hard nogo condition in controls , the effective connectivity from right ( r ) dorsolateral prefrontal cortex ( dlpfc ) to l caudate became more positive , and the effective connectivity from r ventrolateral prefrontal cortex ( vlpfc ) to l caudate became more negative . in cd subjects , the effective connectivity from l acc to l caudate became more negative during the hard nogo conditions . these results indicate that during hard nogo trials in cd subjects , the acc rather than dlpfc or vlpfc influenced caudate during response inhibition .

Introduction Methods Results Discussion

cocaine dependence is associated with increased impulsivity ( chamberlain and sahakian , 2007 ; moeller et al . in addition , cocaine dependent ( cd ) subjects with higher baseline impulsivity predict reduced retention in outpatient treatment trials for cocaine dependence than cd subjects with lower baseline impulsivity ( moeller et al . both cocaine dependence and impulsive behavior are under the regulatory control of cortico - striatal networks ( aron , 2011 ; cunningham and anastasio , 2014 ; dalley et al . however , there is no direct evidence for this putative disruption of directional information flow in cortico - striatal networks in humans , either in cocaine use disorder research or impulsivity research . response inhibition ( ability to withhold a prepotent response ) is one main measure of impulsivity ( moeller et al . most neuroimaging analyses of response inhibition have used either a go / nogo task or a stop - signal task ( colzato et al . , 2011 ) of go / nogo neuroimaging studies have shown activation of frontal , subcortical , parietal , and insular regions with right hemispheric dominance during response inhibition under the nogo condition . it has been hypothesized that the dorsolateral prefrontal cortex ( dlpfc ) , ventrolateral prefrontal cortex ( vlpfc ) , and pre - supplementary motor area are particularly important for response inhibition during nogo conditions ( chikazoe , 2010 ) . the go / nogo task has revealed altered patterns of cortical recruitment under acute demands to curtail a prepotent response in subjects with cocaine dependence . ( 2003 ) conducted a functional magnetic resonance imaging ( fmri ) study with a go / nogo task and found poorer behavioral performance and lower activation in the cingulate , pre - supplementary motor cortex , and insula during response inhibition in active cocaine users compared to cocaine - naive controls . , 2011 ) to test whether cd subjects have altered directional neuronal connectivity underlying their inhibitory behavioral control . we measured response inhibition using the go / nogo task ( lane et al . unique from other analytic techniques , effective ( directional ) connectivity in dcm is modeled at the neuronal level rather than the observed blood oxygen level dependent ( bold ) signal level ( friston et al . ( 2007 ) used a go / nogo task with two - level nogo difficulty ( easy and hard , in terms of similarity between targets and non - targets ) , and found that cd subjects showed poorer behavioral performance than controls only during hard nogo trials rather than easy nogo trials . the dcm analysis in this study was conducted on fmri data acquired from 13 cd subjects and 10 normal healthy cocaine naive controls while they performed a go / nogo task as used in lane et al . based on the hypothesis that cocaine use disorder and inhibitory behavior are regulated through top - down control of the prefrontal cortex reflective system over an amygdala striatum impulsive system ( aron , 2011 ; bechara , 2005 ; cunningham and anastasio , 2014 ; dalley et al . , 2012 ; , we hypothesized that the effective connectivity from prefrontal regions to sub - cortical regions would be altered in cd subjects compared to controls during successful response inhibition . among the 23 subjects included in the final analyses , 15 subjects ( five cd subjects and 10 controls ) were from the first project , and eight subjects ( all cd subjects ) were from the second project . in addition to the 13 completed cd subjects , 13 additional cd subjects were excluded for the following reasons : behavioral performance ( percentage of correct response < 50% ) ( one subject ) ; excessive head motion during the fmri scan ( two subjects ) ; clinically significant abnormal results on flair scan of the brain ( four subjects ) ; subject 's request to terminate the scanning ( three subjects ) ; and current alcohol dependence ( three subjects ) . a rapid - presentation event - related go / nogo task ( lane et al . for all subjects , there were two go / nogo fmri runs . the go / nogo task has been described in detail elsewhere ( lane et al . each fmri run duration was 10 min 40 s , including 156 go trials ( 75% ) , 26 easy nogo trials ( 12.5% ) , and 26 hard nogo trials ( 12.5% ) . , 2007 ) was used to measure behavioral performance on the go / nogo task in the scanner . as an optional feature of spm software , the head motion parameters can be regressed out in the spm level 1 general linear model analysis , and if so , the motion parameters can be then be regressed out from the effects of interest when generating the roi time series for the dcm analysis . the dcm analysis determines which particular nodes in the model exhibit effective ( directional ) connectivity with other specific nodes in the model , which nodes receive driving inputs from experimental conditions into the model , and which specific connectivities between nodes in the model are modulated during experimental conditions . the endogenous ( or fixed ) connectivity in dcm is quantified by the a matrix parameters , which measure the effective connectivity strengths ( in units of hz ) between nodes , regardless of the moment - to - moment switching on and off of inputs . in the present study , the nonlinear option was not applied , and thus the term modulation effects in the present paper denotes bilinear modulation effects , where bilinear refers to the mathematical form of the equations determining the b matrix parameters . ( 2012 , 2014a ) , the regions ( nodes ) for the dcm analysis in the present study were chosen based on simultaneously meeting the following three criteria : ( 1 ) the region must show activation that is at least significant at the uncorrected cluster level in the present univariate spm second - level analysis ; ( 2 ) the region must also show activation ( nogo vs. go or nogo vs. baseline ) in previous fmri studies using go / nogo tasks ( e.g. thus , the following seven nodes were used for the dcm analyses in the present study : ( 1 ) left ( l ) dorsolateral prefrontal cortex ( dlpfc ) ; ( 2 ) right ( r ) dlpfc ; ( 3 ) l anterior cingulate cortex ( acc ) ; ( 4 ) r acc ; ( 5 ) r ventrolateral prefrontal cortex ( vlpfc ) ; ( 6 ) l caudate ( cau ) ; and ( 7 ) r hippocampus ( hipp ) . because of uncertainty about the exact gross anatomical boundaries in humans of the dlpfc ( fuster , 2008 ) , the dlpfc in the present paper was defined by the middle frontal gyrus , which comprises a major portion of the dlpfc in humans ( fuster , 2008 ) . the number of voxels , volume , and center of mass of the seven vois used as nodes for the dcm analysis are shown in table 1 . dcm structure inference , as applied to task - fmri experiments such as this study , searches for a model of the underlying neuronal connectivity among an a priori selected set of brain regions , in which the combined presence of some endogenous connectivities ( and/or modulation / driving input effects ) and the absence of some other endogenous connectivities ( and/or modulation / driving input effects ) best explain the observed fmri data . , go , easy nogo , and hard nogo conditions ) can be a driving input and a modulatory input ; ( 2 ) each of the putative driving inputs entered all of the seven nodes ; ( 3 ) each node was putatively interconnected to all other nodes , and ( 4 ) each of the modulatory inputs putatively modulated all of the 42 interconnectivities between nodes . the between - subjects factor in this analysis was group ( cd and control groups ) , and the within - subjects factor was levels of nogo difficulty ( easy and hard ) . the mean and standard deviation of the discrimination accuracy measure ( d ) and percentage of correct response in each group during easy nogo and hard nogo are shown in table 2 . the main effects of group ( f = 0.014 ; df = 1,35.13 ; p = 0.905 ) and the interaction of group difficulty ( f = 0.111 ; df = 1,35.13 ; p post - hoc comparisons showed that d during easy nogo was significantly greater than during hard nogo for both groups ( cds and controls ) ( p < 0.001 ) , suggesting that the behavioral performance during hard nogo trials was less accurate than that during easy nogo trials . 2 and table 3 ) was found in portions of r middle frontal gyrus ( g ) , r precentral g , r middle cingulate cortex , r superior frontal g , and r paracentral lobule . spm8 second - level random - effects one - sample t - test analysis across both groups combined revealed several clusters for easy , hard , and hard easy activations with the cluster level p less than 0.05 ( uncorrected , two tail ) in portions of frontal , subcortical , and other brain regions . for the cd group , r hipp , l acc , and r vlpfc were reliable ( posterior probability > 0.9999 ) driving input locations for all three driving inputs ( go , easy nogo , and hard nogo ) . in addition , l caudate was a reliable driving input location for easy nogo and go inputs . for the control group , r hipp , l acc , l caudate , and r dlpfc were reliable driving input locations for all three driving inputs ( go , easy nogo , and hard nogo ) . three connectivities ( r dlpfc to r acc , r acc to r dlpfc , and r acc to l dlpfc ) among the 42 connectivities were switched off ( posterior probability = 0 ) by the post - hoc optimization for the cd group . two connectivities ( r vlpfc to r acc , and r acc to r vlpfc ) among the 42 connectivities were switched off ( posterior probability = 0 ) by the post - hoc optimization for the control group . for the cd group , only one ( l acc to l caudate ) of the 42 connectivity was reliably ( posterior probability > 0.9999 ) modulated during nogo conditions , and this connectivity was modulated during both easy ( modulation effect = 0.0584 hz ) and hard ( modulation effect = 0.0822 hz ) nogo conditions . for the control group , three of the 42 connectivity were reliably ( posterior probability > one connectivity ( l acc to l caudate ) was only modulated during the easy nogo condition ( modulation effect = 0.0229 hz ) . , r dlpfc to l caudate , and r vlpfc to l caudate , were only modulated during the hard nogo condition , with modulation effect = 0.2016 hz for the dlpfc caudate connectivity , and modulation effect = 0.2418 hz for the vlpfc caudate connectivity . a post - hoc analysis ( using student 's t - test ) was conducted to determine whether the two groups were significantly different in the modulation effects exerted by the nogo conditions . there was no group difference ( uncorrected p = 0.3640 , 2-tail ) in the modulation effects exerted by the easy nogo condition ( on the connectivity from l acc to l caudate ) . all the modulation effects exerted by the hard nogo condition were significantly different between the groups ( p < 0.0008 , bonferroni corrected ) . these modulation effects were on the connectivities from l acc to l caudate ( cd group : 0.0822 hz ; control group : 0 hz ) , from r dlpfc to l caudate ( cd group : 0 hz ; control group : 0.2016 hz ) , and from r vlpfc to l caudate ( cd group : 0 hz ; control group : 0.2418 hz ) . the present study provides evidence that cortico - striatal circuits activated in cd subjects during inhibition of a prepotent response are distinct from those employed by normal healthy controls responding under the same task with similar performance . when the task demands were high ( hard nogo trials ) , cd subjects demonstrated acc connectivity to the caudate during successful response inhibition instead of the control subjects ' dlpfc or vlpfc connectivity to the caudate which was also during successful response inhibition . in the dcm analysis , a single optimum model with reliable driving input effects and modulatory effects endogenous connectivities , modulatory effects , and driving input effects with high posterior probability were retained , and those with low posterior probability were eliminated by the network discovery analysis . subjects in both groups performed the go / nogo tasks similarly well and demonstrated several commonalities during the nogo conditions . specifically , only prefrontal caudate connectivity was modulated during the nogo condition for both groups . furthermore , the easy nogo condition modulated only l acc to l caudate effective connectivity . these modulation effects were not significantly different between the two groups and support a previous study that reported similar inhibitory behavioral performance in cd and control groups during the easy nogo condition ( lane et al . interestingly , control and cd subjects also achieved similar successful response inhibition during the hard nogo condition . this is in contrast to the poorer performance by cd subjects during hard nogo trials reported previously ( lane et al . we have used dcm to demonstrate differential modulation of effective cortico - striatal networks during hard nogo condition for cd vs. control subjects . specifically , performance during the hard nogo condition was associated with modulation of the effective connectivity of the r vlpfc to l caudate and r dlpfc to l caudate in control subjects only , and the l acc to l caudate in cd subjects only . the dcm analyses showed negative modulation of the effective connectivity from r vlpfc to l caudate during the hard nogo condition in control subjects . thus , our results may reflect that normal healthy subjects achieve successful response inhibition during hard nogo trials due to effective brake the dcm analyses further showed positive modulation of the effective connectivity from r dlpfc to l caudate during the hard nogo condition in control subjects . this result may reflect that healthy subjects achieve executive control through dlpfc activation during the hard nogo condition . while the effective connectivity from the r dlpfc to l caudate was modulated during the hard nogo condition , this pathway was not modulated during the easy nogo condition . further , the modulation of two inter - hemisphere prefrontal striatal connectivities ( r dlpfc to l caudate and r vlpfc to l caudate ) during the hard nogo condition in the control group is consistent with the hypothesis that dlpfc and vlpfc are essential for go / nogo response inhibition tasks and that the right pfc dominates the left pfc during response inhibition in healthy subjects ( chikazoe , 2010 ) . the present study demonstrates the unique finding that the hard nogo condition modulates different prefrontal particularly , the effective connectivity from l acc to l caudate was negatively modulated during the hard nogo condition in the cd group but unaffected in the control group . , 2000 ) , an important aspect of executive control ( garavan and hester , 2007 ) , and emotional response inhibition ( albert et al . , thus , instead of employing the brake functionality of the vlpfc and executive control functionality of the dlpfc as in control subjects , the cd subjects may complete the task through the behavior monitoring functionality of the acc . alternatively , the difficulty level of the hard nogo trials may arouse frustration and consequently activate emotion - responsive neural nodes ( e.g. , 2012 ) during response inhibition , particularly as observed in the cd subjects . the results also may suggest that cd subjects employ an intact intra - hemisphere connectivity ( l acc to l caudate ) to compensate for an altered inter - hemisphere connectivity ( r vlpfc to l caudate ) to achieve successful response inhibition . while the key connectivity underlying response inhibition in cd subjects ( l acc to l caudate ) seems inconsistent with a previous study ( kaufman et al . , 2003 ) that found the acc to be hypoactive in cocaine users during a go / nogo task , it is important to consider that the current study showed no group differences in success during the nogo condition as opposed to the study that demonstrated acc hypoactivity concurrent with impairments in behavioral performance in cd subjects ( kaufman et al . nonetheless , the present study shows group differences in modulation of prefrontal striatal effective connectivity during hard nogo condition consistent with other studies ( e.g. , 2011 ) in cd subjects . normal healthy controls exhibited the ability to adapt dynamic neuronal connectivity dependent upon the difficulty level of the response inhibition task , while cd subjects demonstrated the same intra - hemispheric ( l acc to l caudate ) connectivity regardless of the difficulty level of the response inhibition task . particularly , the 5-ht2ar and 5-ht2cr abundantly localize to pyramidal and gabaergic neurons within the frontal and cingulate cortex ( cornea - hebert et al . , 1997 ) , and ascending dopaminergic mesolimbic neurons innervating the cortico - striatal networks ( bubar and cunningham , 2007 ; doherty and pickel , 2000 ; nocjar et al . future studies with more subjects will be helpful in providing greater insight into the altered neuronal effective connectivity underlying inhibitory control in cd subjects . ( 2 ) all subjects in the present study received a placebo capsule as part of two larger studies in which they were enrolled . ( 3 ) while we have shown modulation of effective connectivity during the nogo conditions , the present study was unable to determine which brain regions mechanistically caused these modulation effects . ( 4 ) although the go / nogo paradigm has been frequently used to investigate response inhibition mechanisms , regional brain activation elicited by go / nogo tasks may not be directly related to response inhibition ( criaud and boulinguez , 2013 ) . therefore , the connectivity observed in the present study may reflect the engagement of other cognitive processes ( e.g. in summary , the control and cd subjects had similar levels of performance on the go / nogo task . given the nodes in our network model , the dcm network discovery analysis revealed that prefrontal striatal connectivities were modulated during the nogo conditions for both groups , consistent with the theory that successful inhibition is related to top - down control by a prefrontal , reflective system over a subcortical , impulsive system . while the effective connectivity from l acc to l caudate was similarly modulated during the easy nogo condition for both groups , differences in connectivity were observed during hard nogo trials between groups . in the control group , the effective connectivity from r vlpfc to l caudate was negatively modulated while the r dlpfc to l caudate was positively modulated during the hard nogo condition ; there were no modulation effects on these two connectivities during the hard nogo condition in the cd group . in the cd group , the effective connectivity from l acc to l caudate was negatively modulated during the hard nogo condition ; there was no modulation effect on this network during the hard nogo condition in the control group . these results indicate that cd subjects use different patterns of connectivity to achieve behavioral performance similar to control subjects during hard nogo trials .

monoamine oxidases are widely distributed enzymes that contain a flavin adenine dinucleotide ( fad ) covalently bounded to a cysteine residue ( 1 ) . many living organisms possess maos and in mammals two isoforms are present , mao - a and mao - b , located in the outer membrane of the mitochondria . these two isoforms are involved in the oxidative deamination of exogenous and endogenous amines , including neurotransmitters , thus modulating their concentrations in the brain and peripheral tissues . physiologically , maos oxidize biogenic neurotransmitters such as dopamine , norepinephrine , 5-hydroxytryptamine ( 5-ht , serotonin ) and -phenethylamine , dietary , and xenobiotic amines such as tyramine and benzylamine ( 2 , 3 ) . mao - a inhibitors are frequently used as antidepressants and anti - anxiety agents while mao - b inhibitors , alone or combined with l - dopa , are relevant tools in the therapy of alzheimer s and parkinson s diseases ( 4 ) . development of mao inhibitors is important not only from the standpoint of symptomatic treatment , but also with regard to the neuroprotective effects ( 5 ) . jun za in traditional chinese - tibetan medicine , have been used as medicament of astriction , choleplania and pyrexia in tibet , china for thousands of years ( 6 ) . in the searching for naturally occurring mao inhibitors from plant , we found that the extract of rheum palmatum showed potential mao inhibition with ic50 of 10.49 g / ml ( 7 ) . further bioactivity - guided fractionation resulted in the isolation of seven stilbenes and one catechin . herein the report , isolation , structure elucidation and mao inhibitory property of these compounds is demonstrated . general experimental procedures nmr spectra were recorded with a varian mercury-400bb nmr spectrometer . semi preparative hplc system consisted of a jasco pu-2086 plus , uv -2075 plus detector and ymc - pack ods - a column ( 5m , 250 10 mm , ymc co. ltd . ) . silica gel 200 - 300 mesh for column chromatography and silica gf254 for tlc were supplied by the qingdao marine chemical inc . , china . mci - gel chp20 ( 75 - 150m ) were from mitsubishi chemical holdings corp . macroporous resin ( hpd-100 ) were purchased from cangzhou baoen chemical inc . , china . polyamide 100 - 200 mesh for column chromatography was purchased from taizhou luqiao sijia biochemical plastic factory , china . rhizomes of rheum palmatum were purchased from bayi herb market in xining , china , which were identified by associate professor , lin yang who majored in plant classification , school of life science and engineering , lanzhou university of technology , lanzhou , china . a voucher specimen ( no . 2011071719 ) is deposited at the school of life science and engineering , lanzhou university of technology . extraction and isolation dried rhizomes of rheum palmatum ( 2.4 kg ) were powdered and refluxed with etoh ( 15 l2 ) for 2 h. the etoh extract was evaporated in vacuo , yielding a extractum . to remove alkaline constituent , the extractum was suspended with distilled water ( 2 l ) and adjusted ph to 10 - 11 , then filtered . the filtrate was adjusted ph to 2 - 3 , and extracted with the etoac 3 times . the etoac extract liquor was evaporated in vacuo , yielding a extractum ( 80 g ) . the extractum was suffered on pre - fractionation using macroporous resin ( hpd-100 , cangzhou bon absorber technology co. ltd . , china ) ( 5 kg ) with a step gradient of etoh - h2o solvent system ( 0:100 , 30:70 , 50:50 , 70:30 , 100:0 , v / v ) as an eluent to yield 6 fractions ( a1~a6 ) . a3 ( 16 g ) was chromatographed on polyamide ( 100 - 200 mesh , 300 g ) using a step gradient of meoh - h2o solvent system ( 0:100 , 30:70 , 50:50 , 70:30 , 100:0 , v / v ) as an eluent to yield 6 fractions ( a3 - 1~a3 - 6 ) . a3 - 5 ( 2 g ) was re - chromatographed on mci gel ( 200 g ) with a stepwise gradient of meoh - h2o solvent system ( 0:100 , 30:70 , 50:50 , 70:30 , 100:0 , v / v ) as an eluent to yield 6 fractions ( a3 - 5 - 1~a3 - 5 - 6 ) . a3 - 5 - 4 ( 350 mg ) was separated successively on silica gel ( 200 - 400 mesh , 350 g ) with a stepwise gradient of chcl3 and meoh ( 20:1 , 15:1 , 10:1 , 5:1 , 1:1 , v / v ) to produce compounds 4 ( 130 mg ) and 2 ( 10 mg ) . a3 - 5 - 3 ( 50 mg ) was purified by polyamide ( 100 - 200 mesh , 50 g ) using meoh - h2o ( 50:50 , v / v ) as a solvent system to give 6 ( 15 mg ) . a3 - 5 - 6 ( 30 mg ) was re - crystallized with meoh to yield 1 ( 20 mg ) . a5 ( 15 g ) was purified by silica gel ( 200 - 400 mesh,150 g ) with a stepwise gradient of chcl3 and meoh ( 20:1 , 15:1 , 10:1 , 5:1 , 1:1 , v / v ) as an eluent to yield 5 fractions ( a5 - 1~a5 - 5 ) , a5 - 5 ( 1 g ) was purified by silica gel(200 - 400 mesh , 150 g ) with a stepwise gradient of petroleum ether - acetone(10:1 - 1:1 , v / v ) to produce 8 ( 30 mg ) . a1(2 g ) was chromatographed on silica gel ( 200 - 400 mesh , 200 g ) with a stepwise gradient of chcl3 and meoh ( 10:1 , 8:1 , 5:1 , 1:1 , v / v ) to produce 7 ( 10 mg ) . a1 - 1 ( 40 mg ) and a1 - 2 ( 100 mg ) were separated successively by semi - preparative hplc ( ymc - pack ods - a , 250 10 mm , 5 m , flow rate 2 ml / min ) eluting with meoh - h2o ( 40:60 , v / v ) to produce 5 ( 15 mg , tr 28 min ) and 3 ( 10 mg , tr 43 min ) , respectively . h - nmr ( 400 mhz , cd3cocd3 ) : ppm 7.07 ( 1h , d , j = 2.0 hz , h-2 ' ) , 6.95 ( 1h , d , j = 16.0 hz , h- ) , 6.90 ( 1h , dd , j = 8.0 , 1.2 hz , h-6 ' ) , 6.80 ( 2h , m , h- , 5 ' ) , 6.53(2h , s , h-2,6 ) , 6.26 ( 1h , s , h-4 ) . c - nmr ( 100 mhz , cd3cocd3 ) : 159.8 ( c-3 , 5 ) , 146.6 ( c-3 ' ) , 141.4 ( c-1 ) , 131.2 ( c-1 ' ) , 129.8 ( c- ) , 127.1 ( c- ) , 120.3 ( c-6 ' ) , 120.3 ( c-6 ' ) , 116.6 ( c-5 ' ) , 114.0 ( c-2 ' ) , 105.9 ( c-2,6 ) , 102.8 ( c-4 ) . ( 8) white powder , c14h12o3.h - nmr ( 400 mhz , cd3cocd3 ) : ppm7.42 ( 2h , d , j = 8.4hz , h-2 ' , 6 ' ) , 7.02 ( 1h , d , j = 16.6 hz , h- ) , 6.88 ( 1h , d , j = 16.6 hz , h- ) , 6.84 ( 2h , d , j = 8.3 hz , h-3 ' , 5 ' ) , 6.54 ( 2h , d , j = 2.0 hz , h-2 , 6 ) , 6.27 ( 1h , d , j = 2.0 hz , h-4 ) . ppm 159.6 ( c-3 , 5 ) , 158.2 ( c-4 ' ) , 141.0 ( c-1 ) , 130.0 ( c-1 ' ) , 129.2 ( c- ) , 128.8 ( c-2 ' , 6 ' ) , 126.9 ( c- ) , 116.5 ( c-3 ' , 5 ' ) , 105.8 ( c-2 , 6 ) , 102.7 ( c-4 ) ( 9 ) . h - nmr ( 400 mhz , cd3od ) : ppm 7.35 ( 2h , d , j = 8.0 hz , h-2 ' , 6 ' ) , 7.02 ( 1h , d , j = 16.0 hz , h- ) , 6.83 ( 1h , d , j = 16.0 hz , h- ) , 6.78 ( 3h , m , h-2 , 3 ' , 5 ' ) , 6.60 ( 1h , brs , h-6 ) , 6.43 ( 1h , brs , h-4 ) , 4.90 ( 1h , d , j = 7.2 hz , h-1 '' ) , 3.303.47 ( 4h , m , h-2 '' , 3 '' , 4 '' and 5 '' ) , 3.70 ( 1h , dd , j = 11.0 , 5.4 hz , h-6b '' ) , 3.90 ( 1h , dd , j = 11.0 , 1.5 hz , h-6a '' ) ( 10 ) . yellow amorphous powder , c20h22o9.h - nmr ( 400 mhz , cd3cocd3 ) : ppm 7.09 ( 1h , brs , h-2 ' ) , 6.86 - 6.99 ( 4h , m , h-5 ' , h-6 ' , h- , h- ) , 6.55 ( 2h , brs , h-2 , 6 ) , 6.27 ( 1h , brs , h-4 ) , 3.84 ( 3h , s , 4'-och3 ) . c - nmr ( 100 mhz , cd3cocd3 ) : ppm 159.5 ( c-3 , 5 ) , 148.3 ( c-3 ' ) , 147.6 ( c-4 ' ) , 140.7 ( c-1 ) , 131.7 ( c-1 ' ) , 129.1 ( c- ) , 127.6 ( c- ) , 119.7 ( c-2 ' ) , 113.2 ( c-5 ' ) , 112.3 ( c-6 ' ) , 105.7 ( c-2 , 6 ) , 102.7 ( c-4 ) , 56.2 ( 4'-och3 ) ( 11 ) . h - nmr ( 400 mhz , cd3od ) : ppm 7.46 ( 1h , d , brs , h-2 ' ) , 7.05 ( 1h , dd , j = 2.0 , 8.6 hz , h-6 ' ) , 6.92 ( 1h , d , j = 16.4 hz , h- ) , 6.81 ( 2h , m , h- , 5 ' ) , 6.45 ( 2h , d , brs , h-2 , 6 ) , 6.16 ( 1h , brs , h-4 ) , 4.80 ( 1h , d , j = 7.6 hz , h-1 '' ) , 3.94 ( 1h , j = 11.0 , 1.5 hz , h-6a '' ) , 3.72 ( 1h , dd , j = 11.0 , 5.4 hz , h-6b '' ) , 3.30 - 3.52 ( 4h , m , h-2 '' toh-5 '' ) . c - nmr ( 400 mhz , cd3od ) ppm : 159.6 ( c-3 , 5 ) , 148.4 ( c-3 ' ) , 147.1 ( c-4 ' ) , 141.1 ( c-1 ) , 131.2 ( c-1 ' ) , 129.2 ( c-),127.7 ( c- ) , 123.6 ( c-6 ' ) , 117.2 ( c-5 ' ) , 116.6 ( c-2 ' ) , 105.8(c-2 , 6 ) , 104.5 ( c-4 ) , 102.7 ( c-1 '' ) , 78.6 ( c-5 '' ) , 77.8 ( c-2 '' ) , 75.1 ( c-3 '' ) , 71.6 ( c-4 '' ) , 62.7 ( c-6 '' ) ( 8) . light yellow amorphous powder , c15h14o4 . h - nmr ( 400 mhz , cd3od ) : ppm 7.00 ( 1h , d , brs , h-2 ' ) , 6.81 - 6.98 ( 5h , m , h-2 , 5 ' , 6 ' , , ) , 6.61 ( 1h , brs , h-2 ) , 6.45 ( 1h , brs , h-4 ) , 3.82 ( 3h , s , 4'-och3 ) , 4.88 ( 1h , d , j = 7.6 hz , h-1 '' ) , 3.92(1h , j = 11.0 , 2.0 hz , glc h-6a '' ) , 3.70 ( 1h , dd , j = 11.0 , 5.6 hz , glc h-6b '' ) , 3.35 - 3.50 ( 4h , m , h-2 '' toh-5 '' ) . c - nmr ( 100 mhz , cd3od ) : ppm 160.4(c-5 ) , 159.6 ( c-3 ) , 149.1 ( c-4 ' ) , 147.9 ( c-3 ' ) , 141.3 ( c-1 ) , 132.4 ( c-1'),129.5 ( c- ) , 128.0 ( c- ) , 120.2 ( c-6 ' ) , 113.8 ( c-2 ' ) , 112.9(c-5 ' ) , 108.3 ( c-6 ) , 107.0 ( c-2 ) , 104.1 ( c-4 ) , 102.4 ( c-1 '' ) , 78.2 ( c-5 '' ) , 78.0 ( c-3 '' ) , 74.9 ( c-2 '' ) , 71.5 ( c-4 '' ) , 62.6 ( c-6 '' ) , 56.5 ( 4'-och3 ) ( 11 ) . h - nmr ( cd3od , 400 mhz ) : ppm 6.83 ( 1h , d , j = 1.8 hz , h-2 ' ) , 6.75 ( 1h , d , j = 8.2 hz , h-5 ' ) , 6.71 ( 1h , dd , j = 8.1,1.8 hz , h-60 ) , 5.92 ( 1h , br s , h-8 ) , 5.85 ( 1h , d , j = 2.2 hz , h-6 ) , 4.56 ( 1h , d , j = 7.4 hz , h-2 ) , 3.97 ( 1h , m , h-3 ) , 2.84 ( 1h , dd , j = 16.0 , 5.2 hz , h-4a ) , 2.50 ( 1h , dd , j = 16.1 , 8.1 hz , h-4b ) ( 12 ) . h - nmr ( cd3od,400 mhz ) : ppm 7.41 ( 2h , d , j = 8.8 hz , h-2 ' , 6 ' ) , 6.97 ( 1h , d , j = 16.0 hz , h- ) , 6.81 - 6.88 ( 3h , m , h-3 ' , 5 ' , ) , 6.46 ( 2h , brs , h-2 , 6 ) , 6.16 ( 1h , brs , h-4 ) , 3.77 ( 3h , s , 4'-och3 ) . c - nmr ( 100 mhz , cd3od ) : ppm : 160.9 ( c-4 ' ) , 159.8 ( c-3 , 5 ) , 141.3 ( c-1 ) , 131.6 ( c-1 ' ) , 129.2 ( c- ) , 128.8 ( c-2 ' , 6 ' ) , 127.9 ( c- ) , 115.2 ( c-3 ' , 5 ' ) , 105.9 ( c-2 , 6 ) 102.9 ( c-4 ) , 55.8 ( 4'-och3 ) ( 9 ) . in - vitro monoamine oxidase inhibition assay preparation of rat liver homogenates mao was partially purified by isolation of mitochondria from rat liver homogenates by a slightly modied with holt s method ( 13 ) . briefly , male wistar rats ( 280300 g ) were euthanised by cervical dislocation and livers dissected out , washed in ice - cold sodium phosphate buffer ( 0.2 m , ph 7.6 ) , liver tissue was homogenized 1:10 ( w / v ) in 0.3 m sucrose . the homogenate was centrifugated at 1000 g for 10 min , the supernatant was draw off , the pellet was centrifugated at 1200 g for 15 min after washed with 20 ml 0.3 m sucrose . supernatants were combined and further centrifuged at 10,000 g for 30 min to obtain mitochondrial pellet . the pellet was resuspended in 4 ml of phosphate buffer ( 0.2 m ; ph 7.6 ) and stored at 4 c . suspensions of mao were diluted eight times before use , and it must be used up in 7 days . monoamine oxidase assay monoamine oxidase inhibition activity was measured in the 96-well microplates according to the method reported by holt et al . with some modifications ( holt et al . , 1997 ) . briey , 40 l enzyme and 40 l sample solution were placed in 96-well microplates and pre - incubated at 37 c for 20 min . the reaction was started by adding 120 l amino substrate ( 2.5 mm tyramine in sodium phosphate buffer ) , 40 l chromogenic solution ( 1 mm vanillic acid , 0.5 mm 4-aminoantipyrine , 4 u / ml peroxidase in sodium phosphate buffer ) , and the total solution was incubated at 37 c for 60 min . blanks were set up by adding 40 l buffer solutions instead of 40 l sample solution . blank negative controls were set up by adding 160 l buffer solutions instead of 40 l sample solution and 120 l substrate solution . sample controls were set up by adding 120 l buffer solution instead of 120 l substrate solution aiming to deduct sample background . the inhibition rate ( % ) was calculated by the following equation : inhibition rate% = 100% inhibitory activity was expressed as the mean of 50% inhibitory concentration ( ic50 ) , obtained by interpolation of concentration - inhibition curves . the selectivity of sample against mao - a and mao - b were also evaluated in this assay . to test specific mao - a activity , the rat liver homogenate was pre - incubated ( 37 c ; 30 min ) with 500 nm pargyline ( a selective inhibitor of mao - b ) to entirely inhibit mao - b . to test specific mao - b activity , the rat liver homogenate was pre - incubated ( 37 c ; 30 min ) with 500 nm clorgyline ( a selective inhibitor of mao - a ) to entirely inhibit mao - a . after the enzyme was pre - incubated , the ic50 values of sample against mao - a and mao - b were determined respectively according above method . in order to further study of structure activity relationship , we have docked three representative compounds 2 , 4 , 8 into the active site of mao . the representative crystal structures of mao - a with harmine ( pdb code : 2z5x ) and mao - b with safinamide ( pdb code : 2v5z ) were obtained from the protein data bank . protein preparation wizard panel tool was used to prepare the protein which include removing crystallographic water molecules , adding hydrogen atoms , assigning partial charges with the opls-2005 force field , assigning protonation states and minimizing the structures . three dimensional structures of compounds 2 , 4 , 8 were built in chembio3d ultra 11.0 and the geometry was optimized with mm2 force field . then the ligprep module ( ligprep , version 2.5 , schrdinger , llc , new york , ny , 2012 ) was used to assign protonation states at a target ph value of 7.0 2.0 . docking grid boxes of mao - a and mao - b were defined by centering on the ligand in 2z5x and 2v5z respectively . the molecular docking was performed using the glide ( glide , version 5.8 , schrdinger , llc , new york , ny , 2012 ) in standard precision ( sp ) mode . as a result , highest scoring docking poses of compound 2 , 4 , 8 in two kinds of protein receptors were selected for further analysis . repeated column chromatography resulted in the isolation of eight compounds 1 - 8 ( figure 1 ) . by comparing nmr data with those published in the literature , 1 - 8 were identified as piceatannol 1 , resveratrol 2 , piceid 3 , rhapontigenin 4 , piceatannol-3'-o--d - glucopyranoside 5 , rhapontigenin 6 , catechin 7 and desoxyrhapontigenin 8 . the inhibitory effects of the isolated compounds 1 - 8 on mao , were showed in table 1 . as shown in table 1 . compounds 2 , 3 , 6 and 7 exhibited weak inhibitory activity against mixed type mao ; compounds 4 , 5 exhibited moderate inhibitory activity ; whereas , compounds 1 and 8 displayed significant inhibitory activity with the ic50 values 16.4 and 11.5 m , respectively , which were close to those of positive control ( ( + ) iproniazid phosphate ic50 = 7.0 m ) . the selectivity of compounds 1 - 8 against mao - a and mao - b were also evaluated . the results ( table 1 . ) showed that compounds 468 preferred to inhibit mao - a rather than mao - b with selectivity values ( [ ic50 of mao - b]/ [ ic50 of mao - a ] ) of 4.74 , 10.01 and 9.42 respectively . the preliminary structure - activity relationships ( sars ) could be drawn from the data of table 1 . as follows : ( 1 ) the activity of stilbenes ( compounds 1 - 6 , 8) was better than that of flavanol ( compound 7 ) . ( 2 ) the methoxyl at the c-4 position of stilbenes plays an important role for mao - a selectivity . for example , compounds 4 , 6 , 8 which all have a methoxyl at the c-4 position exhibited relatively strong inhibition potencies towards mao - a compared to mao - b . ( 3 ) only one methoxyl group at c-4 position and no other substituent groups on b - ring , were favorable for mao ( mixed - type mao , mao - a and mao - b ) inhibition . for example , compound 8 , with only one methoxyl group at c-4 position showed the best inhibitory activity , and the activity of compound 2 ( a demethylated product of 8) decreased almost 8 times for mixed type mao , 20 times for mao - a , 4 times for mao - a , compared to that of 8 . this influence was also reflected between 4 and 8 , 4 with an extra hydroxyl at c-3 position having weaker activity than 8 . the fact that compounds 1 , 3 , 5 with no methoxyl group at c-4 and compound 6 with methoxyl group at c-4 but also with an extra groups at c-3 , showed weaker activity than 8 , further supported the conclusion that only one methoxyl at c-4 position were important for mao inhibition . ( 4 ) the influences of the glycosyls at a - ring on activity have no rules . for example , the activity of 6 ( the glucoside of 4 ) against mixed - type mao and mao - b have a little decrease compared to 4 , but the activity of 3 ( the glucoside of 2 ) against mao - a and mao - b have a little increase compared to 2 . the predicted binding mode of compounds 2 , 4 , 8 to mao - a and mao - b . the side chains of the active site residues ( green or blue sticks ) and compounds 2 , 4 , 8 ( magenta sticks ) were represented as stick model the superimposition of the ligand in mao - a ( orange ) and mao - b ( magentas ) . values are the mean sd of triplicate experiments . the calculated binding free energy of 2 , 4 , 8with mao . compound 2 , 4 , 8 were selected to explore the possible interaction mode between ligand and the active site of mao - a and mao - b . figure 2 . showed the interaction mode of 2 , 4 , 8 with mao - a and mao - b . among the three compounds , 8 had the best binding affinity with both mao - a and mao - b . the two hydroxyl on the ring a of 8 formed hydrogen bonds with thr336 and phe208 residues of mao - a , respectively ( figure 2a ) . the methoxyl on the ring b of 8 had a strong van der waals interaction with fad in mao - a ( figure 2a ) . for the interaction mode between 8 and mao - b , one hydrogen bond formed by the hydroxyl group of the ring a with pro102 was observed , and the amino acid residues ile199leu71tyr326 and phe168 showed relatively strong hydrophobic interaction with compound 8 . compound 4 adopted the same conformation as 8 , as seen in figure 2c . 2d . however , the affinity of 4 with mao - a and mao - b were weaker than those of 8 . in terms of mao - a , the hydroxyl at c-3 position of 4 produce strong steric repulsion with tyr444 which caused some degree of twist of the ring b and also reduced the van der waals interaction between the 4-och3 and fad . for mao - b , the ring a of 4 had some degree of twist which led to - stacking interaction of phe168 and phe103 with ligand decrease . above results indicated the introduction of -oh in ortho position of -och3 would result in decrease of interaction of active pocket of enzyme and ligand . figure 2e and 2f . showed the interaction mode of 2 with mao - a and mao - b . the location of ring a and ring b of compound 2 caused the loss of hydrogen bond interaction with specific residues and decrease of the van der waals interaction with fad . as a result , the activity of 2 against mao - a and mao - b decreased remarkably . the docking results above , revealed that the removal of -och3 at c-4 was disadvantageous to activity . to explain the reason that the methoxyl at the c-4 position of stilbenes plays an important role for mao - a selectivity , we aligned the conformations of compound 2 and 8 in mao - a to those in mao - b . figure 3 . is the superimposition of the location of compound 2 and 8 in mao - a and mao - b binding pocket ( orange is for mao - a and magentas is for mao - b ) . although the structures of mao - a and mao - b have some similarity , there are still some key amino acids different in binding pocket ( mao - a : phe208 , ile335 ; mao - b : ile199 , tyr326 ) which cause ligands are closer to fad in mao - a than in mao - b when binding to the active site . since compound 2 with no -och3 at c-4 only has relatively weak van der waals interaction with fad in mao - a or mao - b , the distance of 2 and fad less affected the mao inhibitory activity . thus , the selectivity of 2 against mao - a and mao - b is weak . however , for compound 8 , due to the presence of a -och3 at c-4 , the van der waals interactions between -och3 and fad tend to play a key role in the interaction of ligand and active pocket . because the distance between 8 and the fad in mao - a pocket is shorter than that in mao - b , the hydrophobic interactions of 8 with mao - a will be stronger than that of 8 with mao - b . this explain the reason that 8 prefer to inhibit mao - a rather than mao - b . in addition , the binding free energy of 2 , 4 and 8 were also calculated which were found to correlate well with experimental results , as shown in table 2 . up to now , many pant extracts have been reported with potential mao inhibitory activity which suggested that plant is a good source for finding new maois . ( 14 ) screened 905 natural extracts for their human mao - b inhibitory activity . the data showed some extracts showed strong inhibitory activity and few of that inhibited mao - b within therapeutic range . in conclusion , seven mao inhibitors with stilbene skeleton ( compound 1 - 6 , 8) have been obtained and their preliminary structure activity relationships ( sars ) were also discussed . the result shows that : ( 1 ) the 4-och3 of stilbenes were important for mao inhibitory activity and selectivity . ( 2 ) except for 4-och3 , the presences of other substituent group at ring b were disadvantageous to mao inhibition .

seven stilbenes and one catechin were bioactivity - guidedly isolated from the rhizomes of rheum palmatem . their structures were identified as piceatannol ( 1 ) , resveratrol ( 2 ) , piceid ( 3 ) , rhapontigenin ( 4 ) , piceatannol-3-o--d - glucopyranoside ( 5 ) , rhaponticin ( 6 ) , catechin ( 7 ) and desoxyrhapontigenin ( 8) . anti - monoamine oxidase ( mao ) activities of compounds 18 were tested . compounds 1 and 8 showed significant mao inhibitory activities with ic50 values 16.4 1.5 m and 11.5 1.1 , respectively , when the ic50 value of iproniazid as a standard was 6.5 0.5 m . the selectivity of compounds 1 - 8 against mao - a and mao - b were also evaluated . the results showed that compounds 468 preferred to inhibit mao - a rather than mao - b with selectivity values ( [ ic50 of mao - b]/ [ ic50 of mao - a ] ) of 4.74 , 10.01 and 9.42 , respectively . the preliminary structure activity relationships ( sars ) of these compounds were discussed and the molecular modeling was also performed to explore the binding mode of inhibitors at the active site of mao - a and mao - b .

Introduction Material and Methods Results and Discussion Conclusion Conflicts of interest

monoamine oxidases are widely distributed enzymes that contain a flavin adenine dinucleotide ( fad ) covalently bounded to a cysteine residue ( 1 ) . many living organisms possess maos and in mammals two isoforms are present , mao - a and mao - b , located in the outer membrane of the mitochondria . physiologically , maos oxidize biogenic neurotransmitters such as dopamine , norepinephrine , 5-hydroxytryptamine ( 5-ht , serotonin ) and -phenethylamine , dietary , and xenobiotic amines such as tyramine and benzylamine ( 2 , 3 ) . mao - a inhibitors are frequently used as antidepressants and anti - anxiety agents while mao - b inhibitors , alone or combined with l - dopa , are relevant tools in the therapy of alzheimer s and parkinson s diseases ( 4 ) . development of mao inhibitors is important not only from the standpoint of symptomatic treatment , but also with regard to the neuroprotective effects ( 5 ) . jun za in traditional chinese - tibetan medicine , have been used as medicament of astriction , choleplania and pyrexia in tibet , china for thousands of years ( 6 ) . in the searching for naturally occurring mao inhibitors from plant , we found that the extract of rheum palmatum showed potential mao inhibition with ic50 of 10.49 g / ml ( 7 ) . further bioactivity - guided fractionation resulted in the isolation of seven stilbenes and one catechin . herein the report , isolation , structure elucidation and mao inhibitory property of these compounds is demonstrated . rhizomes of rheum palmatum were purchased from bayi herb market in xining , china , which were identified by associate professor , lin yang who majored in plant classification , school of life science and engineering , lanzhou university of technology , lanzhou , china . extraction and isolation dried rhizomes of rheum palmatum ( 2.4 kg ) were powdered and refluxed with etoh ( 15 l2 ) for 2 h. the etoh extract was evaporated in vacuo , yielding a extractum . a5 ( 15 g ) was purified by silica gel ( 200 - 400 mesh,150 g ) with a stepwise gradient of chcl3 and meoh ( 20:1 , 15:1 , 10:1 , 5:1 , 1:1 , v / v ) as an eluent to yield 5 fractions ( a5 - 1~a5 - 5 ) , a5 - 5 ( 1 g ) was purified by silica gel(200 - 400 mesh , 150 g ) with a stepwise gradient of petroleum ether - acetone(10:1 - 1:1 , v / v ) to produce 8 ( 30 mg ) . a1 - 1 ( 40 mg ) and a1 - 2 ( 100 mg ) were separated successively by semi - preparative hplc ( ymc - pack ods - a , 250 10 mm , 5 m , flow rate 2 ml / min ) eluting with meoh - h2o ( 40:60 , v / v ) to produce 5 ( 15 mg , tr 28 min ) and 3 ( 10 mg , tr 43 min ) , respectively . ( 8) white powder , c14h12o3.h - nmr ( 400 mhz , cd3cocd3 ) : ppm7.42 ( 2h , d , j = 8.4hz , h-2 ' , 6 ' ) , 7.02 ( 1h , d , j = 16.6 hz , h- ) , 6.88 ( 1h , d , j = 16.6 hz , h- ) , 6.84 ( 2h , d , j = 8.3 hz , h-3 ' , 5 ' ) , 6.54 ( 2h , d , j = 2.0 hz , h-2 , 6 ) , 6.27 ( 1h , d , j = 2.0 hz , h-4 ) . ppm 159.6 ( c-3 , 5 ) , 158.2 ( c-4 ' ) , 141.0 ( c-1 ) , 130.0 ( c-1 ' ) , 129.2 ( c- ) , 128.8 ( c-2 ' , 6 ' ) , 126.9 ( c- ) , 116.5 ( c-3 ' , 5 ' ) , 105.8 ( c-2 , 6 ) , 102.7 ( c-4 ) ( 9 ) . h - nmr ( 400 mhz , cd3od ) : ppm 7.35 ( 2h , d , j = 8.0 hz , h-2 ' , 6 ' ) , 7.02 ( 1h , d , j = 16.0 hz , h- ) , 6.83 ( 1h , d , j = 16.0 hz , h- ) , 6.78 ( 3h , m , h-2 , 3 ' , 5 ' ) , 6.60 ( 1h , brs , h-6 ) , 6.43 ( 1h , brs , h-4 ) , 4.90 ( 1h , d , j = 7.2 hz , h-1 '' ) , 3.303.47 ( 4h , m , h-2 '' , 3 '' , 4 '' and 5 '' ) , 3.70 ( 1h , dd , j = 11.0 , 5.4 hz , h-6b '' ) , 3.90 ( 1h , dd , j = 11.0 , 1.5 hz , h-6a '' ) ( 10 ) . c - nmr ( 100 mhz , cd3cocd3 ) : ppm 159.5 ( c-3 , 5 ) , 148.3 ( c-3 ' ) , 147.6 ( c-4 ' ) , 140.7 ( c-1 ) , 131.7 ( c-1 ' ) , 129.1 ( c- ) , 127.6 ( c- ) , 119.7 ( c-2 ' ) , 113.2 ( c-5 ' ) , 112.3 ( c-6 ' ) , 105.7 ( c-2 , 6 ) , 102.7 ( c-4 ) , 56.2 ( 4'-och3 ) ( 11 ) . h - nmr ( 400 mhz , cd3od ) : ppm 7.46 ( 1h , d , brs , h-2 ' ) , 7.05 ( 1h , dd , j = 2.0 , 8.6 hz , h-6 ' ) , 6.92 ( 1h , d , j = 16.4 hz , h- ) , 6.81 ( 2h , m , h- , 5 ' ) , 6.45 ( 2h , d , brs , h-2 , 6 ) , 6.16 ( 1h , brs , h-4 ) , 4.80 ( 1h , d , j = 7.6 hz , h-1 '' ) , 3.94 ( 1h , j = 11.0 , 1.5 hz , h-6a '' ) , 3.72 ( 1h , dd , j = 11.0 , 5.4 hz , h-6b '' ) , 3.30 - 3.52 ( 4h , m , h-2 '' toh-5 '' ) . c - nmr ( 400 mhz , cd3od ) ppm : 159.6 ( c-3 , 5 ) , 148.4 ( c-3 ' ) , 147.1 ( c-4 ' ) , 141.1 ( c-1 ) , 131.2 ( c-1 ' ) , 129.2 ( c-),127.7 ( c- ) , 123.6 ( c-6 ' ) , 117.2 ( c-5 ' ) , 116.6 ( c-2 ' ) , 105.8(c-2 , 6 ) , 104.5 ( c-4 ) , 102.7 ( c-1 '' ) , 78.6 ( c-5 '' ) , 77.8 ( c-2 '' ) , 75.1 ( c-3 '' ) , 71.6 ( c-4 '' ) , 62.7 ( c-6 '' ) ( 8) . h - nmr ( cd3od , 400 mhz ) : ppm 6.83 ( 1h , d , j = 1.8 hz , h-2 ' ) , 6.75 ( 1h , d , j = 8.2 hz , h-5 ' ) , 6.71 ( 1h , dd , j = 8.1,1.8 hz , h-60 ) , 5.92 ( 1h , br s , h-8 ) , 5.85 ( 1h , d , j = 2.2 hz , h-6 ) , 4.56 ( 1h , d , j = 7.4 hz , h-2 ) , 3.97 ( 1h , m , h-3 ) , 2.84 ( 1h , dd , j = 16.0 , 5.2 hz , h-4a ) , 2.50 ( 1h , dd , j = 16.1 , 8.1 hz , h-4b ) ( 12 ) . h - nmr ( cd3od,400 mhz ) : ppm 7.41 ( 2h , d , j = 8.8 hz , h-2 ' , 6 ' ) , 6.97 ( 1h , d , j = 16.0 hz , h- ) , 6.81 - 6.88 ( 3h , m , h-3 ' , 5 ' , ) , 6.46 ( 2h , brs , h-2 , 6 ) , 6.16 ( 1h , brs , h-4 ) , 3.77 ( 3h , s , 4'-och3 ) . c - nmr ( 100 mhz , cd3od ) : ppm : 160.9 ( c-4 ' ) , 159.8 ( c-3 , 5 ) , 141.3 ( c-1 ) , 131.6 ( c-1 ' ) , 129.2 ( c- ) , 128.8 ( c-2 ' , 6 ' ) , 127.9 ( c- ) , 115.2 ( c-3 ' , 5 ' ) , 105.9 ( c-2 , 6 ) 102.9 ( c-4 ) , 55.8 ( 4'-och3 ) ( 9 ) . the pellet was resuspended in 4 ml of phosphate buffer ( 0.2 m ; ph 7.6 ) and stored at 4 c . the reaction was started by adding 120 l amino substrate ( 2.5 mm tyramine in sodium phosphate buffer ) , 40 l chromogenic solution ( 1 mm vanillic acid , 0.5 mm 4-aminoantipyrine , 4 u / ml peroxidase in sodium phosphate buffer ) , and the total solution was incubated at 37 c for 60 min . the selectivity of sample against mao - a and mao - b were also evaluated in this assay . to test specific mao - a activity , the rat liver homogenate was pre - incubated ( 37 c ; 30 min ) with 500 nm pargyline ( a selective inhibitor of mao - b ) to entirely inhibit mao - b . to test specific mao - b activity , the rat liver homogenate was pre - incubated ( 37 c ; 30 min ) with 500 nm clorgyline ( a selective inhibitor of mao - a ) to entirely inhibit mao - a . after the enzyme was pre - incubated , the ic50 values of sample against mao - a and mao - b were determined respectively according above method . in order to further study of structure activity relationship , we have docked three representative compounds 2 , 4 , 8 into the active site of mao . the representative crystal structures of mao - a with harmine ( pdb code : 2z5x ) and mao - b with safinamide ( pdb code : 2v5z ) were obtained from the protein data bank . three dimensional structures of compounds 2 , 4 , 8 were built in chembio3d ultra 11.0 and the geometry was optimized with mm2 force field . docking grid boxes of mao - a and mao - b were defined by centering on the ligand in 2z5x and 2v5z respectively . repeated column chromatography resulted in the isolation of eight compounds 1 - 8 ( figure 1 ) . by comparing nmr data with those published in the literature , 1 - 8 were identified as piceatannol 1 , resveratrol 2 , piceid 3 , rhapontigenin 4 , piceatannol-3'-o--d - glucopyranoside 5 , rhapontigenin 6 , catechin 7 and desoxyrhapontigenin 8 . the inhibitory effects of the isolated compounds 1 - 8 on mao , were showed in table 1 . compounds 2 , 3 , 6 and 7 exhibited weak inhibitory activity against mixed type mao ; compounds 4 , 5 exhibited moderate inhibitory activity ; whereas , compounds 1 and 8 displayed significant inhibitory activity with the ic50 values 16.4 and 11.5 m , respectively , which were close to those of positive control ( ( + ) iproniazid phosphate ic50 = 7.0 m ) . the selectivity of compounds 1 - 8 against mao - a and mao - b were also evaluated . the results ( table 1 . ) showed that compounds 468 preferred to inhibit mao - a rather than mao - b with selectivity values ( [ ic50 of mao - b]/ [ ic50 of mao - a ] ) of 4.74 , 10.01 and 9.42 respectively . the preliminary structure - activity relationships ( sars ) could be drawn from the data of table 1 . as follows : ( 1 ) the activity of stilbenes ( compounds 1 - 6 , 8) was better than that of flavanol ( compound 7 ) . ( 2 ) the methoxyl at the c-4 position of stilbenes plays an important role for mao - a selectivity . for example , compounds 4 , 6 , 8 which all have a methoxyl at the c-4 position exhibited relatively strong inhibition potencies towards mao - a compared to mao - b . ( 3 ) only one methoxyl group at c-4 position and no other substituent groups on b - ring , were favorable for mao ( mixed - type mao , mao - a and mao - b ) inhibition . for example , compound 8 , with only one methoxyl group at c-4 position showed the best inhibitory activity , and the activity of compound 2 ( a demethylated product of 8) decreased almost 8 times for mixed type mao , 20 times for mao - a , 4 times for mao - a , compared to that of 8 . this influence was also reflected between 4 and 8 , 4 with an extra hydroxyl at c-3 position having weaker activity than 8 . the fact that compounds 1 , 3 , 5 with no methoxyl group at c-4 and compound 6 with methoxyl group at c-4 but also with an extra groups at c-3 , showed weaker activity than 8 , further supported the conclusion that only one methoxyl at c-4 position were important for mao inhibition . ( 4 ) the influences of the glycosyls at a - ring on activity have no rules . for example , the activity of 6 ( the glucoside of 4 ) against mixed - type mao and mao - b have a little decrease compared to 4 , but the activity of 3 ( the glucoside of 2 ) against mao - a and mao - b have a little increase compared to 2 . the predicted binding mode of compounds 2 , 4 , 8 to mao - a and mao - b . the side chains of the active site residues ( green or blue sticks ) and compounds 2 , 4 , 8 ( magenta sticks ) were represented as stick model the superimposition of the ligand in mao - a ( orange ) and mao - b ( magentas ) . compound 2 , 4 , 8 were selected to explore the possible interaction mode between ligand and the active site of mao - a and mao - b . showed the interaction mode of 2 , 4 , 8 with mao - a and mao - b . among the three compounds , 8 had the best binding affinity with both mao - a and mao - b . the two hydroxyl on the ring a of 8 formed hydrogen bonds with thr336 and phe208 residues of mao - a , respectively ( figure 2a ) . for the interaction mode between 8 and mao - b , one hydrogen bond formed by the hydroxyl group of the ring a with pro102 was observed , and the amino acid residues ile199leu71tyr326 and phe168 showed relatively strong hydrophobic interaction with compound 8 . however , the affinity of 4 with mao - a and mao - b were weaker than those of 8 . in terms of mao - a , the hydroxyl at c-3 position of 4 produce strong steric repulsion with tyr444 which caused some degree of twist of the ring b and also reduced the van der waals interaction between the 4-och3 and fad . for mao - b , the ring a of 4 had some degree of twist which led to - stacking interaction of phe168 and phe103 with ligand decrease . showed the interaction mode of 2 with mao - a and mao - b . as a result , the activity of 2 against mao - a and mao - b decreased remarkably . to explain the reason that the methoxyl at the c-4 position of stilbenes plays an important role for mao - a selectivity , we aligned the conformations of compound 2 and 8 in mao - a to those in mao - b . is the superimposition of the location of compound 2 and 8 in mao - a and mao - b binding pocket ( orange is for mao - a and magentas is for mao - b ) . although the structures of mao - a and mao - b have some similarity , there are still some key amino acids different in binding pocket ( mao - a : phe208 , ile335 ; mao - b : ile199 , tyr326 ) which cause ligands are closer to fad in mao - a than in mao - b when binding to the active site . since compound 2 with no -och3 at c-4 only has relatively weak van der waals interaction with fad in mao - a or mao - b , the distance of 2 and fad less affected the mao inhibitory activity . thus , the selectivity of 2 against mao - a and mao - b is weak . because the distance between 8 and the fad in mao - a pocket is shorter than that in mao - b , the hydrophobic interactions of 8 with mao - a will be stronger than that of 8 with mao - b . this explain the reason that 8 prefer to inhibit mao - a rather than mao - b . in addition , the binding free energy of 2 , 4 and 8 were also calculated which were found to correlate well with experimental results , as shown in table 2 . the data showed some extracts showed strong inhibitory activity and few of that inhibited mao - b within therapeutic range . in conclusion , seven mao inhibitors with stilbene skeleton ( compound 1 - 6 , 8) have been obtained and their preliminary structure activity relationships ( sars ) were also discussed . the result shows that : ( 1 ) the 4-och3 of stilbenes were important for mao inhibitory activity and selectivity . ( 2 ) except for 4-och3 , the presences of other substituent group at ring b were disadvantageous to mao inhibition .

cancer is a disease that is difficult to treat , and novel drugs are still highly demanded . synthesis of metal complexes with biologically active ligands is a promising approach in developing anticancer drugs , as metal ions can significantly alter the physical and biological properties of these ligands . indolo[3,2-d]benzazepines , also referred to as paullones , are one class of potential cyclin - dependent kinase ( cdk ) inhibitors , identified in a comparative database search at the national cancer institute ( nci ; nci60 screen ) . thereby , the lead compound kenpaullone exhibited an activity profile similar to that of flavopiridol , the first clinically studied cdk inhibitor . within a series of paullones , however , the antiproliferative activity did not parallel the cdk inhibitory potencies . as a result , other intracellular targets for this class of compounds , e.g. , glycogen synthase kinase 3 ( gsk3 ) or mitochondrial malate dehydrogenase ( mmdh ) , have been suggested . despite marked efforts to develop these compounds as anticancer drugs , paullones remain at an early preclinical stage mainly because of their low aqueous solubility and bioavailability . however , the original paullones did not contain suitable binding sites for metal ions , and these had to be introduced by chemical modification . a library of paullone - based ligands with a broad structural diversity and the respective complexes with copper(ii ) , gallium(iii ) , ruthenium(ii ) , and osmium(ii ) have been reported . in an effort to elucidate novel structure activity relationships ( sars ) , the folded seven - membered azepine ring of paullones has been replaced by a pyridine ring , leading to another class of biologically active compounds , namely , indolo[3,2-c]quinolines , with an essentially planar structure . indolo[3,2-c]quinolines and the structurally related indolo[3,2-b]quinolines are known phytochemicals found in the roots of the west african climbing shrub cryptolepis sanguinolenta that is used in traditional african medicine . both exhibit a broad spectrum of biological properties , including antibacterial , antitumor , as well as anti - inflammatory activity . in contrast to indolo[3,2-b]quinolines , few studies have addressed indolo[3,2-c]quinolines . like paullones , they can , however , be introduced with synthetic tools essentially different from those applied for paullones . the first ruthenium(ii ) , osmium(ii ) , and copper(ii ) complexes with modified indolo[3,2-c]quinoline ligands were derived from structurally related paullone complexes using distinct chemical transformations . in particular , it has been found that complexes of indolo[3,2-c]quinolines exhibit higher cytotoxicity than their paullone counterparts , thus clearly establishing the effect of replacing the azepine ring in paullones by a pyridine ring in indoloquinolines . in addition , it was shown that sars of complexes with modified paullones do not necessarily apply to indolo[3,2-c]quinoline - based compounds . current efforts by us are focused on investigation of the underlying mechanisms of their antiproliferative activity by exploiting the intrinsic fluorescence of indolo[3,2-c]quinolines . recently , we reported on the syntheses of highly antiproliferative copper(ii ) complexes with modified indolo[3,2-c]quinolines . herein , we report on the synthesis of a more elaborate bioconjugate hl with two distinct binding sites and the dinuclear copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively . the new ligand is sufficiently soluble in biological media and intrinsically fluorescent when light irradiated at ex = 395 nm . these properties permitted us to track the intracellular distribution of hl and 2 . moreover , the ligand design led to assembly of homometallic dinuclear complexes with distinct compartments ( scheme 1 ) , a feature not explored by us so far in the development of anticancer metal complexes . hcl ; ( ii ) diethyl-2,2-iminodiacetate , triethylamine , dry thf , room temperature , 3 h ( 95% ) ; ( iii ) methanol , room temperature , 2 h ( 95% ) ; ( iv ) copper(ii ) acetate monohydrate or zinc(ii ) acetate dihydrate , methanol , room temperature , 30 min [ 1 ( 37% ) , 2 ( 33% ) ] . hydrochloric acid , 2-hydroxy-5-methylbenzaldehyde ( a ) , diethyl-2,2-iminodiacetate , 4-((2-hydroxyethyl)-piperazin-1-yl)ethanesulfonic acid ( hepes ) , and guanosine 5-triphosphate were received from sigma - alrdich . l - histidine , formaldehyde solution ( 35% ) , copper(ii ) acetate monohydrate , and zinc(ii ) acetate dihydrate were received from merck , while tetrahydrofuran ( thf ) and methanol ( both analytical reagent grade ) were received from fisher scientific . dimethyl sulfoxide ( dmso ) was received from acros , ammonium bicarbonate , formic acid , and l - glutamic acid were received from fluka , and l - aspartic acid was received from serva . milli - q water ( 18.2 m , millipore advantage a10 , 185 uv ultrapure water system , molsheim , france ) and methanol ( fisher , hplc grade ) were used for esi - ms experiments . 6-hydrazinyl-11h - indolo[3,2-c]quinolone ( d , scheme 1 ) was synthesized according to the published protocol . details of the synthesis and h nmr characterization of d are given in the supporting information . 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) was obtained from 5-methyl-2-hydroxybenzaldehyde ( a ) via a previously described chloromethylation reaction . to a stirred solution of 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( 0.10 g , 0.54 mmol ) in dry thf ( 30 ml ) was added diethyl-2,2-iminodiacetate ( 85 l , 0.48 mmol ) under argon atmosphere . upon addition of triethylamine ( 281 l , 2.0 mmol ) the colorless solution turned bright yellow and a white precipitate formed . after stirring for 3 h at room temperature , the yellow filtrate was concentrated under reduced pressure to give an orange oil , which was dried in vacuo overnight . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 10.60 ( s , 1h , oh ) , 10.23 ( s , 1h , c1 ) , 7.43 ( d , 1h , j(hc5 ) = 2.0 hz , c7 ) , 7.32 ( d , 1h , j(hc7 ) = 2.1 hz , c5 ) , 4.12 ( q , 4h , j(hc16 , c18 ) = 7.1 hz , c15 , c17 ) , 3.90 ( s , 2h , c9 ) , 3.54 ( s , 4h , c11 , c13 ) , 2.26 ( s , 3h , c8 ) , 1.20 ( t , 6h , j(hc15 , c17 ) = 7.1 hz , c16 , c18 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 192.9 ( c1 ) , 171.2 ( c12 , c14 ) , 158.3 ( c3 ) , 137.9 ( c5 ) , 129.1 ( c7 ) , 128.6 ( c6 ) , 125.2 ( c4 ) , 122.3 ( c2 ) , 60.8 ( c15 , c17 ) , 54.4 ( c11 , c13 ) , 53.6 ( c9 ) , 20.3 ( c8 ) , 14.5 ( c16 , c18 ) . to a solution of c ( 0.78 g , 2.3 mmol ) in methanol ( 40 ml ) the reaction mixture turned into a bright yellow suspension , which was stirred for 2 h under argon atmosphere and allowed to stand at 4 c overnight . the yellow product was filtered off , washed with cold methanol ( 2 4 ml ) , and dried in vacuo overnight . calcd for c32h33n5o50.75h2o ( mr = 581.15 ) : c , 66.14 ; h , 5.98 ; n , 12.05 . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.46 ( s , 1h , n11 ) , 10.72 ( s , 1h , n5 ) , 10.63 ( s , 1h , oh ) , 8.77 ( s , 1h , c14 ) , 8.43 ( d , 1h , j(hc8 ) = 7.8 hz , c7 ) , 8.11 ( d , 1h , j(hc2 ) = 7.8 hz , c1 ) , 7.80 ( d , 1h , j(hc3 ) = 8.3 hz , c4 ) , 7.61 ( d , 1h , j(hc9 ) = 8.3 hz , c10 ) , 7.59 ( s , 1h , c20 ) , 7.507.44 ( m , 1h , c3 ) , 7.407.35 ( m , 1h , c9 ) , 7.307.22 ( m , 2h , c8 , c2 ) , 7.10 ( s , 1h , c18 ) , 4.13 ( q , 4h , j(hc29 , c31 ) = 7.1 hz , c28 , c30 ) , 3.94 ( s , 2h , c22 ) , 3.57 ( s , 4h , c24 , c26 ) , 2.30 ( s , 3h , c21 ) , 1.21 ( t , 6h , j(hc28 , c30 ) = 7.1 hz , c29 , c31 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 171.2 ( c25 , c27 ) , 154.0 ( c16 ) , 152.1 ( c14 ) , 150.1 ( c6 ) , 138.6 ( c10a ) , 138.5 ( c11a ) , 138.3 ( c4a ) , 132.1 ( c18 ) , 129.5 ( c3 ) , 129.2 ( c20 ) , 127.8 ( c19 ) , 124.3 ( c9 ) , 124.2 ( c17 ) , 124.1 ( c6b ) , 122.9 ( c7 ) , 122.2 ( c1 ) , 121.9 ( c2 ) , 121.2 ( c8 ) , 120.8 ( c15 ) , 117.1 ( c4 ) , 113.6 ( c11b ) , 112.0 ( c10 ) , 105.2 ( c6a ) , 60.6 ( c28 , c30 ) , 54.5 ( c24 , c26 ) , 53.1 ( c22 ) , 20.7 ( c21 ) , 14.6 ( c29 , c31 ) . esi - ms ( methanol ) , positive m / z 379 [ hl n(ch2cooet)2 ] , 568 [ hl + h ] , 590 [ hl + na ] ; negative m / z 566 [ hl h ] , 603 [ hl + cl ] . vis ( methanol ) , max ( , m cm ) : 226 ( 43 300 ) , 260 ( 32 350 ) , 273 sh ( 25 300 ) , 306 ( 22 400 ) , 348 ( 15 250 ) , 365 sh ( 15 900 ) , 382 ( 17 300 ) . atr - ir , selected bands , cm : 3640 , 3375 , 2976 , 1730 , 1608 , 1461 , 1192 , 1002 . to a suspension of hl ( 0.20 g , 0.35 mmol ) in methanol ( 15 ml ) was added copper(ii ) acetate monohydrate ( 0.16 g , 0.78 mmol ) . after stirring for 30 min the dark - green solution was allowed to stand at 25 c to evaporate slowly . after 3 days , green crystals formed were filtered off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31cu2n5o91.5h2o ( mr = 795.72 ) : c , 49.81 ; h , 4.31 ; n , 8.80 . found : c , 49.57 ; h , 4.30 ; n , 8.64 . esi - ms ( methanol ) : positive m / z 604 unidentified , 648 [ 1 ( hoac ) ( oac ) ] , 680 [ 1 ( oac)2 + ( ch3o ) ] . uv vis ( methanol ) , max ( , m cm ) : 235 ( 60 800 ) , 272 ( 41 000 ) , 296 ( 24 540 ) , 354 ( 18 900 ) , 420 sh ( 20 800 ) , 441 ( 22 700 ) . atr - ir , selected bands , cm : 1737 , 1583 , 1540 , 1385 , 1217 , 1028 . x - ray diffraction - quality single crystals were picked from the reaction vessel prior to filtration . to a suspension of hl ( 0.14 g , 0.25 mmol ) in methanol ( 15 ml ) was added zinc(ii ) acetate dihydrate ( 0.12 g , 0.57 mmol ) . after stirring for 30 min after 4 days cold pentane was added and the mixture allowed to stand at 4 c for 3 h. the yellow precipitate formed was filtered off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31n5o9zn2ch3ohh2o ( mr = 822.46 ) : c , 49.65 ; h , 4.53 ; n , 8.52 . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.3311.60 ( bs , 2h , n11 , n12 ) , 8.626.71 ( bm , 11h , c14 , 710 , 14 , 18 , 20 ) , 4.003.53 ( bm , 9h , c22 , 24 , 26 , 28 ) , 2.22 ( s , 3h , c21 ) , 1.88 ( bs , 6h , ch3coo ) . esi - ms ( methanol ) , positive : m / z 668 [ 2 ( oac)2 ( ch3 ) + ( ch3oh ) ] , 682 [ 2 ( oac)2 + ( ch3o ) ] , 710 [ 2 ( oac ) ] , 724 unidentified , 784 unidentified . uv vis ( methanol ) , max ( , m cm ) : 230 ( 44 400 ) , 258 ( 45 700 ) , 290 ( 27 900 ) , 309 ( 31 000 ) , 330 ( 17 900 ) , 346 ( 18 200 ) , 394 ( 18 900 ) . atr - ir , selected bands , cm : 1744 , 1706 , 1583 , 1407 , 1216 , 1012 . x - ray diffraction - quality single crystals were picked from the reaction vessel prior to addition of pentane . c hsqc , and h c hmbc nmr spectra were recorded on a bruker avance iii spectrometer ( ultrashield magnet ) in dmso - d6 at 25 c using standard pulse programs at 500.13 ( h ) and 125.76 ( c ) mhz . h and c nmr chemical shifts are quoted relative to the residual solvent signals . elemental analyses were carried out at the microanalytical service of the faculty of chemistry , university of vienna . electrospray ionization mass spectrometry was performed on a bruker esquire 3000 instrument ( bruker daltonic , bremen , germany ) on samples dissolved in methanol . vis spectra were recorded with an agilent 8453 spectrophotometer in the 1901000 nm window using samples dissolved in methanol at 10 m concentrations . ir spectra were measured with a bruker vertex 70 fourier transform ir spectrometer by means of the attenuated total reflection ( atr ) technique . fluorescence excitation and emission spectra were recorded with a horiba floromax-4 spectrofluorimeter and processed using the fluoressence v3.5 software package . samples of hl and 2 were prepared from a 1 mm solution of each in dmso and dilution with hepes buffer ( 20 mm , ph = 7.4 ) to give samples at 10 m concentrations with a maximum content of 1% dmso ( v / v ) . x - ray diffraction measurements were performed on a bruker x8 apexii ccd diffractometer . single crystals were positioned at 40 mm from the detector , and 1312 and 722 frames were measured , each for 60 and 90 s over 1 scan width for 13ch3oh and 22ch3oh , correspondingly . crystal data , data collection parameters , and structure refinement details are given in table 1 . structures were solved by direct methods and refined by full - matrix least - squares techniques . non - hydrogen atoms were refined with anisotropic displacement parameters , while h atoms were inserted in calculated positions and refined with a riding model . the following software programs were used : structure solution , shelxs-97 ; refinement , shelxl-97 ; molecular diagrams , ortep ; computer , intel coreduo . wr2 = { [w(fo fc)]/[w(fo)]}. gof = { [w(fo fc)]/(n p ) } , where n is the number of reflections and p is the total number of parameters refined . magnetic measurements were carried out on a microcrystalline sample of 1 with a quantum design squid magnetometer ( mpms - xl ) . variable - temperature ( 2300 k ) direct current ( dc ) magnetic susceptibility was measured under an applied magnetic field of 0.1 t. all data were corrected for the contribution of the sample holder and diamagnetism of the samples estimated from pascal s constants . analysis of the magnetic data was carried out by fitting the mt(t ) and m(t ) thermal variations including temperature - independent paramagnetism ( tip ) , impurity contribution ( ) , and intermolecular interaction ( zj ) according to the expression ( eq 1)1 complex formation was studied by uv vis titration of 10 and 250 m solutions of hl in methanol with 10 l aliquots of 0.5 and 6.25 mm stock solutions of copper(ii ) acetate monohydrate , respectively . one aliquot was added at 2 min intervals followed by homogenization of the solutions as within this period the equilibrium could be reached . an agilent 8453 spectrophotometer was used to record uv vis spectra in the 1901000 nm window . stability constants and molar absorbance spectra of the individual copper(ii ) complexes were calculated by the computer program psequad . electrospray ionization mass spectra were recorded on an amazon sl ion trap mass spectrometer ( bruker daltonics gmbh , bremen , germany ) . experimental data and provided simulations were acquired using compass 1.3 software and processed using data analysis 4.0 ( bruker daltonics gmbh , bremen , germany ) . the experimentally obtained mass signals include a maximum standard deviation of m / z 0.06 for each species . general instrument parameters were set as follows : positive - ion mode ( hv 4.5 kv , rf level 89% , trap drive 74.4 , dry temperature 250 c , nebulizer 8 psi , dry gas 6 l / min and average accumulation time 144 s ) , negative - ion mode ( hv 4.5 kv , rf level 89% , trap drive 63.8 , dry temperature 250 c , nebulizer 8 psi , dry gas 6 l / min and average accumulation time 2 ms ) . samples were diluted with water : methanol ( 50:50 ) or water : methanol : formic acid ( 50:50:0.2 ) to a final metal concentration of 510 m and measured by direct infusion into the mass spectrometer at a flow rate of 4 l / min . stock solutions of 1 and 2 in dmso ( 10 mm ) were prepared and stored at 20 c in the dark . each compound was diluted in ammonium carbonate buffer ( 20 mm , ph = 7.95 ) to give a solution of 100 m of each compound ( with 1% dmso content ) . furthermore , a solution containing l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 5-triphosphate ( gtp ) in equimolar amounts ( 100 m each ) and a solution containing his , asp , glu , and gtp ( each 100 m ) and ascorbic acid ( asc , 400 m ) were prepared in the same buffer . metal - containing solutions were diluted with buffer or mixed with the solutions containing the amino acids and asc at equimolar ratios to give a final metal concentration in each incubation mixture of 50 m . reaction mixtures were incubated at 37 c , and aliquots were measured directly after mixing and after 1 , 3 , 5 , and 24 h after 10-fold dilution of each with water : methanol ( 1:1 ) . the slightly acidic tetramethylammonium acetate buffer ( 20 mm , ph = 6 ) was avoided because partial release of the metal was observed . finally , dilution with water only resulted in a low ionization in the positive- and negative - ion modes . for cytotoxicity determination , three different human cancer cell lines were used : a549 ( nonsmall cell lung cancer ) and sw480 ( colon carcinoma ) from the american type culture collection ( atcc , manassas , va ) , both kindly provided by brigitte marian , institute of cancer research , department of medicine i , medical university vienna , austria , as well as ch1 ( ovarian carcinoma ) , established and kindly provided by the laboratory of lloyd r. kelland , crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . cells were grown as adherent monolayer cultures in 75 cm culture flasks ( starlab , cytoone ) in minimal essential medium supplemented with 10% heat - inactivated fetal bovine serum ( invitrogen ) , 1 mm sodium pyruvate , 1% ( v / v ) nonessential amino acids ( from 100 ready - to - use stock ) , and 4 mm l - glutamine but without antibiotics at 37 c under a moist atmosphere containing 5% co2 and 95% air . all cell culture media and reagents were purchased from sigma - aldrich austria unless indicated otherwise . cytotoxicity was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ) as described previously . briefly , cells were harvested by trypsinization and seeded in medium ( vide supra ) into 96-well plates in volumes of 100 l / well . depending on the cell line , different cell densities were used to ensure exponential growth of the untreated controls during the experiment : 1.0 10 ( ch1 ) , 2.0 10 ( sw480 ) , and 3.0 10 ( a549 ) cells per well . in the first 24 h , the cells were allowed to settle and resume exponential growth . then the test compounds were dissolved in dmso , serially diluted in medium , and added to the plates in volumes of 100 l / well so that the dmso content did not exceed 1% . due to limited solubility of hl and 1 , the highest concentration was applied in volumes of 200 l / well after replacing the original medium . after continuous exposure for 96 h ( in the incubator at 37 c and under 5% co2 ) , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) and mtt solution ( mtt reagent in phosphate - buffered saline , 5 mg / ml ) in a ratio of 6:1 and plates were incubated for further 4 h. then the medium / mtt mixture was removed and the formed formazan was dissolved in dmso ( 150 l / well ) . optical densities at 550 nm were measured ( reference wavelength 690 nm ) with a microplate reader ( elx880 , biotek ) . the quantity of viable cells was expressed as a percentage of untreated controls , and 50% inhibitory concentrations ( ic50 ) were calculated from the concentration effect curves by interpolation . every test was repeated in at least three independent experiments , each consisting of three replicates per concentration level . sw480 cells were seeded in medium on coverslips in 6-well plates and allowed to settle and resume exponential growth for 24 h. then cells were incubated for 12 h with 5 m of 2 or 10 m of hl in medium . co - staining with er - tracker red and lyso - tracker red ( invitrogen ) was performed according to the manufacturer s instructions . after staining , each slide was washed three times in pbs . a fluorescence microscope bx40 ( olympus ) with f - view ccd camera ( olympus ) , cellf fluorescence imaging software ( olympus ) , and 60 magnification oil immersions objective lens were used . syntheses of the ligand hl and the copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively , were carried out as shown in scheme 1 . we prepared a potentially hexadentate nonsymmetric ligand consisting of two chelating arms , one flexible able to provide facial coordination to an octahedral metal ion , while the second is rigid and provides a meridional binding . ester functionalities are frequently introduced into the structure of organic molecules to improve their aqueous solubility and bioavailability . recently , our group reported on the conjugation of l- and d - proline to 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) after chloromethylation of 2-hydroxy-5-methylbenzaldehyde ( a ) ( scheme 1 ) . similarly , we reacted 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) with diethyl-2,2-iminodiacetate and triethylamine in dry thf at room temperature , obtaining diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)-diacetate ( c ) as an orange oil in excellent yield ( 95% ) . the ligand hl was obtained by reacting c with 6-hydrazinyl-11h - indolo[3,2-c]quinoline ( d ) in methanol at room temperature , again in excellent yield ( 95% ) . complexes 1 and 2 were synthesized in 37% and 33% yields starting from the ligand hl and copper(ii ) acetate monohydrate and zinc(ii ) acetate dihydrate , respectively , in methanol at room temperature . the complexation reaction is in both cases accompanied by hydrolysis of one ethyl ester group and transesterification of another ethyl ester function with formation of a new ligand hl . both generated donor arms are involved in coordination to copper(ii ) and zinc(ii ) in 1 and 2 , respectively , via the deprotonated carboxylate group and the carbonyl oxygen of the methyl ester group ( see scheme 1 , figures 1 and 2 ) . the ligand hl and its zinc(ii ) complex 2 have been characterized by one- and two - dimensional nmr spectroscopy , esi mass spectrometry , elemental analysis , uv vis , and atr - ir spectroscopy , while copper(ii ) complex 1 was studied by magnetic susceptibility measurements , esi mass spectrometry , and optical spectroscopy . h and c nmr spectral data of intermediate c , ligand hl , and zinc(ii ) complex 2 along with their assignments are given in the experimental section . the presence of a proton at n in the h nmr spectra and the chemical shift of neighboring c in the c nmr spectra indicate that the ligand adopts a configuration with an exocyclic c = n double bond . moreover , esi mass spectra of 1 and 2 in methanol showed peaks that confirmed formation of dimetal complexes . the most abundant peaks at m / z 680 and 682 for 1 and 2 , correspondingly , were assigned to [ 1/2 ( oac)2 + ( ch3o ) ] . uv vis spectra of hl , 1 , and 2 in methanol are depicted in figure s1 ( supporting information ) . metal coordination led to pronounced changes in the visible range of the ligand spectrum , namely , to evolution of an absorption band at ca . 400 nm for 2 and formation of a broad charge - transfer band at 440 nm for 1 . results of x - ray diffraction studies of 13ch3oh and 22ch3oh shown in figures 1 and 2 , respectively , confirm formation of dinuclear complexes with the two copper(ii ) ions and zinc(ii ) ions bridged by the phenolate oxygen and two exogenous 2-: acetato ligands . both copper(ii ) ions in 1 are distorted square pyramidal with = 0.27 for cu1 with the bridging phenolate oxygen o1 in an apical position and tertiary amine n23 , atoms o6 and o8 of the two bridging acetates , and one aminoacetate o2 in the basal plane . we do not describe the coordination environment around cu1 as octahedral , since the interaction between cu1 and o5 of the dangling methyl ester group is extremely weak ( cu1o5 2.946(2 ) ) . for cu2 a distorted square - pyramidal coordination geometry ( = 0.22 ) was realized with a bridging phenolate oxygen o1 , quinoline nitrogen n5 , hydrazinic nitrogen n13 , one oxygen atom o9 of bridging acetate in a basal plane , and another bridging acetate oxygen atom o7 in apical position . unlike 1 , the coordination environments of zinc(ii ) ions in 2 differ from each other . zn1 has an octahedral environment comprised of the bridging phenolate oxygen o1 , tertiary amine donor n23 , methyl ester oxygen o5 , and atom o6 of the bridging acetate in equatorial positions and two oxygen atoms , one aminoacetate o2 , and a second o8 of a bridging acetate in apical positions . zn2 in contrast to cu2 shows a more pronounced tendency toward a trigonal - bipyramidal coordination geometry ( = 0.47 ) of the same donor atoms . cu2 lies in the mean plane through cu2n5c6n12n13 in 1 , while cu1 comes out from this plane by 1.307 . in 2 the deviation of zn1 from the mean plane through zn2n5c6n12n13 is markedly smaller ( 0.820 ) , while distortion from planarity of the indoloquinoline moiety is more evident than in 1 . the bridging of copper(ii ) ions via phenolate oxygen results in distinct cu1o1 and cu2o1 bond distances . the difference between them ( 0.39 ) is larger than in other nonsymmetrically -phenoxido bridged dicopper(ii ) complexes , in which the two copper(ii ) ions in addition are bridged by at least one exogenous 2-: acetato group . the cu1o6 bond distance is markedly shorter than cu2o7 , and cu1o8 is also shorter than cu2o9 ( see caption to figure 1 ) . the cu1cu2 distance in the complex is 3.2897(6 ) , which is comparable with cucu distances of 3.297(3 ) and 3.263(2 ) in dicopper(ii ) complexes with symmetric dinucleating ligands , containing a di--acetato--phenolatodicopper(ii ) core . ortep view of [ cu2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% probability level . selected bond distances ( angstroms ) and bond angles ( degrees ) : cu1o1 2.323(2 ) , cu1o2 1.937(2 ) , cu1o6 1.946(2 ) , cu1o8 1.936(2 ) , cu1n23 2.093(3 ) , cu1o5 2.946(2 ) , cu2o1 1.913(2 ) , cu2o7 2.194(2 ) , cu2o9 2.002(2 ) , cu2n5 2.038(3 ) , cu2n13 1.956(3 ) , cu1o1cu2 101.47(10 ) . the zn1o1 bond distance is only slightly longer than zn2o1 , as also observed in other complexes with nonsymmetrical dinucleating ligands with a di--acetato--phenolatodizinc(ii ) core . the zn1o6 bond distance is only slightly longer than zn2o7 , while the difference between zn1o8 and the shorter bond zn2o9 is more pronounced ( see caption to figure 2 ) . the interaction between zn1 and o5 of the dangling methyl ester group is markedly stronger than comparable interaction in 1 . the zn1zn2 distance in the complex is at 3.2154(7 ) , which is similar to the znzn distance of 3.29(1 ) in a dizinc(ii ) complex with a nonsymmetrical hybrid ligand . ortep view of [ zn2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% probability level . selected bond distances ( angstroms ) and bond angles ( degrees ) : zn1o1 2.057(3 ) , zn1o2 2.105(3 ) , zn1o6 2.004(3 ) , zn1o8 2.079(3 ) , zn1n23 2.149(4 ) , zn1o5 2.322(3 ) , zn2o1 2.027(3 ) , zn2o7 1.991(3 ) , zn2o9 1.993(3 ) , zn2n5 2.087(4 ) , zn2n13 2.097(4 ) , zn1o1zn2 103.86(14 ) . the magnetic behavior of a polycrystalline sample of 13ch3oh in the temperature range 2300 k in a field of 0.1 t is shown in figure 3 . the value of mt is 0.952 cm k mol at 300 k. this value is slightly higher than the expected mt value ( 0.750 cm k mol ) for two noninteracting copper(ii ) ions ( d , g = 2.0 , s = 1/2 ) . the value of mt continuously increases with decreasing temperature and reaches a value of 1.174 cm k mol at 3 k. this behavior suggests the presence of ferromagnetic interactions in 13ch3oh . according to x - ray diffraction data , complex 13ch3oh has a dinuclear structure , in which the two copper(ii ) ions are connected by a phenolate oxygen atom and two bidentate bridging acetato ligands ( figure 1 ) . therefore , the magnetic behavior can be analyzed by using the classical spin hamiltonian ( eq 2):2where j is the exchange coupling constant and s1 = s2 = 1/2 . plots of mt vs t and magnetization vs h ( inset ) at 2 and 3 k for 13ch3oh . solid lines correspond to the best fit with parameters quoted in the text . in this case , the van vleck equation leads to the following analytical expression ( eq 3)3the fitting procedure results in an excellent agreement between the experimental data and the calculated curve ( r = 1.4 10 ; figure 3 ) . the parameters extracted from the fit are j = 3.49(3 ) cm , g = 2.24(1 ) , and zj = 0.08(1 ) cm and correspond to ferromagnetic interaction between copper(ii ) ions . the temperature - independent paramagnetism ( tip ) and impurity contribution ( ) have values close to zero , and both were fixed at zero in the final fit . the presence of ferromagnetic interaction was confirmed by magnetization measurements at low temperature ( see inset picture in figure 3 ) . the fitting of magnetization vs field using the brillouin function indicates the presence of spin ground state s = 1 ( g = 2.203(2 ) ) in 13ch3oh , which is consistent with the results obtained from analysis of the temperature dependence of magnetic susceptibility . the nature of magnetic interaction in dinuclear copper(ii ) complexes has been extensively studied from both theoretical and experimental points of view . the magnetic interaction in 13ch3oh occurs via three bridges : two 2-: acetato ligands and one bridging phenolate . . according to the literature , the acetate bridges mediate the antiferromagnetic interactions , while the phenolate bridge in dinuclear copper(ii ) complexes can promote both antiferromagnetic as well as ferromagnetic interactions . the character of magnetic interaction depends on geometrical features , especially on the cu o cu angle , out - of - plane deviation angle ( see figure 4 ) , and torsion angle cu o cu o . for angles < 99 and angles > 30 , a strong ferromagnetic interaction can be expected . in the case of 13ch3oh with = 101.47 and = 30.04 , the presence of a weak ferromagnetic interaction ( j = 3.49 cm ) is justified . we can conclude that due to the out - of - plane deviation of the phenol group relative to cu o cu plane the resulting magnetic interaction between the triple - bridged cu(ii ) ions is weakly ferromagnetic . comparable weakly ferromagnetic interactions were reported for other dinuclear copper(ii ) complexes with two or three different bridges . to elucidate whether the two binding sites in hl show different affinities to copper(ii ) , vis titrations of the ligand hl at two different concentrations with copper(ii ) acetate monohydrate in methanol at room temperature ( figures 5 and s2 , supporting information ) . uv vis absorbance spectrum of the ligand hl ( dashed trace ) and its changes by addition of copper(ii ) acetate monohydrate ( solid traces ) in methanol ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm ) . 440 nm was observed upon addition of up to 1.5 mol equiv of copper(ii ) . a wide band with max at 664 nm overlapped partly with the charge - transfer band was seen upon addition of copper(ii ) . this absorption band is slightly red shifted upon addition of more than 1 equiv of copper(ii ) . on the basis of the spectral changes in the wavelength range 230520 nm ( figure 5 ) , overall stability constants have been calculated for the mono- [ cul ] ( log = 7.17 0.08 ) and dinuclear [ cu2l ] species ( log = 13.13 0.24 ; log k = 5.96 ) . the molar absorbance spectra of the ligand , [ cul ] , and [ cu2l ] complexes were also calculated ( figure 5 ) . the goodness - of - fit between measured and calculated absorbance values is shown in figure 6 . d transition bands were in good agreement with those obtained by monitoring the charge - transfer band within 0.2 log unit . stepwise formation constant of the [ cu2l ] species is merely 1 log unit lower than that of the [ cul ] showing the overlapping binding of the metal ions . therefore , it can be concluded that both binding sites in ligand hl coordinate with a similar affinity and no preference for either of them can be perceived . measured and calculated ( dashed lines ) absorbance values at 382 ( ) and 440 nm ( ) at various hl -to - copper(ii ) ratios ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm , methanol ) . the stability of complexes 1 and 2 in aqueous solution and their reactivity toward small biomolecules was studied by esi mass spectrometry ( esi - ms ) since it proved to be effective for characterizing also complex metallodrug interactions with biomolecules . both complexes display a very similar aqueous solution behavior , which is characterized by ester hydrolysis of the ligand and partial metal release over time . products of ester hydrolysis are detected directly after dissolving the compounds in buffer , and the ester is quantitatively hydrolyzed within 24 h. the major thermodynamic products after this period correspond to ions [ m2(l me)(oh ) h ] and [ m(l me ) h ] , where m = cu or zn and l = l , detected in the negative - ion mode ( figure 7 ) . the latter mass signal suggests that release of specifically one metal can occur from both 1 and 2 . interestingly , these signals are detected at 95% and 38% intensities relative to [ m2(l me)(oh ) h ] for 1 and 2 , respectively , i.e. , the cu complex 1 releases the metal to a greater extent . therefore , complex 2 appears to be slightly more stable in aqueous solution , which also seems to be of relevance for the cytotoxicity . additionally , 2 does not ionize in the positive - ion mode , suggesting stable bonds between zn ions and the acetato ligands ( figure s3 , supporting information ) . note that acetato complexes were not detected in the mass spectra of 1 or 2 . furthermore , the isotopic distributions of the major mass signals of 1 and 2 are in good agreement with simulated patterns ( figure s4 , supporting information ) . both complexes were exposed to mixtures containing equimolar amounts of l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 5-triphosphate ( gtp ) . the complexes did not react with any of the biological nucleophiles , and similar mass spectra were observed compared to solutions containing only the respective metal . addition of 4 equiv of ascorbic acid ( asc ) to the amino acids resulted in transient formation of glu and asc adducts with 1 in a small amount ; however , they were only detected immediately after mixing ( figure 7c ) and absent for 2 . free ascorbate was consumed within 1 h but had no impact on the overall reactivity of the complexes . esi mass spectra in negative - ion mode are shown for 1 ( a ) and 2 ( b ) in methanol over a period of 24 h. ( c ) glu- and asc - adducts of 1 , which were only detected directly after mixing . an interesting feature of both compounds is their ability to release a metal ion also in a ph - dependent manner ( figure s5 , supporting information ) . the samples incubated at ph = 7.95 for 24 h displayed only partial metal release . lowering the ph of this incubation solution by dilution with 0.1% formic acid resulted in immediate and quantitative release of one metal from both dimetallic complexes . it is suggested that the carboxylates are prone to protonation under these conditions , leading to release of the coordinated metal . fluorescence spectra of hl and 2 were recorded in hepes - buffered solutions ( 20 mm ; ph = 7.4 ) with a 1% ( v / v ) content of dmso ( figure s6 , supporting information ) . fluorescence excitation spectra ( em = 470 nm ) were measured in the range between 260 and 460 nm and emission spectra ( ex = 395 nm ) in the range from 410 to 710 nm . coordination to zinc(ii ) led to a blue shift of the emission band by 54 nm , with the maximum at 466 nm in the spectra of 2 . hl was found fluorogenic , as excitation and emission spectra strongly increased in intensity upon binding to zinc(ii ) . the cytotoxicity of hl , 1 , and 2 was determined by the mtt assay in three human cancer cell lines , namely , a549 ( nonsmall cell lung carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , all yielding ic50 values in the micromolar concentration range ( table 2 ) . values for a simple copper(ii ) salt , cucl2 , are given for comparison . fifty percent inhibitory concentrations ( means standard deviations from at least three independent experiments ) , as obtained by the mtt assay using exposure times of 96 h. ch1 is the most sensitive cell line to all tested compounds , whereas a549 , a more chemoresistant cell line equipped with multidrug - resistance - mediating proteins , is the least sensitive one , with ic50 values up to 13 times higher than in ch1 cells . whereas complexation with copper(ii ) has either little effect on cytotoxicity ( a549 , sw480 cells ) or yields 3-fold decreased potency ( ch1 cells ) , complexation with zinc(ii ) results in about 2-fold enhancement of cytotoxicity , compared to the metal - free ligand hl in all three cell lines . in comparison to the dicopper(ii ) complex 1 , the dizinc(ii ) complex 2 is up to three times more active in sw480 and four times more active in ch1 cells ( see also figure s7 , supporting information ) . this might be directly related to the lower tendency of 2 to release a metal in aqueous media compared to 1 as observed in the esi - ms experiments . on the basis of these observations , it can be concluded that complexation to zinc(ii ) results in higher cytotoxicity but also better solubility in biocompatible media compared to the metal - free ligand as well as copper(ii ) complex 1 . cytotoxic potency of a simple copper(ii ) salt , cucl2 , is lower , with ic50 values being at least five times higher than those of complex 1 . on the basis of the fluorescence properties of hl and dizinc(ii ) complex 2 , their subcellular localization was studied by fluorescence microscopy in human cancer cells including their colocalization with organelle - specific dyes . for visualization of the compounds in live sw480 cells , the u - mwu2 filter ( olympus japan , excitation filter bp330 - 385 , emission filter ba420 ) was used , while the costaining dyes were recorded using the u - mwg2 filter ( olympus japan , excitation filter bp510 - 550 , emission filter ba590 ) . the compounds do not show interference in the u - mwg2 channel , and autofluorescence of the cells was not observed with the used filters . microscopic images of cells treated with hl and 2 , as shown in figure 8 , revealed localization of fluorescence in diffuse voluminous as well as distinct small cytoplasmic structures but no discernible uptake into the nucleus . the highest accumulation matches with both the er - tracker red and the lyso - tracker red staining , suggesting that the endoplasmic reticulum as well as lysosomes are potential target compartments of hl or that lysosomes are involved in sequestration and/or detoxification of the compound . the same may apply to 2 , provided that the complex is sufficiently stable throughout its passage through the cell , as it can not be ruled out that the fluorescence distribution originates from dissociated ligand molecules . cells were costained with 10 m of hl ( a ) or 5 m of 2 ( b ) and er - tracker red ( 500 nm ) and lyso - tracker red ( 1 m ) , respectively . condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate afforded a new nonsymmetric dinucleating ligand hl with increased aqueous solubility and fluorescence properties . complexes 1 and 2 were obtained upon treatment of the ligand with 2 equiv of cu(ch3coo)2h2o and zn(ch3coo)22h2o in methanol , respectively . complexation reaction in both cases is accompanied by hydrolysis of one ethyl ester group and transesterification of another ethyl ester function with formation of hl . dinuclear structure in 13ch3oh and 23ch3oh is supported by three bridges : two acetato ligands and one phenolato bridge from nonsymmetric hl ligand . the temperature dependence and field dependence magnetic measurements for 13ch3oh indicate a weak ferromagnetic interaction ( j = 3.49 cm ) between copper(ii ) ions . all three compounds show respectable antiproliferative activity in human cancer cell lines ( a549 , ch1 , sw480 ) with ic50 values in the low micromolar concentration range . it seems that the increased resistance of 2 toward metal release in aqueous solution compared to 1 may be responsible for the higher cytotoxicity . localization of hl and 2 in cytoplasmic structures has been found by fluorescence microscopy , suggesting that the endoplasmic reticulum as well as the lysosomes can be potential target compartments of these compounds .

dicopper(ii ) and dizinc(ii ) complexes [ cu2(meooclcoo)(ch3coo)2 ] ( 1 ) and [ zn2(meooclcoo)(ch3coo)2 ] ( 2 ) were synthesized by reaction of cu(ch3coo)2h2o and zn(ch3coo)22h2o with a new nonsymmetric dinucleating ligand etoochlcooet prepared by condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate . the design and synthesis of this elaborate ligand was performed with the aim of increasing the aqueous solubility of indolo[3,2-c]quinolines , known as biologically active compounds , and investigating the antiproliferative activity in human cancer cell lines and the cellular distribution by exploring the intrinsic fluorescence of the indoloquinoline scaffold . the compounds have been comprehensively characterized by elemental analysis , spectroscopic methods ( ir , uv vis , 1h and 13c nmr spectroscopy ) , esi mass spectrometry , magnetic susceptibility measurements , and uv vis complex formation studies ( for 1 ) as well as by x - ray crystallography ( 1 and 2 ) . the antiproliferative activity of etoochlcooet , 1 , and 2 was determined by the mtt assay in three human cancer cell lines , namely , a549 ( nonsmall cell lung carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , yielding ic50 values in the micromolar concentration range and showing dependence on the cell line . the effect of metal coordination on cytotoxicity of etoochlcooet is also discussed . the subcellular distribution of etoochlcooet and 2 was investigated by fluorescence microscopy , revealing similar localization for both compounds in cytoplasmic structures .

Introduction Experimental Section Results and Discussion Conclusion

synthesis of metal complexes with biologically active ligands is a promising approach in developing anticancer drugs , as metal ions can significantly alter the physical and biological properties of these ligands . a library of paullone - based ligands with a broad structural diversity and the respective complexes with copper(ii ) , gallium(iii ) , ruthenium(ii ) , and osmium(ii ) have been reported . in an effort to elucidate novel structure activity relationships ( sars ) , the folded seven - membered azepine ring of paullones has been replaced by a pyridine ring , leading to another class of biologically active compounds , namely , indolo[3,2-c]quinolines , with an essentially planar structure . indolo[3,2-c]quinolines and the structurally related indolo[3,2-b]quinolines are known phytochemicals found in the roots of the west african climbing shrub cryptolepis sanguinolenta that is used in traditional african medicine . the first ruthenium(ii ) , osmium(ii ) , and copper(ii ) complexes with modified indolo[3,2-c]quinoline ligands were derived from structurally related paullone complexes using distinct chemical transformations . in particular , it has been found that complexes of indolo[3,2-c]quinolines exhibit higher cytotoxicity than their paullone counterparts , thus clearly establishing the effect of replacing the azepine ring in paullones by a pyridine ring in indoloquinolines . current efforts by us are focused on investigation of the underlying mechanisms of their antiproliferative activity by exploiting the intrinsic fluorescence of indolo[3,2-c]quinolines . herein , we report on the synthesis of a more elaborate bioconjugate hl with two distinct binding sites and the dinuclear copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively . l - histidine , formaldehyde solution ( 35% ) , copper(ii ) acetate monohydrate , and zinc(ii ) acetate dihydrate were received from merck , while tetrahydrofuran ( thf ) and methanol ( both analytical reagent grade ) were received from fisher scientific . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 10.60 ( s , 1h , oh ) , 10.23 ( s , 1h , c1 ) , 7.43 ( d , 1h , j(hc5 ) = 2.0 hz , c7 ) , 7.32 ( d , 1h , j(hc7 ) = 2.1 hz , c5 ) , 4.12 ( q , 4h , j(hc16 , c18 ) = 7.1 hz , c15 , c17 ) , 3.90 ( s , 2h , c9 ) , 3.54 ( s , 4h , c11 , c13 ) , 2.26 ( s , 3h , c8 ) , 1.20 ( t , 6h , j(hc15 , c17 ) = 7.1 hz , c16 , c18 ) . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.46 ( s , 1h , n11 ) , 10.72 ( s , 1h , n5 ) , 10.63 ( s , 1h , oh ) , 8.77 ( s , 1h , c14 ) , 8.43 ( d , 1h , j(hc8 ) = 7.8 hz , c7 ) , 8.11 ( d , 1h , j(hc2 ) = 7.8 hz , c1 ) , 7.80 ( d , 1h , j(hc3 ) = 8.3 hz , c4 ) , 7.61 ( d , 1h , j(hc9 ) = 8.3 hz , c10 ) , 7.59 ( s , 1h , c20 ) , 7.507.44 ( m , 1h , c3 ) , 7.407.35 ( m , 1h , c9 ) , 7.307.22 ( m , 2h , c8 , c2 ) , 7.10 ( s , 1h , c18 ) , 4.13 ( q , 4h , j(hc29 , c31 ) = 7.1 hz , c28 , c30 ) , 3.94 ( s , 2h , c22 ) , 3.57 ( s , 4h , c24 , c26 ) , 2.30 ( s , 3h , c21 ) , 1.21 ( t , 6h , j(hc28 , c30 ) = 7.1 hz , c29 , c31 ) . analysis of the magnetic data was carried out by fitting the mt(t ) and m(t ) thermal variations including temperature - independent paramagnetism ( tip ) , impurity contribution ( ) , and intermolecular interaction ( zj ) according to the expression ( eq 1)1 complex formation was studied by uv vis titration of 10 and 250 m solutions of hl in methanol with 10 l aliquots of 0.5 and 6.25 mm stock solutions of copper(ii ) acetate monohydrate , respectively . stock solutions of 1 and 2 in dmso ( 10 mm ) were prepared and stored at 20 c in the dark . furthermore , a solution containing l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 5-triphosphate ( gtp ) in equimolar amounts ( 100 m each ) and a solution containing his , asp , glu , and gtp ( each 100 m ) and ascorbic acid ( asc , 400 m ) were prepared in the same buffer . for cytotoxicity determination , three different human cancer cell lines were used : a549 ( nonsmall cell lung cancer ) and sw480 ( colon carcinoma ) from the american type culture collection ( atcc , manassas , va ) , both kindly provided by brigitte marian , institute of cancer research , department of medicine i , medical university vienna , austria , as well as ch1 ( ovarian carcinoma ) , established and kindly provided by the laboratory of lloyd r. kelland , crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . cells were grown as adherent monolayer cultures in 75 cm culture flasks ( starlab , cytoone ) in minimal essential medium supplemented with 10% heat - inactivated fetal bovine serum ( invitrogen ) , 1 mm sodium pyruvate , 1% ( v / v ) nonessential amino acids ( from 100 ready - to - use stock ) , and 4 mm l - glutamine but without antibiotics at 37 c under a moist atmosphere containing 5% co2 and 95% air . cytotoxicity was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ) as described previously . depending on the cell line , different cell densities were used to ensure exponential growth of the untreated controls during the experiment : 1.0 10 ( ch1 ) , 2.0 10 ( sw480 ) , and 3.0 10 ( a549 ) cells per well . after continuous exposure for 96 h ( in the incubator at 37 c and under 5% co2 ) , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) and mtt solution ( mtt reagent in phosphate - buffered saline , 5 mg / ml ) in a ratio of 6:1 and plates were incubated for further 4 h. then the medium / mtt mixture was removed and the formed formazan was dissolved in dmso ( 150 l / well ) . syntheses of the ligand hl and the copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively , were carried out as shown in scheme 1 . complexes 1 and 2 were synthesized in 37% and 33% yields starting from the ligand hl and copper(ii ) acetate monohydrate and zinc(ii ) acetate dihydrate , respectively , in methanol at room temperature . both generated donor arms are involved in coordination to copper(ii ) and zinc(ii ) in 1 and 2 , respectively , via the deprotonated carboxylate group and the carbonyl oxygen of the methyl ester group ( see scheme 1 , figures 1 and 2 ) . the ligand hl and its zinc(ii ) complex 2 have been characterized by one- and two - dimensional nmr spectroscopy , esi mass spectrometry , elemental analysis , uv vis , and atr - ir spectroscopy , while copper(ii ) complex 1 was studied by magnetic susceptibility measurements , esi mass spectrometry , and optical spectroscopy . moreover , esi mass spectra of 1 and 2 in methanol showed peaks that confirmed formation of dimetal complexes . uv vis spectra of hl , 1 , and 2 in methanol are depicted in figure s1 ( supporting information ) . metal coordination led to pronounced changes in the visible range of the ligand spectrum , namely , to evolution of an absorption band at ca . results of x - ray diffraction studies of 13ch3oh and 22ch3oh shown in figures 1 and 2 , respectively , confirm formation of dinuclear complexes with the two copper(ii ) ions and zinc(ii ) ions bridged by the phenolate oxygen and two exogenous 2-: acetato ligands . both copper(ii ) ions in 1 are distorted square pyramidal with = 0.27 for cu1 with the bridging phenolate oxygen o1 in an apical position and tertiary amine n23 , atoms o6 and o8 of the two bridging acetates , and one aminoacetate o2 in the basal plane . in 2 the deviation of zn1 from the mean plane through zn2n5c6n12n13 is markedly smaller ( 0.820 ) , while distortion from planarity of the indoloquinoline moiety is more evident than in 1 . the cu1cu2 distance in the complex is 3.2897(6 ) , which is comparable with cucu distances of 3.297(3 ) and 3.263(2 ) in dicopper(ii ) complexes with symmetric dinucleating ligands , containing a di--acetato--phenolatodicopper(ii ) core . the zn1zn2 distance in the complex is at 3.2154(7 ) , which is similar to the znzn distance of 3.29(1 ) in a dizinc(ii ) complex with a nonsymmetrical hybrid ligand . according to x - ray diffraction data , complex 13ch3oh has a dinuclear structure , in which the two copper(ii ) ions are connected by a phenolate oxygen atom and two bidentate bridging acetato ligands ( figure 1 ) . according to the literature , the acetate bridges mediate the antiferromagnetic interactions , while the phenolate bridge in dinuclear copper(ii ) complexes can promote both antiferromagnetic as well as ferromagnetic interactions . the character of magnetic interaction depends on geometrical features , especially on the cu o cu angle , out - of - plane deviation angle ( see figure 4 ) , and torsion angle cu o cu o . uv vis absorbance spectrum of the ligand hl ( dashed trace ) and its changes by addition of copper(ii ) acetate monohydrate ( solid traces ) in methanol ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm ) . on the basis of the spectral changes in the wavelength range 230520 nm ( figure 5 ) , overall stability constants have been calculated for the mono- [ cul ] ( log = 7.17 0.08 ) and dinuclear [ cu2l ] species ( log = 13.13 0.24 ; log k = 5.96 ) . the molar absorbance spectra of the ligand , [ cul ] , and [ cu2l ] complexes were also calculated ( figure 5 ) . the stability of complexes 1 and 2 in aqueous solution and their reactivity toward small biomolecules was studied by esi mass spectrometry ( esi - ms ) since it proved to be effective for characterizing also complex metallodrug interactions with biomolecules . products of ester hydrolysis are detected directly after dissolving the compounds in buffer , and the ester is quantitatively hydrolyzed within 24 h. the major thermodynamic products after this period correspond to ions [ m2(l me)(oh ) h ] and [ m(l me ) h ] , where m = cu or zn and l = l , detected in the negative - ion mode ( figure 7 ) . furthermore , the isotopic distributions of the major mass signals of 1 and 2 are in good agreement with simulated patterns ( figure s4 , supporting information ) . esi mass spectra in negative - ion mode are shown for 1 ( a ) and 2 ( b ) in methanol over a period of 24 h. ( c ) glu- and asc - adducts of 1 , which were only detected directly after mixing . coordination to zinc(ii ) led to a blue shift of the emission band by 54 nm , with the maximum at 466 nm in the spectra of 2 . the cytotoxicity of hl , 1 , and 2 was determined by the mtt assay in three human cancer cell lines , namely , a549 ( nonsmall cell lung carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , all yielding ic50 values in the micromolar concentration range ( table 2 ) . fifty percent inhibitory concentrations ( means standard deviations from at least three independent experiments ) , as obtained by the mtt assay using exposure times of 96 h. ch1 is the most sensitive cell line to all tested compounds , whereas a549 , a more chemoresistant cell line equipped with multidrug - resistance - mediating proteins , is the least sensitive one , with ic50 values up to 13 times higher than in ch1 cells . on the basis of these observations , it can be concluded that complexation to zinc(ii ) results in higher cytotoxicity but also better solubility in biocompatible media compared to the metal - free ligand as well as copper(ii ) complex 1 . on the basis of the fluorescence properties of hl and dizinc(ii ) complex 2 , their subcellular localization was studied by fluorescence microscopy in human cancer cells including their colocalization with organelle - specific dyes . for visualization of the compounds in live sw480 cells , the u - mwu2 filter ( olympus japan , excitation filter bp330 - 385 , emission filter ba420 ) was used , while the costaining dyes were recorded using the u - mwg2 filter ( olympus japan , excitation filter bp510 - 550 , emission filter ba590 ) . the compounds do not show interference in the u - mwg2 channel , and autofluorescence of the cells was not observed with the used filters . microscopic images of cells treated with hl and 2 , as shown in figure 8 , revealed localization of fluorescence in diffuse voluminous as well as distinct small cytoplasmic structures but no discernible uptake into the nucleus . the highest accumulation matches with both the er - tracker red and the lyso - tracker red staining , suggesting that the endoplasmic reticulum as well as lysosomes are potential target compartments of hl or that lysosomes are involved in sequestration and/or detoxification of the compound . cells were costained with 10 m of hl ( a ) or 5 m of 2 ( b ) and er - tracker red ( 500 nm ) and lyso - tracker red ( 1 m ) , respectively . condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate afforded a new nonsymmetric dinucleating ligand hl with increased aqueous solubility and fluorescence properties . complexes 1 and 2 were obtained upon treatment of the ligand with 2 equiv of cu(ch3coo)2h2o and zn(ch3coo)22h2o in methanol , respectively . all three compounds show respectable antiproliferative activity in human cancer cell lines ( a549 , ch1 , sw480 ) with ic50 values in the low micromolar concentration range . localization of hl and 2 in cytoplasmic structures has been found by fluorescence microscopy , suggesting that the endoplasmic reticulum as well as the lysosomes can be potential target compartments of these compounds .

the health risk assessment of environmental selenium , concerning both abnormally low and high intakes , and the related regulatory guidelines are generally based on old evidence , since they have generally been unable to take into consideration the most recent epidemiologic and biochemical evidence , and particularly the recent results of large and well - designed randomized controlled trials ( rcts ) ( 13 ) . the results of these trials , in connection with biochemical and toxicological studies , have shed new light on this relevant public health issue . this has happened with reference to both the upper and the lower limits of selenium intake , which have been so far based in all the assessment on observational studies carried out in seleniferous chinese areas during the 1980s ( 4,5 ) . the availability of the experimental studies ( the trials ) is of particular importance , since they allowed to rule out the key issue of ( unmeasured ) confounding , typically affecting most of observational studies with the possible only exception of the so called in addition , the recent observational and experimental studies made it possible to investigate different populations with reference to age , genetic background and life - style factors , also allowing to test the health effect of selective exposure to specific selenium compounds , such as inorganic haxavalent selenium ( selenate ) and an organic form , selenomethionine . this is particularly important since there is growing evidence of the key importance of the specific selenium forms in influencing the biological activity of this element , with reference to both its toxicological and nutritional effects ( 711 ) . a very large number of epidemiologic studies assessed the relation between chronic exposure to environmental selenium and human health . the studies on this issue frequently investigated the effect on human health of unusually low or high environmental exposures to selenium , due to an abnormal selenium content in soil , locally produced foods and drinking water , or following combustion of coal with high selenium content ( 5,12,13 ) . in addition , the scientific literature encompasses a large number of nutritional epidemiology studies on the long - term health effects of selenium carried out in populations living in non - seleniferous regions and countries . these studies include experimental investigations ( randomized controlled trials ) and observational studies , the latter characterized by case - control , cohort , cross - sectional and ecologic design and being characterized by a far weaker ability compared with trials in addressing the selenium and health relation ( 1,14,15 ) . while the entire review of this huge literature goes beyond the possibility of this report , we aim at briefly updating the evidence generated by the most recent environmental and nutritional studies on the human health effects of selenium , the biological plausibility of this relation , an overview of the challenges that these studies and their interpretation pose , and finally their implications on the adequacy of current environmental selenium standards . our update of this issue starts from the comprehensive assessments of selenium exposure carried out by the us institute of medicine in 2000 ( 4 ) and by a world health organization ( who ) working group in 2004 ( 16 ) . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . 1 ) , adding relevant data to our understanding of the health effects of selenium in humans . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . this literature includes the investigation of health effects of high - selenium environment in south and north america , india , china , and italy . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . in addition , the observational design of these studies induces in most cases a major concern , the potential bias arising from unmeasured ( dietary and life - style ) confounding , in addition to the potential issue of exposure misclassification . moreover , health endpoints were generally different in these studies , thus not allowing their systematic analysis ( and meta - analysis ) in the different populations . finally , in several cases the small number of exposed subjects made it impossible to compute statistically stable estimates , and this lack of precision hampered the detection of potential health effects of such abnormally low and high exposures to environmental selenium . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . however , most of these studies had an observational design , thus suffering from the potential severe bias due to unmeasured confounding and exposure misclassification even in prospective cohort studies , in addition to the other biases typically effecting studies with case - control , cross - sectional and clearly ecologic design ( 1,14,29 ) . in addition , their results have frequently been conflicting even for the same cancer type , as shown for instance for liver cancer ( 30,31 ) , lung cancer ( 3234 ) or breast cancer ( 3538 ) , though in most cases they supported the occurrence of an inverse relation between selenium status and cancer risk ( 1 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . the aim of these studies has been to investigate the effects on cancer risk of an increased intake of this element ( 1,40 ) . in table ii , we report the main features and results of these human studies with experimental design , including an assessment of the possible or established bias of this study based on our evaluation and the criteria developed within the cochrane collaboration network ( 41 ) . in this overview , old selenium trials carried out in china are also reported , but their scientific interest is very limited , if any , due to their very high risk of bias , as reported in detail in a previous assessment ( 1 ) . fortunately , these rcts have generated a clear and consistent pattern of evidence about the effect of selenium on cancer risk , though partially unexpected given the underlying hypothesis which generated the trials , i.e. a beneficial effect of selenium on cancer risk ( 15 ) . this is even more particularly with reference to the cancer type originally suggested by npc to be most strongly associated with a beneficial effect of selenium , prostate cancer ( 39 ) . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . these results strongly and unexpectedly differ from the results reported in the earliest trial , the npc ( 49,52 ) , which however was small and more importantly was later found to be affected by a detection bias ( 53 ) . as previously mentioned , these trials have also been of fundamental ( and unforeseen ) importance in identifying the early signs , symptoms and diseases associated with chronic or subchronic selenium toxicity . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . such excess diabetes risk linked to selenium overexposure was first discovered in trial carried out in a population with a low baseline selenium status ( 15,22 ) and later confirmed in large trials ( 3,23 ) . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . 1 ) , adding relevant data to our understanding of the health effects of selenium in humans . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . this literature includes the investigation of health effects of high - selenium environment in south and north america , india , china , and italy . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . in addition , the observational design of these studies induces in most cases a major concern , the potential bias arising from unmeasured ( dietary and life - style ) confounding , in addition to the potential issue of exposure misclassification . moreover , health endpoints were generally different in these studies , thus not allowing their systematic analysis ( and meta - analysis ) in the different populations . finally , in several cases the small number of exposed subjects made it impossible to compute statistically stable estimates , and this lack of precision hampered the detection of potential health effects of such abnormally low and high exposures to environmental selenium . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . however , most of these studies had an observational design , thus suffering from the potential severe bias due to unmeasured confounding and exposure misclassification even in prospective cohort studies , in addition to the other biases typically effecting studies with case - control , cross - sectional and clearly ecologic design ( 1,14,29 ) . in addition , their results have frequently been conflicting even for the same cancer type , as shown for instance for liver cancer ( 30,31 ) , lung cancer ( 3234 ) or breast cancer ( 3538 ) , though in most cases they supported the occurrence of an inverse relation between selenium status and cancer risk ( 1 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . the aim of these studies has been to investigate the effects on cancer risk of an increased intake of this element ( 1,40 ) . in table ii , we report the main features and results of these human studies with experimental design , including an assessment of the possible or established bias of this study based on our evaluation and the criteria developed within the cochrane collaboration network ( 41 ) . in this overview , old selenium trials carried out in china are also reported , but their scientific interest is very limited , if any , due to their very high risk of bias , as reported in detail in a previous assessment ( 1 ) . fortunately , these rcts have generated a clear and consistent pattern of evidence about the effect of selenium on cancer risk , though partially unexpected given the underlying hypothesis which generated the trials , i.e. a beneficial effect of selenium on cancer risk ( 15 ) . this is even more particularly with reference to the cancer type originally suggested by npc to be most strongly associated with a beneficial effect of selenium , prostate cancer ( 39 ) . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . these results strongly and unexpectedly differ from the results reported in the earliest trial , the npc ( 49,52 ) , which however was small and more importantly was later found to be affected by a detection bias ( 53 ) . as previously mentioned , these trials have also been of fundamental ( and unforeseen ) importance in identifying the early signs , symptoms and diseases associated with chronic or subchronic selenium toxicity . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . such excess diabetes risk linked to selenium overexposure low baseline selenium status ( 15,22 ) and later confirmed in large trials ( 3,23 ) . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . 1 ) , adding relevant data to our understanding of the health effects of selenium in humans . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . this literature includes the investigation of health effects of high - selenium environment in south and north america , india , china , and italy . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . in addition , the observational design of these studies induces in most cases a major concern , the potential bias arising from unmeasured ( dietary and life - style ) confounding , in addition to the potential issue of exposure misclassification . moreover , health endpoints were generally different in these studies , thus not allowing their systematic analysis ( and meta - analysis ) in the different populations . finally , in several cases the small number of exposed subjects made it impossible to compute statistically stable estimates , and this lack of precision hampered the detection of potential health effects of such abnormally low and high exposures to environmental selenium . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . however , most of these studies had an observational design , thus suffering from the potential severe bias due to unmeasured confounding and exposure misclassification even in prospective cohort studies , in addition to the other biases typically effecting studies with case - control , cross - sectional and clearly ecologic design ( 1,14,29 ) . in addition , their results have frequently been conflicting even for the same cancer type , as shown for instance for liver cancer ( 30,31 ) , lung cancer ( 3234 ) or breast cancer ( 3538 ) , though in most cases they supported the occurrence of an inverse relation between selenium status and cancer risk ( 1 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . the aim of these studies has been to investigate the effects on cancer risk of an increased intake of this element ( 1,40 ) . in table ii , we report the main features and results of these human studies with experimental design , including an assessment of the possible or established bias of this study based on our evaluation and the criteria developed within the cochrane collaboration network ( 41 ) . in this overview , old selenium trials carried out in china are also reported , but their scientific interest is very limited , if any , due to their very high risk of bias , as reported in detail in a previous assessment ( 1 ) . fortunately , these rcts have generated a clear and consistent pattern of evidence about the effect of selenium on cancer risk , though partially unexpected given the underlying hypothesis which generated the trials , i.e. a beneficial effect of selenium on cancer risk ( 15 ) . this is even more particularly with reference to the cancer type originally suggested by npc to be most strongly associated with a beneficial effect of selenium , prostate cancer ( 39 ) . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . these results strongly and unexpectedly differ from the results reported in the earliest trial , the npc ( 49,52 ) , which however was small and more importantly was later found to be affected by a detection bias ( 53 ) . as previously mentioned , these trials have also been of fundamental ( and unforeseen ) importance in identifying the early signs , symptoms and diseases associated with chronic or subchronic selenium toxicity . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . such excess diabetes risk linked to selenium overexposure low baseline selenium status ( 15,22 ) and later confirmed in large trials ( 3,23 ) . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . an issue therefore arises about the adequacy of current standards for environmental risk assessment of selenium in the human , for both abnormally low and high exposures . these standards have been defined by a number of agencies since 2000 to 2014 , and as summarized in fig . 2 they encompass minimal recommended values ranging from 30 to 70 g / day , and upper doses ranging from 300 to 400 g / day ( in adults ) for overall selenium exposure ( 4,16,5861 ) . on the contrary , specific guidelines for single selenium species have not been unfortunately set , despite the clear evidence that the various chemical forms of selenium have different biological properties , i.e. nutritional and toxicological activities ( 7,9,10,62 ) . so far , the adequacy of the selenium standards has been mainly based on biochemical endpoints ( for the lowest recommended intake ) and on the occurrence of adverse health outcomes ( for the upper level ) , as identified in old studies carried out in seleniferous areas from china . however , the newly available data from the clinical trials indicate the need of a substantial reassessment of the dose of selenium toxicity , though they unfortunately do not allow to clearly identify a noael and probably also a reliable loael , since only one supplemental dose ( 200 g / selenium / day ) have been used in these trials and dose - response data are lacking . however , using an uncertainty factor as little as 3 , i.e. lower that the uncertainty factors usually adopted in risk assessment ( 10 or more ) also in light of the peculiar nature of this element and its nutritional relevance , selenium intake should not exceed 90 g / day taking into account the signs of toxicity yielded by the npc trial ( an excess diabetes and skin cancer risk ) and by the select trial ( an excess incidence of diabetes , advanced prostate cancer , dermatitis and alopecia ) ( 1 ) , as shown in fig . 2 . however , this estimate may be still inadequate to protect human health from chronic selenium toxicity , and in addition it appears to apply only to organic selenium , and to selenomethionine in particular [ whose toxicity has bene recently much better elucidated ( 6365 ) ] . for inorganic selenium , typically selenate such as those found in underground and drinking waters , the epidemiologic evidence points to a much higher toxicity compared with organic selenium and exactly as expected on the basis of experimental studies ( 10 ) , therefore suggesting much lower acceptable environmental standards ( 57 ) , tentatively 1 g / new standards should also be considered for occupational exposure to selenium , given the limited data available and the potential for toxicity of this source of exposure ( 12,13,66,67 ) . finally , air selenium might represent a so far overlooked risk factor for chronic diseases , taking into account that its outdoor air concentrations have been positively associated with cardiovascular mortality ( 68 ) and with childhood leukemia risk ( 69 ) , though more evidence is clearly required to confirm such possible associations mainly due to the inherent risk of unmeasured confounding in these observational studies . two approaches have been used to define such lowest safe level of exposure : the proteomic change induced by the trace element , and the avoidance of adverse health effects . concerning the latter point ( health issues ) , still limited and inconclusive evidence is available on the large number of diseases tentatively ascribed to a deficiency of environmental selenium ( 4,70 ) , such as the chronic degenerative osteoarthropathy with unclear etiology named kashin - beck disease ( 71,72 ) and an increased susceptibility to viral infections ( 73,74 ) . in addition , the hypothesis of an effect of low environmental selenium exposure in increasing cancer risk may now be ruled out , thanks to the consistent evidence yielded by the recent large and well - conducted randomized trials , which ruled out any preventive effect of selenium on cancer risk . on the converse , evidence exists on the involvement of selenium deficiency on the etiology of a rare but severe cardiomyopathy named keshan disease and endemic in some chinese areas ( 17,18,20,7577 ) , and this observation has played a key role in the identification of the minimal amount of selenium which appears to be required in humans ( 4,78 ) . such involvement has been suggested mainly on the basis of observational evidence , i.e. a lower selenium status in the populations more affected by this disease , and following the beneficial effects of a selenium supplementation trial on disease incidence . however , some epidemiologic features of the disease have since the discovery of the disease suggested alternative etiologic hypotheses ( 79 ) , particularly a cardiotropic infectious agent such as a coxsackie virus , selenium deficiency possibly being a cofactor in disease etiology or simply an innocent bystander ( 19,20,54,77 ) . under this perspective , the beneficial effect of selenium supplementation in a chinese trial might be interpreted as an indication of antiviral effects of the selenium compound used ( inorganic tetravalent selenium , i.e. selenite ) , as suggested by laboratory studies ( 40,80 ) . in any case , while still investigating the cause of keshan disease and the possible involvement of selenium status , it is prudent to avoid a too low intake of selenium under the hypothesis of a role in keshan disease etiology , and therefore average population intake must be higher than that shown to be required to avoid disease incidence , i.e. 13.3 g / day in females and 19.1 in males ( 16 ) . finally , recent evidence has suggested adverse health effects of mutations affecting sec insertion sequence - binding protein 2 or the selenoprotein n1 gene ( 8183 ) , though such abnormalities might not be strictly related to a selenium deficiency neither were they corrected by its supplementation ( 84 ) , thus being of limited interest in the setting of minimal dietary selenium requirements . alternatively , to the use of health endpoints , and considerably more frequently , the amount of the selenium needed to induce the maximization of selenoprotein synthesis ( particularly glutathione - peroxidase and plasma selenoprotein p ) has been proposed to set the minimal requirement of selenium in the human . this approach has been based on the assumption that achievement of this biochemical endpoint , i.e. upregulation ( frequently defined as optimization ) of selenoprotein synthesis indicates the achievement of an adequate supply of this trace element to the human ( 40,85 ) . this would point to adequate dietary intake ( considering this as only source of selenium exposure ) of amount in the order of 70 g / day ( 85 ) , thus reaching or even exceeding the upper limit definable on the basis of the select trial results using an uncertainty factor of 3 and clearly even more , of course , when using an uncertainty factor of 10 ( fig . 2 ) . in addition , this biochemical approach does not take into account that selenoprotein maximization which follows selenium species administration may derive not just from the correction of a nutritional deficiency of the trace element , but as a compensatory response of these proteins ( all characterized by antioxidant properties ) to the pro - oxidant activity of selenium species ( 40,54,8694 ) . there is also little evidence showing that selenoprotein activity , and particularly its maximization , are beneficial to human health , and therefore ( as more generally levels for antioxidant enzymes ) this should not be regarded as an objective unless more evidence in humans are provided ( 40,54 ) . this approach is further strengthened when taking into account that these enzymes are physiologically induced and inducible by oxidative stress ( for selenoproteins , even in the absence of any change in selenium supply ) ( 40,54 ) , as long recognized since the discovery of the selenium - containing antioxidant enzyme glutathione - peroxidase ( 9597 ) . overall , it seems therefore prudent to avoid a maximal expression of selenoproteins ( 54,98 ) , setting as standard a lower amount of their activity , such as proposed by who when suggesting a nutritionally adequate target ( recommended nutrient intake ) the achievement of two thirds of the maximal selenoprotein activity , corresponding to a daily selenium intake of 2534 g in adults ( fig . 2 ) . however , more research is clearly required to set reliable lower and upper safe selenium levels , though the current standards need to be quickly updated with reference to the upper levels taking into account the above - mentioned recent results of the epidemiologic studies , i.e. the high - quality rcts and the environmental studies , and also considering the opportunity to set species - specific standards for this element .

new data have been accumulated in the scientific literature in recent years which allow a more adequate risk assessment of selenium with reference to human health . this new evidence comes from environmental studies , carried out in populations characterized by abnormally high or low selenium intakes , and from high - quality and large randomized controlled trials with selenium recently carried out in the us and in other countries . these trials have consistently shown no beneficial effect on cancer and cardiovascular risk , and have yielded indications of unexpected toxic effects of selenium exposure . overall , these studies indicate that the minimal amount of environmental selenium which is source of risk to human health is much lower than anticipated on the basis of older studies , since toxic effects were shown at levels of intake as low as around 260 g / day for organic selenium and around 16 g / day for inorganic selenium . conversely , populations with average selenium intake of less than 1319 g / day appear to be at risk of a severe cardiomyopathy , keshan disease . overall , there is the need to reconsider the selenium standards for dietary intake , drinking water , outdoor and indoor air levels , taking into account the recently discovered adverse health effects of low - dose selenium overexposure , and carefully assessing the significance of selenium - induced proteomic changes .

Introduction The epidemiologic evidence None Studies in populations living in unusually high and low selenium environments Adequacy of environmental standards

the health risk assessment of environmental selenium , concerning both abnormally low and high intakes , and the related regulatory guidelines are generally based on old evidence , since they have generally been unable to take into consideration the most recent epidemiologic and biochemical evidence , and particularly the recent results of large and well - designed randomized controlled trials ( rcts ) ( 13 ) . this has happened with reference to both the upper and the lower limits of selenium intake , which have been so far based in all the assessment on observational studies carried out in seleniferous chinese areas during the 1980s ( 4,5 ) . in addition , the scientific literature encompasses a large number of nutritional epidemiology studies on the long - term health effects of selenium carried out in populations living in non - seleniferous regions and countries . these studies include experimental investigations ( randomized controlled trials ) and observational studies , the latter characterized by case - control , cohort , cross - sectional and ecologic design and being characterized by a far weaker ability compared with trials in addressing the selenium and health relation ( 1,14,15 ) . while the entire review of this huge literature goes beyond the possibility of this report , we aim at briefly updating the evidence generated by the most recent environmental and nutritional studies on the human health effects of selenium , the biological plausibility of this relation , an overview of the challenges that these studies and their interpretation pose , and finally their implications on the adequacy of current environmental selenium standards . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . this is even more particularly with reference to the cancer type originally suggested by npc to be most strongly associated with a beneficial effect of selenium , prostate cancer ( 39 ) . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . this is even more particularly with reference to the cancer type originally suggested by npc to be most strongly associated with a beneficial effect of selenium , prostate cancer ( 39 ) . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . a large number of environmental studies which investigated the health effects of unusually high or low selenium areas have been published , as summarized in table i. these studies have also substantially contributed to the pubmed - indexed papers on the epidemiology of selenium and human health in addition to the previous papers ( fig . some of these studies have been published after the 2000 institute of medicine selenium assessment ( 4 ) , considerably extending the limited evidence previously available on the basis of a few old chinese studies . the high content of selenium in these areas , in most cases of geological origin , has induced unusually high levels of selenium in locally grown foodstuffs and occasionally in outdoor air and in drinking water , thus increasing human exposure to the element . however , systematic investigations of the health effects of such exposures are unfortunately limited , and in most cases they came from cross - sectional studies , and very rarely from studies with a more adequate design , such as case - control and particularly cohort studies . overall , these studies have yielded an indication that the extremely low selenium intake , in the order of < 10 - 15 g / day , may increase the risk of a severe cardiomyopathy named keshan disease ( 1720 ) , while high selenium intake may have unfavorable effects on the endocrine system and particularly on the thyroid status ( 21 ) , and increase the risk of type 2 diabetes ( 3,22,23 ) , some specific cancers such as melanoma and lymphoid cancers ( 2426 ) , and nervous system disturbances including alterations in visual evoked potentials ( 27 ) and excess risk of amyotrophic lateral sclerosis ( 26,28 ) . several studies have investigated the effects on human health of even limited changes in exposure to environmental selenium , which occurs through different environmental sources ( primarily diet , but also air pollution , occupational environment , smoking and drinking water ) , in populations characterized by exposure levels not considered a priori to be unusually these studies , generally carried out in western populations , have investigated a broad number of health outcomes , but in the majority of cases they focused on cancer risk ( 14,15 ) . luckily and rather unexpectedly for a nutrient with also was known to exert a powerful toxicity , the nutritional interest in this metalloid as well as the extremely attractive preliminary results of the first selenium trial carried out in western countries , the nutrition prevention of cancer ( npc ) trial ( 39 ) , a large number of randomized controlled trials have been conducted during the last two decades . in addition , these studies contributed in elucidating the relation between selenium and cardiovascular risk , another major issue of interest ( 42 ) . moreover , these trials have been fundamental in our understanding of the adverse effects of environmental selenium , rather unexpectedly since they encompassed supplementation of selenium doses considered a priori to be entirely safe ( 1,15 ) . therefore , and differently from other elements of comparable toxicity and of less nutritional interest , the risk assessment of environmental selenium has benefitted from the implementation of experimental studies originally designed for a setting of potential selenium deficiency , but later found to be able to show and identify the early signs and symptoms related to the toxicity of this element . overall , all the recent trials have consistently shown that selenium does not modify risk of overall cancer , prostate cancer and other specific cancers ( 2,3,23,4345 ) , while it may even increase risk of cancers such as advanced ( 46,47 ) or overall prostate cancer ( 48 ) , non - melanoma skin cancer ( 49,50 ) and possibly breast cancer in high - risk women ( 51 ) . in fact , they have shown that already at amount of selenium exposure ( baseline dietary intake plus supplementation ) of around 250300 g / day there is an increased risk of type-2 diabetes . finally , the largest of the selenium rcts , select ( 23 ) , whose overall selenium intake in the supplemented group averaged 300 g / day ( 15 ) , has shown that such amount of exposure induces minor adverse effects such as dermatitis and alopecia [ a long - recognized sign of selenium toxicity ( 12 ) ] . these effects indicate that the selenium lower - observed - adverse - effect - level ( loael ) is much lower than previously considered by regulatory agencies ( 5,54 ) , which could base their assessment on the scarce data yielded by a few old chinese environmental studies ( 55 ) , calling for an update of the risk assessment of this element ( 5,15,56,57 ) . an issue therefore arises about the adequacy of current standards for environmental risk assessment of selenium in the human , for both abnormally low and high exposures . so far , the adequacy of the selenium standards has been mainly based on biochemical endpoints ( for the lowest recommended intake ) and on the occurrence of adverse health outcomes ( for the upper level ) , as identified in old studies carried out in seleniferous areas from china . however , the newly available data from the clinical trials indicate the need of a substantial reassessment of the dose of selenium toxicity , though they unfortunately do not allow to clearly identify a noael and probably also a reliable loael , since only one supplemental dose ( 200 g / selenium / day ) have been used in these trials and dose - response data are lacking . lower that the uncertainty factors usually adopted in risk assessment ( 10 or more ) also in light of the peculiar nature of this element and its nutritional relevance , selenium intake should not exceed 90 g / day taking into account the signs of toxicity yielded by the npc trial ( an excess diabetes and skin cancer risk ) and by the select trial ( an excess incidence of diabetes , advanced prostate cancer , dermatitis and alopecia ) ( 1 ) , as shown in fig . for inorganic selenium , typically selenate such as those found in underground and drinking waters , the epidemiologic evidence points to a much higher toxicity compared with organic selenium and exactly as expected on the basis of experimental studies ( 10 ) , therefore suggesting much lower acceptable environmental standards ( 57 ) , tentatively 1 g / new standards should also be considered for occupational exposure to selenium , given the limited data available and the potential for toxicity of this source of exposure ( 12,13,66,67 ) . finally , air selenium might represent a so far overlooked risk factor for chronic diseases , taking into account that its outdoor air concentrations have been positively associated with cardiovascular mortality ( 68 ) and with childhood leukemia risk ( 69 ) , though more evidence is clearly required to confirm such possible associations mainly due to the inherent risk of unmeasured confounding in these observational studies . on the converse , evidence exists on the involvement of selenium deficiency on the etiology of a rare but severe cardiomyopathy named keshan disease and endemic in some chinese areas ( 17,18,20,7577 ) , and this observation has played a key role in the identification of the minimal amount of selenium which appears to be required in humans ( 4,78 ) . in any case , while still investigating the cause of keshan disease and the possible involvement of selenium status , it is prudent to avoid a too low intake of selenium under the hypothesis of a role in keshan disease etiology , and therefore average population intake must be higher than that shown to be required to avoid disease incidence , i.e. alternatively , to the use of health endpoints , and considerably more frequently , the amount of the selenium needed to induce the maximization of selenoprotein synthesis ( particularly glutathione - peroxidase and plasma selenoprotein p ) has been proposed to set the minimal requirement of selenium in the human . this would point to adequate dietary intake ( considering this as only source of selenium exposure ) of amount in the order of 70 g / day ( 85 ) , thus reaching or even exceeding the upper limit definable on the basis of the select trial results using an uncertainty factor of 3 and clearly even more , of course , when using an uncertainty factor of 10 ( fig . this approach is further strengthened when taking into account that these enzymes are physiologically induced and inducible by oxidative stress ( for selenoproteins , even in the absence of any change in selenium supply ) ( 40,54 ) , as long recognized since the discovery of the selenium - containing antioxidant enzyme glutathione - peroxidase ( 9597 ) . however , more research is clearly required to set reliable lower and upper safe selenium levels , though the current standards need to be quickly updated with reference to the upper levels taking into account the above - mentioned recent results of the epidemiologic studies , i.e. the high - quality rcts and the environmental studies , and also considering the opportunity to set species - specific standards for this element .

about five to ten percent of all cancers are hereditary and due to germline mutations in cancer predisposition genes . advancements in molecular biology and genetics in the last 1 - 2 decades have enabled the cloning of key cancer predisposition genes , resulting in better characterization of major hereditary cancer syndromes . these include hereditary breast and ovarian cancer syndrome due to the brca1/2 genes , hereditary non - polyposis colorectal cancer syndrome due to the mismatch repair genes [ 4 - 6 ] , and familial adenomatous polyposis due to mutations in the apc gene . the recognition of these syndromes and the establishment of management guidelines have led to the development of cancer genetics as a subspecialty in oncology [ 8 - 13 ] . cancer genetics services have been incorporated into routine cancer services in major oncology centres in the west since the late 1990s . such services represent an important primary prevention arm of oncology in its role of identifying high - risk individuals through family history assessment or genetic testing , and to enrol them into early cancer detection and prevention programmes , with the ultimate goal of reducing cancer burden and mortality . singapore is a small country of 699 kmin south east asia and home to 4 million people . cancer is one of the major causes of mortality , and about 7800 cancer cases are diagnosed every year , with the leading cancers being lung , colorectal and breast cancers . health care in singapore is provided through 9 public hospitals , 7 private hospitals , 16 government outpatient polyclinics , and some 1900 private medical clinics . of the 9 public hospitals , two are tertiary hospitals that offer comprehensive cancer services , encompassing surgical , medical , and radiation oncology specialties . cancer genetics services have been formally incorporated into the cancer services of both tertiary hospitals since 2001 . singapore is a multi - ethnic country comprising three major asian ethnic populations : chinese ( 77% ) , malay ( 14% ) , and indian ( 8% ) . the chinese and indians are largely first- or second - generation migrants from china and southern india , while the malays are indigenous to the regions including malaya , sumatra , java , and the other islands of the indonesian archipelago . english is the official language , but mandarin and chinese dialects are the preferred languages used at home among 35% and 24% of singaporeans respectively , followed by english ( 23% ) , malay ( 14% ) and tamil ( 3% ) . the average monthly household income in singapore is s$4,943 ( us$2,907 ) , with 27% earning less than s$1,999 ( us$1,176 ) . health cost is made affordable to the singaporean public through government subsidized medical services at the public hospitals and outpatient government polyclinics through a co - payment system , where citizens pay a percentage of the medical bill , with the remainder subsidized by the government . this is aided by compulsory medical savings for every working adult , who contributes 6 - 8% of his or her monthly salary to a personal medisave account that can be utilized to pay inpatient medical costs and outpatient cancer treatment . the cancer genetics programme at the national university hospital , singapore , is directed by a medical oncologist ( scl ) , who trained in cancer genetics at the john hopkins university school of medicine , usa , and was credentialed in ' familial cancer risk assessment and management ' by the institute for clinical evaluation in the usa . the programme is assisted by a full - time cancer genetics counsellor ( wsc ) , who is a graduate in biomedical science and who trained on the job . the clinic receives patient referrals from specialists within the hospital and from other government hospitals , specialists practising in the private sector , as well as primary care physicians . the national university hospital cancer service sees about 1500 new cancer cases every year , including approximately 600 new breast and colorectal cancers . three - generation family cancer history forms are given routinely to all cancer patients at the hospital cancer centre . these forms are screened by the genetics counsellor to identify high - risk patients , such as those with young onset cancer , familial cancer clustering , and multiple primary cancers , who would warrant genetics risk assessment . high - risk cases are flagged to the primary cancer physician for referral to the cancer genetics programme . the programme currently runs a weekly clinic , evaluating 1 - 5 patients per session . patients who are referred from the government hospitals and polyclinics pay the subsidized rate of s$21 ( us$12 ) for both first and follow - up visits . patients who are self - referred , or referred by private hospitals and general practitioners , pay the full consultation fee of s$75 ( us$44 ) and s$50 ( us$29 ) for first and follow - up visits respectively . the duration of each new case consultation is 45 - 60 minutes , while a typical follow - up visit lasts 15 - 20 minutes . patients are evaluated and counselled individually or with their family members . during each new consultation , patients are classified as low , modest , moderate , or high risk of having a hereditary cancer syndrome , and are counselled accordingly . patients assessed to be at low risk are given cancer screening recommendations pertaining to themselves and their family members based on their cancer and family history . those assessed to have at least 10% chance of having a hereditary cancer syndrome are given genetic counselling for the particular syndrome , including the mode of inheritance , projected lifetime cancer risks , genetic testing including test interpretation and potential benefits and disadvantages of testing , and screening and preventive options . the counselling session is conducted using picture aids , in english , mandarin , a chinese dialect , or malay through an interpreter , according to the patient 's preference . at the end of the session , a pamphlet in english or mandarin summarizing the pertinent features of the hereditary cancer syndrome discussed is given to patients to facilitate retention of information . pamphlets in malay and tamil are currently not available but may be developed in the future . patients are given 1 - 2 weeks to assimilate and share the information with their family members . they are then followed up by a phone call or a separate clinic visit to address queries that may arise . genetic testing that is offered through the programme includes brca1/2 comprehensive sequencing and single site mutation analysis for hereditary breast and ovarian cancer syndrome , mlh1/msh2 comprehensive sequencing and single site mutation analysis and tumour microsatellite instability testing for hereditary non - polyposis colorectal cancer , apc protein truncation test for familial adenomatous polyposis , and sequencing of exons 10 , 11 , 13 - 16 of the ret proto - oncogene for multiple endocrine neoplasias 2a and 2b . the latter three tests are offered by local laboratories , and cost s$260 - 370 ( us$153 - 218 ) per test . brca1/2 and mlh1/msh2 comprehensive sequencing and respective single site mutation analysis are performed at myriad genetics laboratories ( salt lake city , utah , usa ) at the cost of s$5,058 ( us$2,975 ) , s$3,315 ( us$1,950 ) , and s$595 ( us$350 ) respectively . karyotyping for chromosomal abnormalities is offered locally , while genetic testing for rare conditions such as von hippel lindau syndrome and e - cadherin gene analysis for hereditary diffuse gastric cancer syndrome is performed in overseas laboratories . costs for genetic testing are all out - of - pocket expenses as they are not subsidized by the singapore government nor payable using medisave . since the initiation of the programme , colorectal cancer patients fulfilling eligibility criteria may opt to be tested for germline mlh1/msh2 mutations free of charge as part of a research protocol . since september 2003 , patients assessed to have at least 30% chance of carrying a brca1/2 mutation are eligible to receive a 100% subsidy for brca1/2 sequencing , while their family members are eligible to a 50% subsidy for predictive testing through a special government subsidy programme . demographic information , cancer history , family history , genetic risk assessment information , screening recommendations , and genetic test results of patients evaluated in the cancer genetics programme are collected and stored in a user - defined and password - protected database . as cancer genetics is a new field in singapore , we conducted a questionnaire survey in 2002 , shortly after we started the programme , to assess the level of knowledge on breast cancer risk factors and hereditary breast cancer syndrome . among 284 health professionals and 221 medical students surveyed , less than a quarter recognized that paternal family history of cancer is as important as maternal family history in evaluating for hereditary breast cancer syndrome , and less than half were aware that genetic testing for hereditary breast cancer is clinically available , or that prophylactic mastectomy is a preventive option for women at high risk for breast cancer ( table 1 ) . this general lack of awareness on emerging diagnostic and preventive options for hereditary breast cancer syndrome was identified as an important potential barrier to optimal utilization of the cancer genetics service , and active steps were taken to promote awareness through continuing medical education . awareness of breast cancer risk factors and genetics among health professionals and medical students ( n = 505 ) respondents were given the options ' true ' , ' false ' , or ' do n't know ' ; respondents were given the options ' < 1% ' , ' 5 - 10% ' , ' 20 - 30% ' , ' 50 - 60% ' , or ' do n't know ' we next conducted a questionnaire survey to evaluate the acceptance and potential motivators and barriers of breast cancer genetic counselling among breast cancer patients and cancer - free women . about 70% of the 313 respondents indicated interest in attending genetic counselling when medically indicated and perceived the potential benefits . important motivators were learning about cancer risk and cancer detection , helping the family , and the doctor 's recommendation . important barriers were the misperception that cancer patients could not gain personally , cost issues , fears of bad news , and concerns of inability to make use of the information ( table 2 ) . acceptance , potential motivators and barriers of breast cancer genetic counselling among breast cancer patients and cancer - free women ( n = 313 ) responses of breast cancer patients only from january 2001 to march 2006 , 367 new patients were evaluated at the cancer genetics clinic . 72% of referrals were from within the institution , while 28% were from other hospitals or clinics outside the institution . patients with suspected hereditary breast cancer ( 60% ) and colorectal cancer syndromes ( 31% ) formed the majority of cases ( table 3 ) . 87% of patients were assessed to have at least 10% chance of having a hereditary cancer syndrome and therefore offered genetic counselling and testing . of these , 40% underwent genetic testing , and 23% of those tested were found to carry deleterious germline mutations ( table 4 ) . characteristics of patients reviewed in the cancer genetics clinic ( n = 367 ) include 10 patients counselled for predictive testing for a familial mutation ; include 1 patient counselled for predictive testing for a familial mutation ; 18 patients were deemed to have at least 10% chance of having a known hereditary cancer syndrome and offered genetic counselling and testing ; familial clustering of or young nasopharyngeal cancers ( 9% ) , familial clustering of renal cancers ( 9% ) , suspected li fraumeni syndrome ( 6% ) , familial clustering of paragangliomas suspicious of sdhd mutations ( 3% ) , multiple endocrine neoplasia iia ( 3% ) , suspected neurofibromatosis ( 3% ) , turner 's syndrome with young endometrial cancer ( 3% ) genetic testing in cancer genetics programme , national university hospital , singapore others : comprehensive sequencing and rearrangement analysis for von hippel lindau ( 2 ) , karyotyping for turner 's syndrome ( 1 ) , sequencing for e - cadherin gene mutation ( 1 ) , sequencing for sdhd gene mutation ( 1 ) ; others : turner 's syndrome ( 1 ) , sdhd deleterious mutation ( 1 ) from july 2003 , cancer patients and their accompanying family members were surveyed after the genetic counselling session ( table 5 ) . the age distribution of the 110 cancer patients ( median 37 , range 23 - 77 ) and 95 cancer - free family members ( median 39 , range 16 - 65 ) surveyed were similar . cancer - free family members who attended genetic counselling were more educated and more likely to express interest in genetic testing than cancer patients , with 81% indicating that they would definitely or probably take up genetic testing if medically indicated compared to 61% of cancer patients . for both groups , the most common motivators for genetic testing were to help their children and family members . concerns about the cost of testing and belief that the information could not prevent another cancer were the most common reasons cited for not undertaking genetic testing . more than 80% of respondents indicated willingness to share genetic information with siblings and spouses , but only about 70% were willing to share the information with their parents . after the counselling session , 30% and 22% of all respondents felt ' interested ' and ' empowered or informed ' respectively . cancer patients were more likely to experience negative feelings after the session compared to cancer - free family members . survey on genetic testing ( n = 205 ) statistical analysis was performed using chi - square test for categorical variables , and student 's t - test for age ; respondents were allowed to cite more than one reason ; respondents may describe more than one kind of feelings ; respondents may describe more than one kind of negative feelings ; negative feelings : anxious ( 19% ) , burdensome ( 8% ) , upset ( 8% ) , confused ( 7% ) , distracted ( 4% ) , afraid ( 4% ) , numb ( 3% ) , shocked ( 3% ) , angry ( 2% ) , stressed ( 2% ) , depressed ( 1% ) ; negative feelings : anxious ( 13% ) , afraid ( 5% ) , confused ( 4% ) , upset ( 4% ) , stressed ( 4% ) , burdensome ( 3% ) , depressed ( 3% ) , distracted ( 3% ) , numb ( 1% ) a total of 182 index patients were evaluated to have at least 10% chance of carrying a brca1/2 mutation and offered genetic testing . 48% were young onset breast cancer patients without significant family history , 38% were from breast cancer families , 12% were from breast - ovarian cancer families , and 2% were patients with male or bilateral breast cancers . there was no significant difference in ethnic group , age , and marital status between the acceptors and decliners . 28% of young breast cancer patients and 17% of patients from breast cancer families underwent testing , while 62% of index patients from breast - ovarian cancer families were tested . seventeen patients ( 17/53 , 32% ) were found to carry deleterious germline brca1/2 mutations . family histories of breast or breast - ovarian cancer were the strongest predictors of finding a deleterious mutation ( table 6 ) . of the 17 index patients found to carry brca1/2 mutations , five had bilateral mastectomy for breast cancers , and three had bilateral oophorectomy for ovarian cancer . among the 12 carriers with intact breast(s ) , two ( 17% ) opted for prophylactic mastectomy , and two ( 17% ) are considering the option . among the 14 carriers with intact ovaries , three ( 21% ) opted for prophylactic oophorectomy and three ( 21% ) are considering the option . compliance to breast cancer screening with mammography among the high - risk breast cancer patients evaluated at the cancer genetics programme is 84% , while that of brca1/2 mutation carriers is 94% . nature of brca1/2 deleterious mutations and factors associated with deleterious brca1/2 mutation ( n = 17 ) twenty cancer - free family members were counselled for brca1/2 predictive testing . of these , 55% were siblings and 25% adult children of the index patient , while the remaining were second- or third - degree relatives ( 15% nieces , 5% cousins ) . 67% of cancer - free family members who attended counselling for predictive testing were married , and 50% had more than 10 years of formal education . seven eventually underwent testing , of whom six ( 86% ) were found to carry deleterious mutations and are now on surveillance programmes . sixty - six high - risk colorectal cancer patients were tested for mlh1/msh2 mutations , predominantly as part of a research protocol free of charge . fourteen family members had undergone predictive testing , and 6 were found to carry deleterious mutations and are on surveillance . factors that were associated with deleterious germline mutations include family history fulfilling amsterdam i / ii criteria ( 60% ) , proband with early onset colorectal cancer and family history of colorectal cancer or extracolonic cancers ( 46% ) , proband with colorectal cancer and family history of stomach cancer ( 40% ) , and proband with colorectal cancer demonstrating high microsatellite instability ( 36% ) . in contrast to what was reported in the west , family history of endometrial cancer predicted poorly for a deleterious mutation . compliance to screening colonoscopy among mutation carriers is 77% . among the potential barriers uncovered through our previous hypothetical questionnaire survey prior to the initiation of the government subsidy programme for brca1/2 testing in 2003 , 67/70 ( 96% ) breast cancer patients offered genetic testing declined the test , with the majority citing cost as a major barrier . after the initiation of the subsidy programme , 47/106 ( 44% ) eligible patients underwent brca1/2 testing at 100% government subsidy . however , 59/106 patients ( 56% ) still declined genetic testing despite removal of the cost barrier . a telephone survey of 39/59 ( 66% ) patients who declined testing revealed the following major reasons : siblings / family members were not keen to know of such information or to be tested ( 41% ) , perception that testing would not prevent cancer recurrence or alter medical management ( 39% ) , and concerns about negative feelings associated with genetic test results ( 31% ) ( table 7 ) . demographic characteristics of index patients who declined brca1/2 testing despite test cost subsidies and reasons for declining ( n = 39 ) patients were allowed to indicate more than one reason for declining test ; cost of single site mutation analysis in predictive testing is s$595 ( us$350 ) and the singapore government provides 50% reimbursement cancer is a leading cause of morbidity and mortality in singapore , and cancer genetics and risk assessment programmes represent the primary prevention arm of oncology . as a new programme in a mature comprehensive cancer centre , our current workload of approximately 75 new cases annually is close to the target 60 - 120 new referrals that are expected to arise from our institution . this has in part been attributed to our standard procedure to obtain family cancer history from each new cancer patient to identify high - risk patients for referral into the programme . in addition , the success of continuing medical education to increase awareness among physicians has been reflected by increasing referrals from outside the institution , now accounting for about 30% of our new cases . in contrast to developed nations in the west , where lay press and lay media routinely report new medical advances and provide patients and community physicians easy access to new medical knowledge , health providers in singapore generally rely on medical journals and seminars for information on medical advances , while patients rely on their health providers for pertinent medical information . consequently it comes as no surprise to find only a handful of health providers in singapore who are aware of brca1/2 genetic testing or prophylactic surgery for high - risk individuals . more importantly , lack of awareness of the mode of inheritance of the brca1/2 gene has led many health providers to have the mistaken notion that paternal family history is not as important as maternal family history in evaluating for hereditary breast cancer syndrome . such information has been critical for us to focus continuing medical education efforts on filling important knowledge gaps . cost has been cited as an important barrier to genetic counselling and testing in many prior studies , including our own . while genetic counselling is available to singaporeans at a subsidized and affordable rate , genetic testing is not . in fact , although a significant proportion of index patients expressed interest in brca1/2 testing , the uptake rate was a dismal 4% prior to 2003 when the cost of testing was not subsidized . this posed a significant barrier to downstream work of risk segregating individuals and tailoring screening and preventive recommendations using genetic testing . under the special government subsidy programme that provided free brca1/2 testing for index patients , we observed an eleven - fold increase in genetic testing uptake rate to 44% , allowing mutation carriers to be identified and facilitating predictive testing in cancer - free family members . this highlights the importance of overcoming cost as a barrier to allow the full realization of the potential of a cancer genetics programme . despite removing the cost barrier , we found more than half of eligible patients to still decline genetic testing , suggesting that other barriers exist . the central role of the family in asian culture is underscored by the fact that more than 80% of respondents in a hypothetical situation , and more than 50% of high - risk patients who underwent genetic counselling in our population , cited ' helping the family ' to be an important motivator to attend genetic counselling and undertake genetic testing respectively . yet , when it comes to actual genetic testing and the real possibility of involving family members for predictive testing , two - fifths of decliners cited ' siblings / family members not keen ' to be the reason . even among families with deleterious brca1/2 mutations , fewer than 2 family members per index patient have attended genetic counselling , and less than 1 family member per index patient has opted for predictive testing , highlighting the complexity of involving cancer - free family members in cancer predisposition testing . this low uptake in predictive testing has similarly been reported both in the west and in asia . it was also noteworthy that while over 80% of patients counselled were willing to share genetic information with spouses and siblings , only about 70% were willing to involve the older generation such as their parents . these behaviours may stem from traditional asian beliefs that cancer is a curse that is associated with a stigma and therefore shameful to discuss , causing some cancer patients to be unwilling to broach the subject with cancer - free family members . cancer is also viewed as a taboo in traditional chinese beliefs , and many may feel that discussing it freely in the family or testing for cancer predisposition constitutes bad luck [ 28 - 31 ] . indeed , one - third of patients described ' negative feelings ' after receiving genetic counselling , and about 30% of high - risk breast cancer patients who declined genetic testing were worried about ' negative feelings ' that the test results may incite . the low uptake of predictive testing is contrary to our survey finding of high interest in genetic testing among cancer - free family members who attended genetic counselling . one reason could be that family members needed more time to decide on predictive testing , since many index patients had only been confirmed to carry mutations in the last 2 - 3 years . another reason for this discrepancy is the possibility that cancer - free family members who attended genetic counselling represent a select and more health - conscious population , but who may ultimately not have access to genetic testing because the index patient opted not to be tested or was not found to carry a mutation . in addition , while many family members may express interest in the hypothetical situation , when faced with the real prospect of genetic testing , they may ultimately decline testing because of the fear of being labelled a gene carrier in a traditional society that views cancer as a stigma . we found encouragingly high cancer screening compliance rates among the high - risk breast and colorectal cancer patients in our programme , while the uptake rate for prophylactic surgery among brca1/2 mutation carriers is comparable to those reported in other centres , with 47% of carriers undergoing or contemplating prophylactic mastectomy and/or oophorectomy in our programme . singapore is a developed nation with a comprehensive public healthcare system , and we have successfully initiated a cancer genetics programme in the context of a tertiary hospital . by increasing awareness and level of knowledge among health providers in singapore , we hope to extend the programme to the community in the future . certain elements unique to singapore could enhance the success of our programme , including easy access to medical records and family members , affordable health screening services , and good doctor - patient relationships . at the same time , we have identified potential barriers that are actively being addressed . these include overcoming the cost issue of genetic testing through government assistance plans or health policy changes , continuing medical and public education to increase awareness and knowledge , and being culturally sensitive when dealing with the subject of cancer and cancer predisposition testing with the asian family . complex medical , social , ethical and legal aspects surround genetic testing , and we hope in the future to integrate other specialists , such as surgeons , psychiatrists , and social workers into a more comprehensive programme . we thank ms may - chin yong and ms robyn yip for assisting in genetic counselling and data collection at the cancer genetics clinic .

cancer genetics is now an established oncology subspecialty with the primary prevention role of identifying high - risk individuals through genetic information for enrolment into screening and preventive programmes . integrated into major western centres since the late 1990s , such a programme has been established in singapore since 2001 . our programme has evaluated 367 index patients comprising mainly breast and colorectal cancer cases . cancer patients were receptive to genetic counselling , but cost posed a major barrier to genetic testing . however , when the cost barrier was removed through government subsidy plans , more than half of high - risk patients still declined testing . the major barriers were reluctance to involve family members , perception that the information would not change management , and fears of negative feelings . confirmed mutation carriers were compliant to screening and receptive to prophylactic surgery . uptake of predictive testing among cancer - free family members has been low , possibly arising from the stigma associated with cancer in our asian culture . these potential barriers are being addressed through government subsidy plans , continuing education to increase awareness , and being culturally sensitive when dealing with the asian family .

Introduction Cancer Genetics Programme at the National University Hospital, Singapore Results Discussion Conclusion Acknowledgements

these include hereditary breast and ovarian cancer syndrome due to the brca1/2 genes , hereditary non - polyposis colorectal cancer syndrome due to the mismatch repair genes [ 4 - 6 ] , and familial adenomatous polyposis due to mutations in the apc gene . the recognition of these syndromes and the establishment of management guidelines have led to the development of cancer genetics as a subspecialty in oncology [ 8 - 13 ] . cancer genetics services have been incorporated into routine cancer services in major oncology centres in the west since the late 1990s . such services represent an important primary prevention arm of oncology in its role of identifying high - risk individuals through family history assessment or genetic testing , and to enrol them into early cancer detection and prevention programmes , with the ultimate goal of reducing cancer burden and mortality . cancer is one of the major causes of mortality , and about 7800 cancer cases are diagnosed every year , with the leading cancers being lung , colorectal and breast cancers . health care in singapore is provided through 9 public hospitals , 7 private hospitals , 16 government outpatient polyclinics , and some 1900 private medical clinics . of the 9 public hospitals , two are tertiary hospitals that offer comprehensive cancer services , encompassing surgical , medical , and radiation oncology specialties . cancer genetics services have been formally incorporated into the cancer services of both tertiary hospitals since 2001 . the chinese and indians are largely first- or second - generation migrants from china and southern india , while the malays are indigenous to the regions including malaya , sumatra , java , and the other islands of the indonesian archipelago . health cost is made affordable to the singaporean public through government subsidized medical services at the public hospitals and outpatient government polyclinics through a co - payment system , where citizens pay a percentage of the medical bill , with the remainder subsidized by the government . the cancer genetics programme at the national university hospital , singapore , is directed by a medical oncologist ( scl ) , who trained in cancer genetics at the john hopkins university school of medicine , usa , and was credentialed in ' familial cancer risk assessment and management ' by the institute for clinical evaluation in the usa . the national university hospital cancer service sees about 1500 new cancer cases every year , including approximately 600 new breast and colorectal cancers . these forms are screened by the genetics counsellor to identify high - risk patients , such as those with young onset cancer , familial cancer clustering , and multiple primary cancers , who would warrant genetics risk assessment . high - risk cases are flagged to the primary cancer physician for referral to the cancer genetics programme . during each new consultation , patients are classified as low , modest , moderate , or high risk of having a hereditary cancer syndrome , and are counselled accordingly . those assessed to have at least 10% chance of having a hereditary cancer syndrome are given genetic counselling for the particular syndrome , including the mode of inheritance , projected lifetime cancer risks , genetic testing including test interpretation and potential benefits and disadvantages of testing , and screening and preventive options . patients are given 1 - 2 weeks to assimilate and share the information with their family members . genetic testing that is offered through the programme includes brca1/2 comprehensive sequencing and single site mutation analysis for hereditary breast and ovarian cancer syndrome , mlh1/msh2 comprehensive sequencing and single site mutation analysis and tumour microsatellite instability testing for hereditary non - polyposis colorectal cancer , apc protein truncation test for familial adenomatous polyposis , and sequencing of exons 10 , 11 , 13 - 16 of the ret proto - oncogene for multiple endocrine neoplasias 2a and 2b . the latter three tests are offered by local laboratories , and cost s$260 - 370 ( us$153 - 218 ) per test . brca1/2 and mlh1/msh2 comprehensive sequencing and respective single site mutation analysis are performed at myriad genetics laboratories ( salt lake city , utah , usa ) at the cost of s$5,058 ( us$2,975 ) , s$3,315 ( us$1,950 ) , and s$595 ( us$350 ) respectively . costs for genetic testing are all out - of - pocket expenses as they are not subsidized by the singapore government nor payable using medisave . since the initiation of the programme , colorectal cancer patients fulfilling eligibility criteria may opt to be tested for germline mlh1/msh2 mutations free of charge as part of a research protocol . since september 2003 , patients assessed to have at least 30% chance of carrying a brca1/2 mutation are eligible to receive a 100% subsidy for brca1/2 sequencing , while their family members are eligible to a 50% subsidy for predictive testing through a special government subsidy programme . demographic information , cancer history , family history , genetic risk assessment information , screening recommendations , and genetic test results of patients evaluated in the cancer genetics programme are collected and stored in a user - defined and password - protected database . as cancer genetics is a new field in singapore , we conducted a questionnaire survey in 2002 , shortly after we started the programme , to assess the level of knowledge on breast cancer risk factors and hereditary breast cancer syndrome . among 284 health professionals and 221 medical students surveyed , less than a quarter recognized that paternal family history of cancer is as important as maternal family history in evaluating for hereditary breast cancer syndrome , and less than half were aware that genetic testing for hereditary breast cancer is clinically available , or that prophylactic mastectomy is a preventive option for women at high risk for breast cancer ( table 1 ) . this general lack of awareness on emerging diagnostic and preventive options for hereditary breast cancer syndrome was identified as an important potential barrier to optimal utilization of the cancer genetics service , and active steps were taken to promote awareness through continuing medical education . awareness of breast cancer risk factors and genetics among health professionals and medical students ( n = 505 ) respondents were given the options ' true ' , ' false ' , or ' do n't know ' ; respondents were given the options ' < 1% ' , ' 5 - 10% ' , ' 20 - 30% ' , ' 50 - 60% ' , or ' do n't know ' we next conducted a questionnaire survey to evaluate the acceptance and potential motivators and barriers of breast cancer genetic counselling among breast cancer patients and cancer - free women . about 70% of the 313 respondents indicated interest in attending genetic counselling when medically indicated and perceived the potential benefits . important motivators were learning about cancer risk and cancer detection , helping the family , and the doctor 's recommendation . important barriers were the misperception that cancer patients could not gain personally , cost issues , fears of bad news , and concerns of inability to make use of the information ( table 2 ) . acceptance , potential motivators and barriers of breast cancer genetic counselling among breast cancer patients and cancer - free women ( n = 313 ) responses of breast cancer patients only from january 2001 to march 2006 , 367 new patients were evaluated at the cancer genetics clinic . patients with suspected hereditary breast cancer ( 60% ) and colorectal cancer syndromes ( 31% ) formed the majority of cases ( table 3 ) . 87% of patients were assessed to have at least 10% chance of having a hereditary cancer syndrome and therefore offered genetic counselling and testing . of these , 40% underwent genetic testing , and 23% of those tested were found to carry deleterious germline mutations ( table 4 ) . characteristics of patients reviewed in the cancer genetics clinic ( n = 367 ) include 10 patients counselled for predictive testing for a familial mutation ; include 1 patient counselled for predictive testing for a familial mutation ; 18 patients were deemed to have at least 10% chance of having a known hereditary cancer syndrome and offered genetic counselling and testing ; familial clustering of or young nasopharyngeal cancers ( 9% ) , familial clustering of renal cancers ( 9% ) , suspected li fraumeni syndrome ( 6% ) , familial clustering of paragangliomas suspicious of sdhd mutations ( 3% ) , multiple endocrine neoplasia iia ( 3% ) , suspected neurofibromatosis ( 3% ) , turner 's syndrome with young endometrial cancer ( 3% ) genetic testing in cancer genetics programme , national university hospital , singapore others : comprehensive sequencing and rearrangement analysis for von hippel lindau ( 2 ) , karyotyping for turner 's syndrome ( 1 ) , sequencing for e - cadherin gene mutation ( 1 ) , sequencing for sdhd gene mutation ( 1 ) ; others : turner 's syndrome ( 1 ) , sdhd deleterious mutation ( 1 ) from july 2003 , cancer patients and their accompanying family members were surveyed after the genetic counselling session ( table 5 ) . the age distribution of the 110 cancer patients ( median 37 , range 23 - 77 ) and 95 cancer - free family members ( median 39 , range 16 - 65 ) surveyed were similar . cancer - free family members who attended genetic counselling were more educated and more likely to express interest in genetic testing than cancer patients , with 81% indicating that they would definitely or probably take up genetic testing if medically indicated compared to 61% of cancer patients . for both groups , the most common motivators for genetic testing were to help their children and family members . concerns about the cost of testing and belief that the information could not prevent another cancer were the most common reasons cited for not undertaking genetic testing . more than 80% of respondents indicated willingness to share genetic information with siblings and spouses , but only about 70% were willing to share the information with their parents . cancer patients were more likely to experience negative feelings after the session compared to cancer - free family members . survey on genetic testing ( n = 205 ) statistical analysis was performed using chi - square test for categorical variables , and student 's t - test for age ; respondents were allowed to cite more than one reason ; respondents may describe more than one kind of feelings ; respondents may describe more than one kind of negative feelings ; negative feelings : anxious ( 19% ) , burdensome ( 8% ) , upset ( 8% ) , confused ( 7% ) , distracted ( 4% ) , afraid ( 4% ) , numb ( 3% ) , shocked ( 3% ) , angry ( 2% ) , stressed ( 2% ) , depressed ( 1% ) ; negative feelings : anxious ( 13% ) , afraid ( 5% ) , confused ( 4% ) , upset ( 4% ) , stressed ( 4% ) , burdensome ( 3% ) , depressed ( 3% ) , distracted ( 3% ) , numb ( 1% ) a total of 182 index patients were evaluated to have at least 10% chance of carrying a brca1/2 mutation and offered genetic testing . 48% were young onset breast cancer patients without significant family history , 38% were from breast cancer families , 12% were from breast - ovarian cancer families , and 2% were patients with male or bilateral breast cancers . 28% of young breast cancer patients and 17% of patients from breast cancer families underwent testing , while 62% of index patients from breast - ovarian cancer families were tested . of the 17 index patients found to carry brca1/2 mutations , five had bilateral mastectomy for breast cancers , and three had bilateral oophorectomy for ovarian cancer . compliance to breast cancer screening with mammography among the high - risk breast cancer patients evaluated at the cancer genetics programme is 84% , while that of brca1/2 mutation carriers is 94% . nature of brca1/2 deleterious mutations and factors associated with deleterious brca1/2 mutation ( n = 17 ) twenty cancer - free family members were counselled for brca1/2 predictive testing . 67% of cancer - free family members who attended counselling for predictive testing were married , and 50% had more than 10 years of formal education . sixty - six high - risk colorectal cancer patients were tested for mlh1/msh2 mutations , predominantly as part of a research protocol free of charge . fourteen family members had undergone predictive testing , and 6 were found to carry deleterious mutations and are on surveillance . factors that were associated with deleterious germline mutations include family history fulfilling amsterdam i / ii criteria ( 60% ) , proband with early onset colorectal cancer and family history of colorectal cancer or extracolonic cancers ( 46% ) , proband with colorectal cancer and family history of stomach cancer ( 40% ) , and proband with colorectal cancer demonstrating high microsatellite instability ( 36% ) . compliance to screening colonoscopy among mutation carriers is 77% . among the potential barriers uncovered through our previous hypothetical questionnaire survey prior to the initiation of the government subsidy programme for brca1/2 testing in 2003 , 67/70 ( 96% ) breast cancer patients offered genetic testing declined the test , with the majority citing cost as a major barrier . after the initiation of the subsidy programme , 47/106 ( 44% ) eligible patients underwent brca1/2 testing at 100% government subsidy . however , 59/106 patients ( 56% ) still declined genetic testing despite removal of the cost barrier . a telephone survey of 39/59 ( 66% ) patients who declined testing revealed the following major reasons : siblings / family members were not keen to know of such information or to be tested ( 41% ) , perception that testing would not prevent cancer recurrence or alter medical management ( 39% ) , and concerns about negative feelings associated with genetic test results ( 31% ) ( table 7 ) . demographic characteristics of index patients who declined brca1/2 testing despite test cost subsidies and reasons for declining ( n = 39 ) patients were allowed to indicate more than one reason for declining test ; cost of single site mutation analysis in predictive testing is s$595 ( us$350 ) and the singapore government provides 50% reimbursement cancer is a leading cause of morbidity and mortality in singapore , and cancer genetics and risk assessment programmes represent the primary prevention arm of oncology . this has in part been attributed to our standard procedure to obtain family cancer history from each new cancer patient to identify high - risk patients for referral into the programme . in addition , the success of continuing medical education to increase awareness among physicians has been reflected by increasing referrals from outside the institution , now accounting for about 30% of our new cases . in contrast to developed nations in the west , where lay press and lay media routinely report new medical advances and provide patients and community physicians easy access to new medical knowledge , health providers in singapore generally rely on medical journals and seminars for information on medical advances , while patients rely on their health providers for pertinent medical information . consequently it comes as no surprise to find only a handful of health providers in singapore who are aware of brca1/2 genetic testing or prophylactic surgery for high - risk individuals . such information has been critical for us to focus continuing medical education efforts on filling important knowledge gaps . cost has been cited as an important barrier to genetic counselling and testing in many prior studies , including our own . while genetic counselling is available to singaporeans at a subsidized and affordable rate , genetic testing is not . in fact , although a significant proportion of index patients expressed interest in brca1/2 testing , the uptake rate was a dismal 4% prior to 2003 when the cost of testing was not subsidized . this posed a significant barrier to downstream work of risk segregating individuals and tailoring screening and preventive recommendations using genetic testing . under the special government subsidy programme that provided free brca1/2 testing for index patients , we observed an eleven - fold increase in genetic testing uptake rate to 44% , allowing mutation carriers to be identified and facilitating predictive testing in cancer - free family members . this highlights the importance of overcoming cost as a barrier to allow the full realization of the potential of a cancer genetics programme . despite removing the cost barrier , we found more than half of eligible patients to still decline genetic testing , suggesting that other barriers exist . the central role of the family in asian culture is underscored by the fact that more than 80% of respondents in a hypothetical situation , and more than 50% of high - risk patients who underwent genetic counselling in our population , cited ' helping the family ' to be an important motivator to attend genetic counselling and undertake genetic testing respectively . yet , when it comes to actual genetic testing and the real possibility of involving family members for predictive testing , two - fifths of decliners cited ' siblings / family members not keen ' to be the reason . even among families with deleterious brca1/2 mutations , fewer than 2 family members per index patient have attended genetic counselling , and less than 1 family member per index patient has opted for predictive testing , highlighting the complexity of involving cancer - free family members in cancer predisposition testing . this low uptake in predictive testing has similarly been reported both in the west and in asia . it was also noteworthy that while over 80% of patients counselled were willing to share genetic information with spouses and siblings , only about 70% were willing to involve the older generation such as their parents . these behaviours may stem from traditional asian beliefs that cancer is a curse that is associated with a stigma and therefore shameful to discuss , causing some cancer patients to be unwilling to broach the subject with cancer - free family members . indeed , one - third of patients described ' negative feelings ' after receiving genetic counselling , and about 30% of high - risk breast cancer patients who declined genetic testing were worried about ' negative feelings ' that the test results may incite . the low uptake of predictive testing is contrary to our survey finding of high interest in genetic testing among cancer - free family members who attended genetic counselling . one reason could be that family members needed more time to decide on predictive testing , since many index patients had only been confirmed to carry mutations in the last 2 - 3 years . another reason for this discrepancy is the possibility that cancer - free family members who attended genetic counselling represent a select and more health - conscious population , but who may ultimately not have access to genetic testing because the index patient opted not to be tested or was not found to carry a mutation . in addition , while many family members may express interest in the hypothetical situation , when faced with the real prospect of genetic testing , they may ultimately decline testing because of the fear of being labelled a gene carrier in a traditional society that views cancer as a stigma . we found encouragingly high cancer screening compliance rates among the high - risk breast and colorectal cancer patients in our programme , while the uptake rate for prophylactic surgery among brca1/2 mutation carriers is comparable to those reported in other centres , with 47% of carriers undergoing or contemplating prophylactic mastectomy and/or oophorectomy in our programme . singapore is a developed nation with a comprehensive public healthcare system , and we have successfully initiated a cancer genetics programme in the context of a tertiary hospital . certain elements unique to singapore could enhance the success of our programme , including easy access to medical records and family members , affordable health screening services , and good doctor - patient relationships . at the same time , we have identified potential barriers that are actively being addressed . these include overcoming the cost issue of genetic testing through government assistance plans or health policy changes , continuing medical and public education to increase awareness and knowledge , and being culturally sensitive when dealing with the subject of cancer and cancer predisposition testing with the asian family . complex medical , social , ethical and legal aspects surround genetic testing , and we hope in the future to integrate other specialists , such as surgeons , psychiatrists , and social workers into a more comprehensive programme . we thank ms may - chin yong and ms robyn yip for assisting in genetic counselling and data collection at the cancer genetics clinic .

comprehension difficulty features different levels of misconception : ability to recall information and awareness of comprehension deficits , ie , ability to identify the misconceived information . higher prevalence of multiple illnesses and transient or chronic cognitive impairment among older patients pose a risk of comprehension difficulties , which may lead to medication errors and unplanned readmissions.14 knowledge is lacking on older patients potential comprehension difficulties in a quick diagnostic unit ( qdu ) . inadequate health literacy level influences patient comprehension and is more prevalent among older patients , likely due to age - related cognitive decline.5,6 at the same time , increasing age and patient comorbidities have been associated with unplanned hospital readmissions.3,7 insufficient communication and lack of patient education by hospital personnel have been identified as causes of posthospital medication errors , likewise resulting in unplanned hospital readmissions.1,2 although studies have shown that older patients have impaired ability to recall discharge information compared to younger patients,8 results on awareness of these comprehension deficits are limited . however , in general emergency departments ( eds ) it has been shown that patients had compromised awareness of comprehension deficits when evaluating their discharge information.9,10 a qdu is a relatively new type of ward integrated with the ed . patients admitted to the qdu have surgical and internal medicine conditions with an expected hospitalization of less than 2 days . patient flow and need of quick diagnostic procedures in this setting may challenge older patients comprehension . therefore , the objective of the present study was to investigate patient comprehension of discharge information among older patients and their awareness of their comprehension deficits in a qdu setting . we conducted a cross - sectional questionnaire study inviting at least 100 patients discharged from the qdu at holbk university hospital in a 2-month period , june and july 2012 . exclusion criteria were age below 18 years , patients being discharged from the qdu to another facility or unit , inability to speak or hear , need of a translator , not awake - aware - oriented , glasgow coma scale below 15 , and known dementia . the population was divided into two groups , an older group ( age 65 years ) and a younger group ( age < 65 years ) . the medical staff were informed about the objective and study design including the questionnaire before the beginning of the study . however , patients were not informed about the study until after the discharge interview was completed . patients did not know the specific contents of the questionnaire but were informed about the overall aim of the study . they were not able to consult their discharge paper or medication list when answering the questionnaire . the discharge interview was conducted in the qdu , typically in a room shared by two patients . answered questionnaires the questionnaire covered basic characteristics , self - assessed comprehension of discharge information , ability to recall discharge information , subjective evaluation of the communication , and patient satisfaction with the admission ( scale 110 with maximum score 10 ) . questions addressed admission diagnosis , diagnostic procedures , treatment , follow - up instructions , and when to seek emergency care . binary yes / no answers were used to evaluate self - assessed comprehension , eg , did you understand what was wrong with you / your diagnosis ? the same questions reformulated to obtain descriptive answers were compared to the discharge document in order to evaluate correct recall , eg , write in your own words what was wrong with you / your diagnosis . finally , the two answers were compared to evaluate the patients awareness of comprehension deficits , eg , if a patient answered yes to have understood the admission diagnosis but did not recall the correct answer , then the patient was not aware of his comprehension deficit . in order to validate the questionnaire , authors reviewed answers from the initial ten questionnaires , and since patient answers were as anticipated , the wording of the questions was not changed . however , all uncertainties were discussed and evaluated by the entire group of authors . to minimize bias , objective criteria in scoring the patient responses were established before evaluation . patients were given credit for correct recall if they stated the main diagnosis causing their admission in appropriate lay terms , the main diagnostic tests essential for verifying the diagnosis ( eg , ultrasound examination for deep venous thrombosis ) , the newly prescribed medication name or group ( eg , antibiotics ) , and how to take the medication ( eg , once daily or twice daily ) . if these were specified incorrectly , the answer was considered wrong . particularly in regard to return instructions concerning when to seek emergency care , a relevant answer ( eg , shortness of breath after pneumonia ) was accepted , even though not documented in the discharge document . statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc , usa ) . statistical significance was set as a p - value below 0.05 on two - sided tests . categorical differences between the younger and older group were analyzed with chi - squared or fisher s exact test if an expected frequency was less than five in any cell of the contingency table . differences in ordinal categorical variables between groups were analyzed with cochran armitage trend tests , whereas differences in continuous variables were calculated with wilcoxon rank sum tests and reported as median ( interquartile range [ iqr ] ) due to skewed distributions in the younger and older group . in multiple logistic regression analyses , group differences ( older versus younger group ) in recall and awareness of comprehension deficits were adjusted for sex and education . only analyses with a significant likelihood ratio test and no interaction of group with either sex or education likewise , the associations between age ( continuous variable ) and recall as well as awareness of comprehension deficits were investigated when adjusting for sex and education . models were checked for linearity and potential interaction between age and both sex and education . only models with a significant likelihood ratio test , no interaction , and linearity were reported . we conducted a cross - sectional questionnaire study inviting at least 100 patients discharged from the qdu at holbk university hospital in a 2-month period , june and july 2012 . exclusion criteria were age below 18 years , patients being discharged from the qdu to another facility or unit , inability to speak or hear , need of a translator , not awake - aware - oriented , glasgow coma scale below 15 , and known dementia . the population was divided into two groups , an older group ( age 65 years ) and a younger group ( age < 65 years ) . the medical staff were informed about the objective and study design including the questionnaire before the beginning of the study . however , patients were not informed about the study until after the discharge interview was completed . patients did not know the specific contents of the questionnaire but were informed about the overall aim of the study . they were not able to consult their discharge paper or medication list when answering the questionnaire . the discharge interview was conducted in the qdu , typically in a room shared by two patients . answered questionnaires the questionnaire covered basic characteristics , self - assessed comprehension of discharge information , ability to recall discharge information , subjective evaluation of the communication , and patient satisfaction with the admission ( scale 110 with maximum score 10 ) . questions addressed admission diagnosis , diagnostic procedures , treatment , follow - up instructions , and when to seek emergency care . binary yes / no answers were used to evaluate self - assessed comprehension , eg , did you understand what was wrong with you / your diagnosis ? the same questions reformulated to obtain descriptive answers were compared to the discharge document in order to evaluate correct recall , eg , write in your own words what was wrong with you / your diagnosis . finally , the two answers were compared to evaluate the patients awareness of comprehension deficits , eg , if a patient answered yes to have understood the admission diagnosis but did not recall the correct answer , then the patient was not aware of his comprehension deficit . in order to validate the questionnaire , authors reviewed answers from the initial ten questionnaires , and since patient answers were as anticipated , the wording of the questions was not changed . however , all uncertainties were discussed and evaluated by the entire group of authors . to minimize bias , objective criteria in scoring the patient responses were established before evaluation . patients were given credit for correct recall if they stated the main diagnosis causing their admission in appropriate lay terms , the main diagnostic tests essential for verifying the diagnosis ( eg , ultrasound examination for deep venous thrombosis ) , the newly prescribed medication name or group ( eg , antibiotics ) , and how to take the medication ( eg , once daily or twice daily ) . if these were specified incorrectly , the answer was considered wrong . particularly in regard to return instructions concerning when to seek emergency care , a relevant answer ( eg , shortness of breath after pneumonia ) was accepted , even though not documented in the discharge document . statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc , usa ) . statistical significance was set as a p - value below 0.05 on two - sided tests . categorical differences between the younger and older group were analyzed with chi - squared or fisher s exact test if an expected frequency was less than five in any cell of the contingency table . differences in ordinal categorical variables between groups were analyzed with cochran armitage trend tests , whereas differences in continuous variables were calculated with wilcoxon rank sum tests and reported as median ( interquartile range [ iqr ] ) due to skewed distributions in the younger and older group . in multiple logistic regression analyses , group differences ( older versus younger group ) in recall and awareness of comprehension deficits were adjusted for sex and education . only analyses with a significant likelihood ratio test and no interaction of group with either sex or education were reported . likewise , the associations between age ( continuous variable ) and recall as well as awareness of comprehension deficits were investigated when adjusting for sex and education . models were checked for linearity and potential interaction between age and both sex and education . only models with a significant likelihood ratio test , forty patients were allocated to the older group and 62 to the younger group ( age : 72.0 [ iqr : 6979 ] years versus 48.5 [ iqr : 4258 ] years ; p<0.0001 ) . the two groups were comparable in regard to sex ( male : n=18 , 45.0% versus n=32 , 51.6% ; p=0.51 ) , highest level of education : primary school , high school , university ( p=0.06 ) , other diseases ( older group : 56.3% versus younger group : 58.2% ; p=0.86 ) , length of admission ( 1.0 [ 03 ] days versus 1.0 [ 02 ] days ; p=0.97 ) , and patient satisfaction ( 10.0 [ 910 ] versus 9.5 [ 810 ] ; p=0.13 ) . more prior admissions , however , were found in the older group ( p=0.027 ) . admission diagnosis in the older group was mainly infectious illness ( 22.5% ) , musculoskeletal illness ( 22.5% ) , anemia ( 20.0% ) , neurological illness ( 10.0% ) , and cardiovascular illness ( 7.5% ) . in the younger group , infectious illness ( 21.5% ) , musculoskeletal illness ( 18.5% ) , neurological illness ( 12.3% ) , back illness ( 12.3% ) , and cardiovascular illness ( 9.2% ) there was no difference in self - assessed comprehension of discharge information between the older and younger group . most patients answered that they fully understood the information ( table 1 ) and that the medical staff had used an intelligible language ( nolder=36 , 97.3% versus nyounger=56 , 94.9% ; p=1.0 ) . only a few patients thought that their current illness affected their usual understanding ( nolder=3 , 9.3% versus nyounger=7 , 12.1% ; p=1.0 ) . the older group had more difficulties recalling correct medication instructions ( correct recall : 54.3% versus 78.0% ; p=0.02 ) and diagnostic procedures ( 71.8% versus 91.9% ; p=0.007 ) compared to the younger group , respectively ( table 2 ) . the estimated odds for recalling correct medication instructions were 4.2 times higher for the younger group compared to the older group ( odds ratio [ or ] 4.2 , 95% confidence interval [ ci ] 1.511.9 , p=0.007 ) when adjusted for sex and education . furthermore , for each additional year of age the estimated odds for recalling correct medication instructions decreased 6.1% ( or 0.939 , 95% ci 0.900.98 , p=0.001 ) independent of sex and education . there were no differences between the two groups in recall of the remaining questions , yet several patients in both groups were not able to recall correct information ( table 2 ) . the older group was less aware of their comprehension deficits in four out of seven questions when compared to the younger group ; awareness of comprehension deficits with regard to diagnostic tests ( older group : 71.1% versus younger group : 91.9% ; p=0.006 ) , preventive measures ( 53.3% versus 78.6% ; p=0.02 ) , medication instructions ( 51.4% versus 73.3% ; p=0.03 ) , and when to seek emergency care ( 60.6% versus 80.8% ; p=0.04 ) ( figure 1 ) . the remaining questions showed the same tendency , though not significant : admission diagnosis ( 76.9% versus 88.5% ; p=0.12 ) , treatment ( 60.5% versus 77.4% ; p=0.07 ) , and follow - up at general practitioner / specialist ( 54.1% versus 67.7% ; p=0.17 ) . awareness of comprehension deficits in diagnostic tests was 6.8 times higher among the younger group compared to the older group when adjusted for sex and education ( or 6.8 , 95% ci 1.629.2 , p=0.01 ) . awareness of comprehension deficits in medication instructions and diagnostic tests decreased 6.1% ( or 0.939 , 95% ci 0.9040.98 , p=0.001 ) and 5.8% ( or 0.94 , 95% ci 0.900.99 , p=0.02 ) per 1-year increase in age , respectively , when adjusted for sex and education . the remaining multiple logistic regression analyses investigating recall and awareness of comprehension deficits were insignificant . there was no difference in self - assessed comprehension of discharge information between the older and younger group . most patients answered that they fully understood the information ( table 1 ) and that the medical staff had used an intelligible language ( nolder=36 , 97.3% versus nyounger=56 , 94.9% ; p=1.0 ) . only a few patients thought that their current illness affected their usual understanding ( nolder=3 , 9.3% versus nyounger=7 , 12.1% ; p=1.0 ) . the older group had more difficulties recalling correct medication instructions ( correct recall : 54.3% versus 78.0% ; p=0.02 ) and diagnostic procedures ( 71.8% versus 91.9% ; p=0.007 ) compared to the younger group , respectively ( table 2 ) . the estimated odds for recalling correct medication instructions were 4.2 times higher for the younger group compared to the older group ( odds ratio [ or ] 4.2 , 95% confidence interval [ ci ] 1.511.9 , p=0.007 ) when adjusted for sex and education . furthermore , for each additional year of age the estimated odds for recalling correct medication instructions decreased 6.1% ( or 0.939 , 95% ci 0.900.98 , p=0.001 ) independent of sex and education . there were no differences between the two groups in recall of the remaining questions , yet several patients in both groups were not able to recall correct information ( table 2 ) . the older group was less aware of their comprehension deficits in four out of seven questions when compared to the younger group ; awareness of comprehension deficits with regard to diagnostic tests ( older group : 71.1% versus younger group : 91.9% ; p=0.006 ) , preventive measures ( 53.3% versus 78.6% ; p=0.02 ) , medication instructions ( 51.4% versus 73.3% ; p=0.03 ) , and when to seek emergency care ( 60.6% versus 80.8% ; p=0.04 ) ( figure 1 ) . the remaining questions showed the same tendency , though not significant : admission diagnosis ( 76.9% versus 88.5% ; p=0.12 ) , treatment ( 60.5% versus 77.4% ; p=0.07 ) , and follow - up at general practitioner / specialist ( 54.1% versus 67.7% ; p=0.17 ) . awareness of comprehension deficits in diagnostic tests was 6.8 times higher among the younger group compared to the older group when adjusted for sex and education ( or 6.8 , 95% ci 1.629.2 , p=0.01 ) . awareness of comprehension deficits in medication instructions and diagnostic tests decreased 6.1% ( or 0.939 , 95% ci 0.9040.98 , p=0.001 ) and 5.8% ( or 0.94 , 95% ci 0.900.99 , p=0.02 ) per 1-year increase in age , respectively , when adjusted for sex and education . the remaining multiple logistic regression analyses investigating recall and awareness of comprehension deficits were insignificant . the main finding of the present study was that older patients were less aware of their comprehension deficits when compared to the younger patients . the older patients were unable to identify the misconceived information in four out of seven questions : medication instructions , diagnostic tests , preventive measures , and when to seek emergency care . at the same time , the older patients were less able to recall correct medication instructions and diagnostic tests . in an internal medicine department and an ed it has been found that patients had difficulties recalling newly prescribed medication.8,10 contrary to the present study , patients were not age divided , and these studies were conducted in different settings . in a qdu setting , we found similar recall difficulties , however , most frequently in the older group . this might lead to noncompliance and hereby relapse of illnesses and complications , as potential medication errors have been shown to increase the number of unplanned readmissions.14 especially older patients are at risk of medication discrepancies and unplanned readmissions.24 despite the comprehension difficulties found in the present study , no qdu readmissions were registered during the 2 months of data collection . however , the limited sample size , along with the relatively short follow - up , limits strong conclusions regarding readmissions . furthermore , readmission within a short period after discharge indicates more severe illness that could require admission to a more specialized department . knowledge is lacking on older patients awareness of comprehension deficits , ie , ability to identify the misconceived information . in the present study , we found significant differences between the older and younger patients in four out of eight questions : awareness of comprehension deficits with regard to diagnostic tests , preventive measures , medication instructions , and when to seek emergency care ( figure 1 ) . at the same time , the tendency was that the older group had a lower awareness of comprehension in the remaining questions . a study from an ed found that patients could identify only 20% of their comprehension difficulties.9 these results are difficult to compare to those of the present study since the study design differed considerably . firstly , only one sample population was described , and no subgroup analysis was conducted . secondly , the questions were broader , covering major domains such as ed care and post - ed care . thirdly , analyses were conducted for all questions pooled together and not for each question separately , as in the present study . the recall difficulties along with the unawareness of comprehension deficits found in the present study emphasize the importance of communicative strategies to confirm patient comprehension before discharge . we did not investigate potential communication failures ; however , in a study from an ed11 it was identified that important health information was often missed by the physicians , that patients were rarely asked whether they had questions about the information given , and patient comprehension was never confirmed . patients are limited in regard to how much they can process and recall during hospitalization,12,13 and many older patients experience a temporary , although recoverable , cognitive dysfunction at discharge.14 improvements in this hospitalized related cognitive dysfunction have been found 2 to 4 weeks after discharge,13,14 whereas one study found further improvements after 1 year.12 in the present study , only 9% of the older patients thought their illness had affected their comprehension abilities , even though they showed severe difficulties recalling correct discharge information . in this respect , one quarter of the older patients did not recall their potential follow - up at a general practitioner or specialist . at the same time , they were highly unaware of these comprehension deficits . in this respect , we can not simply rely on the patients to ask the questions needed for clarification or to contact the health care provider in order to secure a sufficient information level . optimizing patient comprehension could involve closed loops communication in which the patient repeats the given information . further , the setting of the discharge interview could be optimized using a single patient room , inviting relatives to join the discharge interview , and implementation of follow - up strategies such as home visits or follow - up calls the day after discharge . firstly , the medical staff were not blinded to the questionnaire , potentially improving the performance of the staff and thereby patient responses . however , patients were approached before leaving the department to avoid recall bias , and it is likely that they may have done better if they had the medication to look at , even though prescription label instructions can be challenging.15 secondly , we did not collect data on mini - mental state examination scores or hearing status at time of admission . potentially , patients with unknown cognitive and perception impairments could have been included , and this might partly explain the differences in comprehension between the older and younger group . fourthly , we did not collect data from other health care levels ( eg , primary care ) after discharge , and due to the cross - sectional design , we are not aware of the clinical consequence of the present results . no readmissions to the qdu were registered during the data - collecting period , but further research is needed to explore this matter . in a demographic perspective , the older population and number of patients with multiple illnesses are growing.16,17 health care resources are limited , stressing cost - effectiveness and decreasing the length of hospital admissions.18 as demonstrated in the present study , older patients represent a vulnerable group in the transition between health care levels . these involve home visits and telephone follow - up , aimed to encourage patients to assert a more active role during care transitions . although there are immediate costs , from an economic prospective , such interventions have shown promising results in lowering the rates of readmission.7,19,20 older patients were less able to recall correct medication instructions and diagnostic tests when compared to younger patients . furthermore , the older patients were less aware of their comprehension deficits with respect to medication instructions , diagnostic tests , preventive measures , and when to seek emergency care . in our perspective , the findings of the present study suggest that communication with the expanding population of older patients requires particular attention .

backgroundhigher prevalence of multiple illnesses and cognitive impairment among older patients pose a risk of comprehension difficulties , potentially leading to medication errors . therefore , the objective of this study was to investigate comprehension of discharge instructions among older patients admitted to a quick diagnostic unit ( qdu).methodsone hundred and two patients discharged from the qdu answered a questionnaire covering understanding of their hospitalization and discharge plan . patients ability to recall discharge instructions and awareness of comprehension deficits , ie , ability to identify the misconceived information , were evaluated by comparing the questionnaires with the discharge letters . the population was divided into an older group ( age 65 years ) and a younger group.resultsthe older group ( n=40 ) was less able to recall correct medication instructions when compared to the younger group ( 54% versus 78% , respectively ; p=0.02 ) . in multiple logistic regression analysis , correct recall of medication instructions was 4.2 times higher for the younger group compared to the older group ( odds ratio 4.2 , 95% confidence interval 1.511.9 , p=0.007 ) when adjusted for sex and education . the older patients were less aware of their own comprehension deficits , and in respect to medication instructions awareness decreased 6.1% for each additional year of age ( odds ratio 0.939 , 95% confidence interval 0.9040.98 , p=0.001 ) when adjusted for sex and education.conclusionolder patients were less able to recall correct medication instructions and less aware of their comprehension deficits after discharge from a qdu . the findings of the present study emphasize the importance of thorough communication and follow - up when treating older patients .

Introduction Materials and methods The survey The questionnaire Analysis Statistics Results Self-assessed comprehension Recall of discharge information Awareness of comprehension deficits Discussion Conclusion

comprehension difficulty features different levels of misconception : ability to recall information and awareness of comprehension deficits , ie , ability to identify the misconceived information . higher prevalence of multiple illnesses and transient or chronic cognitive impairment among older patients pose a risk of comprehension difficulties , which may lead to medication errors and unplanned readmissions.14 knowledge is lacking on older patients potential comprehension difficulties in a quick diagnostic unit ( qdu ) . inadequate health literacy level influences patient comprehension and is more prevalent among older patients , likely due to age - related cognitive decline.5,6 at the same time , increasing age and patient comorbidities have been associated with unplanned hospital readmissions.3,7 insufficient communication and lack of patient education by hospital personnel have been identified as causes of posthospital medication errors , likewise resulting in unplanned hospital readmissions.1,2 although studies have shown that older patients have impaired ability to recall discharge information compared to younger patients,8 results on awareness of these comprehension deficits are limited . however , in general emergency departments ( eds ) it has been shown that patients had compromised awareness of comprehension deficits when evaluating their discharge information.9,10 a qdu is a relatively new type of ward integrated with the ed . patients admitted to the qdu have surgical and internal medicine conditions with an expected hospitalization of less than 2 days . patient flow and need of quick diagnostic procedures in this setting may challenge older patients comprehension . therefore , the objective of the present study was to investigate patient comprehension of discharge information among older patients and their awareness of their comprehension deficits in a qdu setting . we conducted a cross - sectional questionnaire study inviting at least 100 patients discharged from the qdu at holbk university hospital in a 2-month period , june and july 2012 . exclusion criteria were age below 18 years , patients being discharged from the qdu to another facility or unit , inability to speak or hear , need of a translator , not awake - aware - oriented , glasgow coma scale below 15 , and known dementia . the population was divided into two groups , an older group ( age 65 years ) and a younger group ( age < 65 years ) . the discharge interview was conducted in the qdu , typically in a room shared by two patients . answered questionnaires the questionnaire covered basic characteristics , self - assessed comprehension of discharge information , ability to recall discharge information , subjective evaluation of the communication , and patient satisfaction with the admission ( scale 110 with maximum score 10 ) . questions addressed admission diagnosis , diagnostic procedures , treatment , follow - up instructions , and when to seek emergency care . the same questions reformulated to obtain descriptive answers were compared to the discharge document in order to evaluate correct recall , eg , write in your own words what was wrong with you / your diagnosis . finally , the two answers were compared to evaluate the patients awareness of comprehension deficits , eg , if a patient answered yes to have understood the admission diagnosis but did not recall the correct answer , then the patient was not aware of his comprehension deficit . in order to validate the questionnaire , authors reviewed answers from the initial ten questionnaires , and since patient answers were as anticipated , the wording of the questions was not changed . patients were given credit for correct recall if they stated the main diagnosis causing their admission in appropriate lay terms , the main diagnostic tests essential for verifying the diagnosis ( eg , ultrasound examination for deep venous thrombosis ) , the newly prescribed medication name or group ( eg , antibiotics ) , and how to take the medication ( eg , once daily or twice daily ) . particularly in regard to return instructions concerning when to seek emergency care , a relevant answer ( eg , shortness of breath after pneumonia ) was accepted , even though not documented in the discharge document . categorical differences between the younger and older group were analyzed with chi - squared or fisher s exact test if an expected frequency was less than five in any cell of the contingency table . differences in ordinal categorical variables between groups were analyzed with cochran armitage trend tests , whereas differences in continuous variables were calculated with wilcoxon rank sum tests and reported as median ( interquartile range [ iqr ] ) due to skewed distributions in the younger and older group . in multiple logistic regression analyses , group differences ( older versus younger group ) in recall and awareness of comprehension deficits were adjusted for sex and education . only analyses with a significant likelihood ratio test and no interaction of group with either sex or education likewise , the associations between age ( continuous variable ) and recall as well as awareness of comprehension deficits were investigated when adjusting for sex and education . models were checked for linearity and potential interaction between age and both sex and education . we conducted a cross - sectional questionnaire study inviting at least 100 patients discharged from the qdu at holbk university hospital in a 2-month period , june and july 2012 . exclusion criteria were age below 18 years , patients being discharged from the qdu to another facility or unit , inability to speak or hear , need of a translator , not awake - aware - oriented , glasgow coma scale below 15 , and known dementia . the population was divided into two groups , an older group ( age 65 years ) and a younger group ( age < 65 years ) . however , patients were not informed about the study until after the discharge interview was completed . the discharge interview was conducted in the qdu , typically in a room shared by two patients . answered questionnaires the questionnaire covered basic characteristics , self - assessed comprehension of discharge information , ability to recall discharge information , subjective evaluation of the communication , and patient satisfaction with the admission ( scale 110 with maximum score 10 ) . questions addressed admission diagnosis , diagnostic procedures , treatment , follow - up instructions , and when to seek emergency care . the same questions reformulated to obtain descriptive answers were compared to the discharge document in order to evaluate correct recall , eg , write in your own words what was wrong with you / your diagnosis . finally , the two answers were compared to evaluate the patients awareness of comprehension deficits , eg , if a patient answered yes to have understood the admission diagnosis but did not recall the correct answer , then the patient was not aware of his comprehension deficit . in order to validate the questionnaire , authors reviewed answers from the initial ten questionnaires , and since patient answers were as anticipated , the wording of the questions was not changed . patients were given credit for correct recall if they stated the main diagnosis causing their admission in appropriate lay terms , the main diagnostic tests essential for verifying the diagnosis ( eg , ultrasound examination for deep venous thrombosis ) , the newly prescribed medication name or group ( eg , antibiotics ) , and how to take the medication ( eg , once daily or twice daily ) . particularly in regard to return instructions concerning when to seek emergency care , a relevant answer ( eg , shortness of breath after pneumonia ) was accepted , even though not documented in the discharge document . categorical differences between the younger and older group were analyzed with chi - squared or fisher s exact test if an expected frequency was less than five in any cell of the contingency table . differences in ordinal categorical variables between groups were analyzed with cochran armitage trend tests , whereas differences in continuous variables were calculated with wilcoxon rank sum tests and reported as median ( interquartile range [ iqr ] ) due to skewed distributions in the younger and older group . in multiple logistic regression analyses , group differences ( older versus younger group ) in recall and awareness of comprehension deficits were adjusted for sex and education . likewise , the associations between age ( continuous variable ) and recall as well as awareness of comprehension deficits were investigated when adjusting for sex and education . models were checked for linearity and potential interaction between age and both sex and education . only models with a significant likelihood ratio test , forty patients were allocated to the older group and 62 to the younger group ( age : 72.0 [ iqr : 6979 ] years versus 48.5 [ iqr : 4258 ] years ; p<0.0001 ) . the two groups were comparable in regard to sex ( male : n=18 , 45.0% versus n=32 , 51.6% ; p=0.51 ) , highest level of education : primary school , high school , university ( p=0.06 ) , other diseases ( older group : 56.3% versus younger group : 58.2% ; p=0.86 ) , length of admission ( 1.0 [ 03 ] days versus 1.0 [ 02 ] days ; p=0.97 ) , and patient satisfaction ( 10.0 [ 910 ] versus 9.5 [ 810 ] ; p=0.13 ) . more prior admissions , however , were found in the older group ( p=0.027 ) . admission diagnosis in the older group was mainly infectious illness ( 22.5% ) , musculoskeletal illness ( 22.5% ) , anemia ( 20.0% ) , neurological illness ( 10.0% ) , and cardiovascular illness ( 7.5% ) . in the younger group , infectious illness ( 21.5% ) , musculoskeletal illness ( 18.5% ) , neurological illness ( 12.3% ) , back illness ( 12.3% ) , and cardiovascular illness ( 9.2% ) there was no difference in self - assessed comprehension of discharge information between the older and younger group . the older group had more difficulties recalling correct medication instructions ( correct recall : 54.3% versus 78.0% ; p=0.02 ) and diagnostic procedures ( 71.8% versus 91.9% ; p=0.007 ) compared to the younger group , respectively ( table 2 ) . the estimated odds for recalling correct medication instructions were 4.2 times higher for the younger group compared to the older group ( odds ratio [ or ] 4.2 , 95% confidence interval [ ci ] 1.511.9 , p=0.007 ) when adjusted for sex and education . furthermore , for each additional year of age the estimated odds for recalling correct medication instructions decreased 6.1% ( or 0.939 , 95% ci 0.900.98 , p=0.001 ) independent of sex and education . there were no differences between the two groups in recall of the remaining questions , yet several patients in both groups were not able to recall correct information ( table 2 ) . the older group was less aware of their comprehension deficits in four out of seven questions when compared to the younger group ; awareness of comprehension deficits with regard to diagnostic tests ( older group : 71.1% versus younger group : 91.9% ; p=0.006 ) , preventive measures ( 53.3% versus 78.6% ; p=0.02 ) , medication instructions ( 51.4% versus 73.3% ; p=0.03 ) , and when to seek emergency care ( 60.6% versus 80.8% ; p=0.04 ) ( figure 1 ) . the remaining questions showed the same tendency , though not significant : admission diagnosis ( 76.9% versus 88.5% ; p=0.12 ) , treatment ( 60.5% versus 77.4% ; p=0.07 ) , and follow - up at general practitioner / specialist ( 54.1% versus 67.7% ; p=0.17 ) . awareness of comprehension deficits in diagnostic tests was 6.8 times higher among the younger group compared to the older group when adjusted for sex and education ( or 6.8 , 95% ci 1.629.2 , p=0.01 ) . awareness of comprehension deficits in medication instructions and diagnostic tests decreased 6.1% ( or 0.939 , 95% ci 0.9040.98 , p=0.001 ) and 5.8% ( or 0.94 , 95% ci 0.900.99 , p=0.02 ) per 1-year increase in age , respectively , when adjusted for sex and education . the remaining multiple logistic regression analyses investigating recall and awareness of comprehension deficits were insignificant . there was no difference in self - assessed comprehension of discharge information between the older and younger group . the older group had more difficulties recalling correct medication instructions ( correct recall : 54.3% versus 78.0% ; p=0.02 ) and diagnostic procedures ( 71.8% versus 91.9% ; p=0.007 ) compared to the younger group , respectively ( table 2 ) . the estimated odds for recalling correct medication instructions were 4.2 times higher for the younger group compared to the older group ( odds ratio [ or ] 4.2 , 95% confidence interval [ ci ] 1.511.9 , p=0.007 ) when adjusted for sex and education . furthermore , for each additional year of age the estimated odds for recalling correct medication instructions decreased 6.1% ( or 0.939 , 95% ci 0.900.98 , p=0.001 ) independent of sex and education . there were no differences between the two groups in recall of the remaining questions , yet several patients in both groups were not able to recall correct information ( table 2 ) . the older group was less aware of their comprehension deficits in four out of seven questions when compared to the younger group ; awareness of comprehension deficits with regard to diagnostic tests ( older group : 71.1% versus younger group : 91.9% ; p=0.006 ) , preventive measures ( 53.3% versus 78.6% ; p=0.02 ) , medication instructions ( 51.4% versus 73.3% ; p=0.03 ) , and when to seek emergency care ( 60.6% versus 80.8% ; p=0.04 ) ( figure 1 ) . the remaining questions showed the same tendency , though not significant : admission diagnosis ( 76.9% versus 88.5% ; p=0.12 ) , treatment ( 60.5% versus 77.4% ; p=0.07 ) , and follow - up at general practitioner / specialist ( 54.1% versus 67.7% ; p=0.17 ) . awareness of comprehension deficits in diagnostic tests was 6.8 times higher among the younger group compared to the older group when adjusted for sex and education ( or 6.8 , 95% ci 1.629.2 , p=0.01 ) . awareness of comprehension deficits in medication instructions and diagnostic tests decreased 6.1% ( or 0.939 , 95% ci 0.9040.98 , p=0.001 ) and 5.8% ( or 0.94 , 95% ci 0.900.99 , p=0.02 ) per 1-year increase in age , respectively , when adjusted for sex and education . the remaining multiple logistic regression analyses investigating recall and awareness of comprehension deficits were insignificant . the main finding of the present study was that older patients were less aware of their comprehension deficits when compared to the younger patients . the older patients were unable to identify the misconceived information in four out of seven questions : medication instructions , diagnostic tests , preventive measures , and when to seek emergency care . at the same time , the older patients were less able to recall correct medication instructions and diagnostic tests . in an internal medicine department and an ed it has been found that patients had difficulties recalling newly prescribed medication.8,10 contrary to the present study , patients were not age divided , and these studies were conducted in different settings . in a qdu setting , we found similar recall difficulties , however , most frequently in the older group . this might lead to noncompliance and hereby relapse of illnesses and complications , as potential medication errors have been shown to increase the number of unplanned readmissions.14 especially older patients are at risk of medication discrepancies and unplanned readmissions.24 despite the comprehension difficulties found in the present study , no qdu readmissions were registered during the 2 months of data collection . however , the limited sample size , along with the relatively short follow - up , limits strong conclusions regarding readmissions . knowledge is lacking on older patients awareness of comprehension deficits , ie , ability to identify the misconceived information . in the present study , we found significant differences between the older and younger patients in four out of eight questions : awareness of comprehension deficits with regard to diagnostic tests , preventive measures , medication instructions , and when to seek emergency care ( figure 1 ) . at the same time , the tendency was that the older group had a lower awareness of comprehension in the remaining questions . a study from an ed found that patients could identify only 20% of their comprehension difficulties.9 these results are difficult to compare to those of the present study since the study design differed considerably . firstly , only one sample population was described , and no subgroup analysis was conducted . thirdly , analyses were conducted for all questions pooled together and not for each question separately , as in the present study . the recall difficulties along with the unawareness of comprehension deficits found in the present study emphasize the importance of communicative strategies to confirm patient comprehension before discharge . we did not investigate potential communication failures ; however , in a study from an ed11 it was identified that important health information was often missed by the physicians , that patients were rarely asked whether they had questions about the information given , and patient comprehension was never confirmed . patients are limited in regard to how much they can process and recall during hospitalization,12,13 and many older patients experience a temporary , although recoverable , cognitive dysfunction at discharge.14 improvements in this hospitalized related cognitive dysfunction have been found 2 to 4 weeks after discharge,13,14 whereas one study found further improvements after 1 year.12 in the present study , only 9% of the older patients thought their illness had affected their comprehension abilities , even though they showed severe difficulties recalling correct discharge information . in this respect , one quarter of the older patients did not recall their potential follow - up at a general practitioner or specialist . further , the setting of the discharge interview could be optimized using a single patient room , inviting relatives to join the discharge interview , and implementation of follow - up strategies such as home visits or follow - up calls the day after discharge . firstly , the medical staff were not blinded to the questionnaire , potentially improving the performance of the staff and thereby patient responses . however , patients were approached before leaving the department to avoid recall bias , and it is likely that they may have done better if they had the medication to look at , even though prescription label instructions can be challenging.15 secondly , we did not collect data on mini - mental state examination scores or hearing status at time of admission . potentially , patients with unknown cognitive and perception impairments could have been included , and this might partly explain the differences in comprehension between the older and younger group . fourthly , we did not collect data from other health care levels ( eg , primary care ) after discharge , and due to the cross - sectional design , we are not aware of the clinical consequence of the present results . in a demographic perspective , the older population and number of patients with multiple illnesses are growing.16,17 health care resources are limited , stressing cost - effectiveness and decreasing the length of hospital admissions.18 as demonstrated in the present study , older patients represent a vulnerable group in the transition between health care levels . although there are immediate costs , from an economic prospective , such interventions have shown promising results in lowering the rates of readmission.7,19,20 older patients were less able to recall correct medication instructions and diagnostic tests when compared to younger patients . furthermore , the older patients were less aware of their comprehension deficits with respect to medication instructions , diagnostic tests , preventive measures , and when to seek emergency care . in our perspective , the findings of the present study suggest that communication with the expanding population of older patients requires particular attention .

, this chemotherapeutic agent is well - known to generate adverse effects such as nephrotoxicity [ 15 ] and ototoxicity [ 613 ] . high - dose cisplatin treatment typically induces a high - frequency hearing loss , which can gradually or suddenly extend to lower frequencies during subsequent courses [ 1416 ] . in animal studies , cisplatin produces multiple toxic effects on the guinea pig and rat cochlea , which are characterized by lesions to the outer hair cells [ 13,1719 ] the stria vascularis [ 8,9,12,2023 ] and the auditory neurons . protection from these adverse effects has been widely studied , because hearing preservation is crucial for the patient s quality of life . however , no effective otoprotective treatment for cisplatin ototoxicity is currently in clinical use . among the numerous agents that have been proposed as effective oto - protectors are d - methionine , n - l - acetylcysteine and ebselen . d - methionine ( d - met ) , whether administered systemically or through the round window membrane , has been shown to protect from cisplatin - induced ototoxicity [ 13,2330 ] . d - met is a sulphur - containing nucleophile and antioxidant with multiple protective mechanisms . d - met may protect against cisplatin ototoxicity by reversing cellular platinum - thiol complexes , protecting the essential amino acid l - methionine , and also functioning as an antioxidant . campbell reported d - met pre - treatment provided complete protection in vivo against cisplatin - induced hearing loss and outer hair cell loss in the rat at 72 hours post - administration . gabaizadeh demonstrated that in combination with brain - derived neurotrophic factor , d - met also protects against cisplatin - induced loss of auditory neurons . further , d - met does not interfere with cisplatin s anti - tumor action . ekborn found that pre - treatment with d - met in guinea pigs affects the concentration of free cisplatin in the systemic circulation . ekborn assumed that a cisplatin - methionine complex would not be cytotoxic for cancer cells , but deegan documented that a cisplatin - methionine complex is significantly cytotoxic for cancer cells in vitro but lacks the associated renal toxicity . n - l - acetylcysteine ( nac ) , a precursor of glutathione , is an antioxidant that limits the extent of the oxidative stress damage to the cell and is able to improve the oxidant / antioxidant cellular balance . the antioxidative effect has been widely documented in a number of experimental studies using various stressors . pre - treatment with nac was shown by dickey to protect against cisplatin ototoxicity in the long - evans rat model . the major mechanism of nac cyto - protection seems to be mediated via inhibition of the effects induced by reactive oxygen species . ebselen , an anti - inflammatory agent , has been shown to reduce cisplatin ototoxicity in wistar rats after high doses ( 16 mg / kg ) . it has also been demonstrated in fisher 344 rats that ebselen alone ( 16 mg / kg ) or in combination with allopurinol ( each compound was given at 8 mg / kg ) can protect against cisplatin - induced ototoxicity . however , a narrow range for otoprotection of ebselen has been documented in guinea pigs exposed to acoustic trauma . based on the experience in this laboratory that many pharmacological agents show a species dependence [ 4244 ] , the sprague - dawley rat was used as an acute cisplatin ototoxicity model [ 4547 ] to compare the otoprotective efficacy of 2 sulphur - containing antioxidants , d - methionine , n - l - acetylcysteine , and 1 seleno - organic compound , ebselen . each putative protective agent was tested at 3 different dosages in order to assess the influence of dose on auditory preservation . the dosages were derived from the literature or from data collected in this laboratory ( d - met , nac ) . forty male albino sprague - dawley rats ( charles river , italy ) were divided into 10 groups ; 3 groups for each protective agent and a cisplatin - treated control group . the experimental protocol ( applied to all animals ) included these steps : anesthesia with a ketamine - xylazine cocktail . assessment of the auditory function , including auditory brainstem response ( abr ) and distortion product otoacoustic emissions ( dpoae ) recordings . injection of the tested protector ( d - met , nac , ebselen , and saline ) after 1 hour delay , cisplatin ( 14 mg / kg ) was administered to each animal by a slow i.v . infusion ( for details see the next section on cisplatin ) . abr and dpoae data ( ps96 recordings ) were acquired after 96 hours from the time cisplatin was administered . in previous papers the authors have shown that a cisplatin dosage of 16 mg / kg damages the rat cochlea . this dosage serves for an acute ototoxicity model , but it is not well related to the dosages administered in humans . furthermore , the higher the dosage of cisplatin the higher the required dosage of the oto - protector . in order to better evaluate the behavior of the tested otoprotectors in the sprague - dawley rat , a lower cisplatin dosage ( 14 mg / kg ) was administered to the tested animals . cisplatin ( cisplatino , ebewe , italy ) was delivered by slow infusion ( 0.1 ml / min . ) in the caudal vein ( i.v . ) at a concentration of 1 mg / ml . a 1-hour interval ( between the i.p . injection of each protective agent and the i.v . the otoprotectors were administered as an intraperitoneal injection ( i.p . ) in all 10 experimental groups , 1 hour before cisplatin administration . d - methionine ( d - met ; sigma chemical co. ) was dissolved in saline ( 50 mg / ml ) and administered as a bolus i.p . d - met treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 1 , 2 and 3 received 300 , 350 and 400 mg / kg d - met , respectively ( dosages derived from previous pilot studies in this laboratory ) . n - l - acetylcysteine ( nac ; sigma chemical co. ) was dissolved in saline ( 100 mg / ml ) adjusted to ph 7.0 and administered as a bolus i.p . injection . nac - treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 4 , 5 and 6 received 275 , 375 and 475 mg / kg nac , respectively ( dosages derived from previous pilot studies in this laboratory ) . ebselen ( sigma chemical co. ) was dissolved in pure dimethylsulfoxide ( dmso ) at 20 mg / ml and stored at 20c . ebselen - treated animals were divided in 3 groups ( 4 animals per group ) according to dosage : groups 7 , 8 and 9 received 4 , 8 and 12 mg / kg ebselen , respectively , by i.p . the dosages for the ebselen groups were derived from data in the literature , taken with some conservation . for example , lynch reported that 16 mg / kg of ebselen can provide protection against 16 mg / kg of cisplatin in the fischer-344 rat . the same authors also suggested that 8 mg / kg of ebselen and 8 mg / kg of allopurinol can provide similar effects . pourbakht and yamasoba have shown that a lower concentration of ebselen ( 10 mg / kg ) performs better than a higher concentration ( 30 mg / kg ) of ebselen in protecting guinea pigs exposed to noise . considering this information , it was decided that it was safer for the objectives of the study ( ie , using cisplatin at a lower concentration ) to set 12 mg / kg as the maximum ebselen dose . the last group ( n=4 ) received an equivalent volume saline solution and served as the control group . to partially compensate for the small number of animals in each tested group , the abr threshold recordings at low stimulus levels and all dpoae responses were recorded twice and the responses were averaged . abr responses were recorded by 3 platinum - iridium needle electrodes , placed subdermally over the vertex ( positive ) , the mastoid ( negative ) and the dorsum area ( reference / ground ) of the animal . the sound transducer of a motorola tweeter ( flat response 1.5 db from 4.0 to 35 khz ) , was placed at a distance of 4 cm from the rat s ear . the abrs were amplified 20 000 times and filtered from 20 to 5000 hz . the abrs were elicited by 8 , 12 and 16 khz tone pips ( 1 ms rise - fall time , 10 ms plateau ) , over the intensity range of 30110 db spl . the electrophysiological hearing threshold was defined as the lowest intensity at which a replicable abr wave was seen in 2 averaged runs . as in previous studies , the threshold level of the sprague - dawley rat at frequencies up to 16 khz ear plugs were used to occlude the contralateral ear in order to avoid binaural stimulation . the recordings of the distortion product otoacoustic emissions ( dpoae ) were conducted by a starkey 2000 ( starkey labs , usa ) device . the dpoae amplitudes were analyzed in the frequencies from 6.0 to 17.0 khz ( referred to as f2 ) and 5 frequency points were sampled . the frequency ratio between primaries was fixed to 1.21 . each recording was made on the average from 4 seconds of data sampling , and the noise tolerance was fixed at 15 db spl . the recordings were elicited by asymmetrical dpoae protocol where l1>l2 ( l1=50 and l2=40 db spl ) . asymmetric protocols are generally considered a better choice to identify cochlear dysfunction . during the electrophysiological recordings ( abr and dpoae ) the body temperature of the animal was maintained at 370.5c by the use of a temperature control device ( harvard apparatus , usa ) . a rectal probe was placed in order to assess the rat s body temperature changes , and a homoeothermic blanket under the rat s body regulated the heating to keep the body temperature constant for the time needed for the acquisition of recordings . all measurements were conducted at the right ear of each tested animal in a soundproof chamber . the levels of all the stimuli used in the present study were checked by the use of a bruel and kjaer impulse precision sound level meter type 2209 , coupled with an 1-inch condenser microphone bruel and kjaer type 4145 for free field use , which had a normal incidence - free field response linear from 1 to 2 hz ( 3 db ) to 18 khz ( 1.5 db ) and meets the requirement of the ansi ( american national standards institute ) for laboratory standard type l microphone . in addition , a bruel and kjaer 1/3 octave filter set ( type 1616 for 1/3 octave analysis in the range 18 hz44 khz covered by 34-pass band filters ) was used in conjunction with the precision sound level meter type 2209 . each animal was weighed on the day before the pre recordings and 4 days after cisplatin administration before the post recordings . the abr and dpoae variables were evaluated for statistical significance and post - pre differences were evaluated . a 1-way anova , with treatment as the factor , was fit for each protocol and response variable . estimates and confidence intervals were obtained for mean differences per treatment and for pairwise differences between mean differences for different treatments . tukey intervals were used to maintain an overall confidence level for each variable , and a bonferroni adjustment was made to ensure an overall 0.05 level for all intervals per interval type ( mean difference or pairwise difference ) and protocol . for the analysis of body weight alterations , a paired t - test was performed between the body weight at pre - and post - cisplatin administration of each experimental group . forty male albino sprague - dawley rats ( charles river , italy ) were divided into 10 groups ; 3 groups for each protective agent and a cisplatin - treated control group . the experimental protocol ( applied to all animals ) included these steps : anesthesia with a ketamine - xylazine cocktail . assessment of the auditory function , including auditory brainstem response ( abr ) and distortion product otoacoustic emissions ( dpoae ) recordings . injection of the tested protector ( d - met , nac , ebselen , and saline ) after 1 hour delay , cisplatin ( 14 mg / kg ) was administered to each animal by a slow i.v . infusion ( for details see the next section on cisplatin ) . abr and dpoae data ( ps96 recordings ) were acquired after 96 hours from the time cisplatin was administered . in previous papers the authors have shown that a cisplatin dosage of 16 mg / kg damages the rat cochlea . this dosage serves for an acute ototoxicity model , but it is not well related to the dosages administered in humans . furthermore , the higher the dosage of cisplatin the higher the required dosage of the oto - protector . in order to better evaluate the behavior of the tested otoprotectors in the sprague - dawley rat , a lower cisplatin dosage ( 14 mg / kg ) was administered to the tested animals . cisplatin ( cisplatino , ebewe , italy ) was delivered by slow infusion ( 0.1 ml / min . ) in the caudal vein ( i.v . ) at a concentration of 1 mg / ml . a 1-hour interval ( between the i.p . the otoprotectors were administered as an intraperitoneal injection ( i.p . ) in all 10 experimental groups , 1 hour before cisplatin administration . d - methionine ( d - met ; sigma chemical co. ) was dissolved in saline ( 50 mg / ml ) and administered as a bolus i.p . injection . d - met treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 1 , 2 and 3 received 300 , 350 and 400 mg / kg d - met , respectively ( dosages derived from previous pilot studies in this laboratory ) . n - l - acetylcysteine ( nac ; sigma chemical co. ) was dissolved in saline ( 100 mg / ml ) adjusted to ph 7.0 and administered as a bolus i.p . injection . nac - treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 4 , 5 and 6 received 275 , 375 and 475 mg / kg nac , respectively ( dosages derived from previous pilot studies in this laboratory ) . ebselen ( sigma chemical co. ) was dissolved in pure dimethylsulfoxide ( dmso ) at 20 mg / ml and stored at 20c . ebselen - treated animals were divided in 3 groups ( 4 animals per group ) according to dosage : groups 7 , 8 and 9 received 4 , 8 and 12 mg / kg ebselen , respectively , by i.p . the dosages for the ebselen groups were derived from data in the literature , taken with some conservation . for example , lynch reported that 16 mg / kg of ebselen can provide protection against 16 mg / kg of cisplatin in the fischer-344 rat . the same authors also suggested that 8 mg / kg of ebselen and 8 mg / kg of allopurinol can provide similar effects . pourbakht and yamasoba have shown that a lower concentration of ebselen ( 10 mg / kg ) performs better than a higher concentration ( 30 mg / kg ) of ebselen in protecting guinea pigs exposed to noise . considering this information , it was decided that it was safer for the objectives of the study ( ie , using cisplatin at a lower concentration ) to set 12 mg / kg as the maximum ebselen dose . the last group ( n=4 ) received an equivalent volume saline solution and served as the control group . to partially compensate for the small number of animals in each tested group , the abr threshold recordings at low stimulus levels and all dpoae responses were recorded twice and the responses were averaged . abr responses were recorded by 3 platinum - iridium needle electrodes , placed subdermally over the vertex ( positive ) , the mastoid ( negative ) and the dorsum area ( reference / ground ) of the animal . the sound transducer of a motorola tweeter ( flat response 1.5 db from 4.0 to 35 khz ) , was placed at a distance of 4 cm from the rat s ear . the abrs were elicited by 8 , 12 and 16 khz tone pips ( 1 ms rise - fall time , 10 ms plateau ) , over the intensity range of 30110 db spl . the electrophysiological hearing threshold was defined as the lowest intensity at which a replicable abr wave was seen in 2 averaged runs . as in previous studies , the threshold level of the sprague - dawley rat at frequencies up to 16 khz was found to be approximately at 3540 db spl . ear plugs were used to occlude the contralateral ear in order to avoid binaural stimulation . the recordings of the distortion product otoacoustic emissions ( dpoae ) were conducted by a starkey 2000 ( starkey labs , usa ) device . the dpoae amplitudes were analyzed in the frequencies from 6.0 to 17.0 khz ( referred to as f2 ) and 5 frequency points were sampled . the frequency ratio between primaries was fixed to 1.21 . each recording was made on the average from 4 seconds of data sampling , and the noise tolerance was fixed at 15 db spl . the recordings were elicited by asymmetrical dpoae protocol where l1>l2 ( l1=50 and l2=40 db spl ) . asymmetric protocols are generally considered a better choice to identify cochlear dysfunction . during the electrophysiological recordings ( abr and dpoae ) the body temperature of the animal was maintained at 370.5c by the use of a temperature control device ( harvard apparatus , usa ) . a rectal probe was placed in order to assess the rat s body temperature changes , and a homoeothermic blanket under the rat s body regulated the heating to keep the body temperature constant for the time needed for the acquisition of recordings . all measurements were conducted at the right ear of each tested animal in a soundproof chamber . the levels of all the stimuli used in the present study were checked by the use of a bruel and kjaer impulse precision sound level meter type 2209 , coupled with an 1-inch condenser microphone bruel and kjaer type 4145 for free field use , which had a normal incidence - free field response linear from 1 to 2 hz ( 3 db ) to 18 khz ( 1.5 db ) and meets the requirement of the ansi ( american national standards institute ) for laboratory standard type l microphone . in addition , a bruel and kjaer 1/3 octave filter set ( type 1616 for 1/3 octave analysis in the range 18 hz44 khz covered by 34-pass band filters ) was used in conjunction with the precision sound level meter type 2209 . each animal was weighed on the day before the pre recordings and 4 days after cisplatin administration before the post recordings . the abr and dpoae variables were evaluated for statistical significance and post - pre differences were evaluated . a 1-way anova , with treatment as the factor , was fit for each protocol and response variable . estimates and confidence intervals were obtained for mean differences per treatment and for pairwise differences between mean differences for different treatments . tukey intervals were used to maintain an overall confidence level for each variable , and a bonferroni adjustment was made to ensure an overall 0.05 level for all intervals per interval type ( mean difference or pairwise difference ) and protocol . for the analysis of body weight alterations , a paired t - test was performed between the body weight at pre - and post - cisplatin administration of each experimental group . figure 1 shows the pre - treatment hearing levels of all animals , divided per group at 8 , 12 and 16 khz . the hearing levels at 8 khz were consistently lower than the other 2 tested frequencies , across all groups . the hearing levels at 12 and 16 khz were very similar across all groups with the exception of the 275 mg / kg nac group . at 8 khz no significant threshold change was found in the d - met- and nac - treated animals . in contrast , in the 3 groups pre - treated with ebselen significant hearing threshold elevations were observed . the animals treated with 12 mg / kg of ebselen presented the lowest threshold shifts among the ebselen groups , which were statistically significant in comparison to the pre - treatment data . at 12 and 16 khz , only 3 groups presented comparable thresholds to the pre - treatment data the animals that received 350 and 400 mg / kg of d - met and 475 mg / kg of nac . the ebselen - treated animals presented significant threshold shifts and showed the highest threshold elevations in the treated groups . at these frequencies the hearing thresholds across the 3 ebselen groups were similar , although the protector doses were increased 2- and 3-fold ( from 4 mg / kg to 12 mg / kg ) . figures 24 and table 1 summarize the abr data at the 3 tested frequencies across the 10 treatment groups . the dpoae analysis showed that cisplatin causes an average negative shift in the dpoae amplitude in the order of 20 db in the frequency span from 6.5 to 17 khz . figures 5 and 6 summarize the dp - gram information from untreated and treated animals with cisplatin and otoprotectors . only the animals from the group treated with 350 mg / kg of d - met presented lack of statistical differences between the pre and post dpoae recordings . all other groups showed significant alterations in dpoae amplitude , including groups 3 and 6 ( 400 mg / kg d - met and 475 mg / kg nac ) . the 3 ebselen - treated groups showed statistical differences at all tested frequencies , and in a number of animals it was not possible to record a post - treatment dpoae response . the data indicate that only the 375 mg nac treatment provides partial protection against cisplatin - induced weight loss . animals treated by 275 mg of nac had a borderline ( p=0.058 ) weight loss , whereas animals included in all the remaining treatment groups showed a significant cddp - dependant weight loss . we used the sprague dawley rat model to compare the efficacy of different dosages of 3 pharmacological agents that have been reported to exert a protective effect against cisplatin ototoxicity in other strains of rats . experimental studies have shown that cisplatin administration causes the formation of reactive oxygen species in the inner ear , leading to lipid peroxydation , triggering of apoptosis and a significant alteration of the auditory function . the ototoxic effect in experimental animals is dose - dependent and is manifested as an increase of the electrophysiological hearing threshold . there are numerous reports in the literature [ 13,2330,47,52 ] describing the protective effects of various oto - protectors against cisplatin , usually evaluated in a short time - period ( e.g. , 72 hours ) . in the majority of studies the abr has been utilized to assess the hearing threshold of the tested animals . in this study the efficacy of the tested pharmacological agents was additionally evaluated with distortion product otoacoustic emissions . the combined information from both measurements provides an enhanced understanding of the events related to inner ear and neural fiber otoptotection . the abr data from the d - met group confirm the previous findings of campbell in the wister rat , suggesting that dosages of 300400 mg / kg have the potential to protect the inner ear . in terms of performance , the higher dosages ( 350 and 400 mg / kg ) showed better auditory preservation across all the tested abr frequencies . of the 3 tested protocols , only the moderate dose ( 350 mg / kg ) group generated responses with no significant pre - post differences , and in light of this only the moderate dose can be considered as a candidate for auditory preservation . the data from the d - met dpoae responses suggest that increasing the amount of otoprotector does not necessarily increase the index of auditory preservation . the abr data from the nac group showed a partial auditory preservation in the 275 and 375 mg / kg groups and a complete preservation in the 475 mg / kg group . these findings are similar to those of dickey in which a pre - treatment injection of 400 mg / kg nac was able to prevent ototoxicity at a considerably lower dose of cisplatin ( 6 mg / kg ) in the long - evans rat . in their study , nac administration was given 30 minutes and 4 hours before cisplatin injection to reduce ototoxicity . the dpoae responses from the nac treated animals showed significant amplitude changes across many frequencies . in terms of performance , the best results were observed in the 375 mg / kg group . by integrating the information from the abr and dpoae data , we conclude that the nac protocols do not seem to offer complete auditory preservation in dosages of up to 400 mg / kg . an explanation for the observed discrepancy between the abr and dpoae findings in the d - met and nac groups can not be given only from the electrophysiological findings ; however , it is reasonable to speculate that the effect on the dpoae recordings could be a result of minor loss of outer hair cells in the treated animals . considering the time window of observation ( 96 hours ) the dpoae data might indicate cell death of the outer hair cell population . to elucidate this argument further additional studies utilizing longer observation windows ( 168 hours or longer ) are required in order to verify the assumed apoptosis scenario . results of the present study clearly show that ebselen pre - treatment did not protect the cochlea in the sprague dawley albino male rat . this finding is contradictory to earlier reports , showing amelioration of cisplatin ototoxicity in the fischer 344 rat given a 16 mg / kg dose of cisplatin . moreover , rybak showed that the hearing of the wistar rat was protected by ebselen ( 16 mg / kg ) in connection to cisplatin treatment . have shown that in the fischer 344 rat , 8 mg / kg of ebselen administered orally with 8 mg / kg of allopurinol offers protection similar to a single 16 mg / kg ebselen dose . the abr and dpoae data from this study show that in the sprague dawley rat there is no significant otoprotection from an i.p . administration of ebselen in dosages of up to 12 mg / kg . the maximum ebselen dosage used in this study is lower than the dosages reported in the literature ( 16 mg / kg ) to compensate for the lower cisplatin dosage employed ( 14 vs. 16 mg / kg ) . in addition , the pattern of the abr / dpoae data from the ebselen - treated animals was different than the data from the other 2 protectors , even at dosages that did not offer protection at all frequencies . non - optimized dosages of d - met or nac presented some protection in 1 of the tested abr / dpoae frequencies , but this was not the case for ebselen . one may speculate whether less favorable pharmacokinetics or pharmacodynamic parameters influenced the putative protective effect of ebselen in the cochlea after the i.p . the earlier studies reporting positive inner - ear protection effects have all used the same administration route for both ebselen and cisplatin . the findings of amelioration of ototoxicity in these studies might be explained by a direct drug interaction in the blood compartment not obtained by the present treatment protocol . possible interactions between ebselen and cisplatin in the systemic circulation could lower the level of free cisplatin and thereby result in less cochlear injury . the key element of this hypothesis is the absorption time of ebselen from the intraperitoneal cavity of the employed animal model . longer absorption times would promote stronger interaction effects between the 2 pharmacological agents , ebselen and cisplatin , whereas rapid uptake of ebselen and a fast elimination would promote higher concentrations of free cisplatin to reach the inner ear . another explanation is that the levels of ebselen could be affected by first - pass hepatic losses . strain - specific differences for otoprotection are less plausible in explanation of lack of otoprotection , although species specific differences in otoprotection are not uncommon . for example , duan has reported species otoprotection variability against impulse noise using nac . the data from the present study strongly suggest that additional studies are needed on strain - specific otoprotection to better define the benefits with systemic ebselen otoprotection . one of the methodological objectives of the study was the minimization of the drug interaction between cisplatin and the otoprotectors . the reasons behind this objective are rooted in the clinical environment , where it is essential to obtain maximum chemotherapeutic effect while preserving the hearing of patients treated with cisplatin - based chemotherapy . there are grounds for speculation that the time interval between administration of an otoprotector and administration of cisplatin , as well as the mode of drug administration , affect the outcome . the risk of systemic drug interaction suggests that an otoprotector and cisplatin should be given separately , not only in time , but also , ideally , by separating the routes of administration . in the sprague - dawley albino male rats . in humans , 2 modes of systemic administration can be achieved by using i.v . and intra - arterial infusions of cisplatin have been given to patients with advanced hypopharyngeal cancer concomitant with i.v . pre - administration of the thiol - containing antioxidant was used allow the protective agent to accumulate in the inner ear before cisplatin reached the target cells . no pharmacokinetics analysis was undertaken and therefore no such data can be given to explain our findings . administration of the otoprotector was used , one can speculate that the peak concentration in the blood of otoprotector and cisplatin was reached within 1 hour . another to consider in otoprotection is the transport of the drugs over the blood - labyrinth barrier . cisplatin is reported to have a peak concentration in the scala tympani perilymph 20 minutes after an i.v . no information can be found in the literature on the cochlear kinetics of the used otoprotectors after the i.v . thio - sulfate , another thiol - containing antioxidant , is readily transported to the inner ear and reaches its peak concentration in scala tympani perilymph within 10 minutes after an i.v . the terminal half - life of d - met in scala tympani perilymph has been estimated to 0.6 hour . to obtain maximal otoprotection and minimal drug interaction between a thiol - containing antioxidant and cisplatin , it is necessary to acquire data from additional studies on blood and inner ear pharmacokinetics . the abr data show that animals given 350 and 400 mg / kg of d - met and 475 mg / kg of nac presented significantly better auditory preservation than the other groups . the dpoae data show that only animals receiving 350 mg / kg of d - met presented no significant differences between the pre and the post recordings . combining this information with the abr data , it can be concluded that only d - met shows a complete auditory preservation 96 hours after cisplatin administration . findings from ebselen pre - treated sprague - dawley albino male rats demonstrate that ebselen dosages up to 12 mg / kg given by i.p .

summarybackgroundsprague - dawley rats were used as an acute cisplatin ototoxicity model to compare the chemo - protective efficacy of 2 sulphur - containing antioxidants ( d - methionine , n - l - acetylcysteine ) and 1 seleno - organic compound ( ebselen ) . each putative chemo - protective agent was tested at 3 different dosages in order to assess the influence of dose on auditory preservation.material/methodsa total of 40 sprague - dawley albino male rats were used in the study . animals were divided into 10 groups , 3 groups of different doses for each protective agent and a cisplatin - treated control group . the animals were weight - matched before drug exposure to ensure similar weights in all groups . auditory function was assessed with auditory brainstem responses and distortion product otoacoustic emissions at time zero and at 96 hours post-treatment.resultsat the post - treatment follow - up no significant threshold change at 8 khz was found in the d - met- and nac - treated groups . all ebselen - treated animals presented significant threshold elevations . at 12 and 16 khz , only the groups treated with 300 , 450 mg / kg of d - met and 475 mg / kg of nac presented thresholds comparable to the pre - treatment abr data . the ebselen - treated animals presented significant threshold shifts and showed the highest threshold elevations . the dpoae data analysis showed that only the animals from the 350 mg / kg d - met group presented lack of statistical differences between the pre and post recordings.conclusionsconsidering the outcome from the abr and dpoae analyses together , only the 350 mg / kg d - met group presented a complete auditory preservation against the 14 mg / kg cisplatin administered i.v . data from ebselen pre - treated sprague - dawley albino male rats demonstrate that ebselen dosages up to 12 mg / kg given by i.p . administration lack auditory preservation in this species .

Background Material and Methods Animals Experimental protocol Cisplatin Otoprotectors Acoustical and electrophysiological measurements Measurement of weight Statistical analysis Results Discussion Conclusions

among the numerous agents that have been proposed as effective oto - protectors are d - methionine , n - l - acetylcysteine and ebselen . d - methionine ( d - met ) , whether administered systemically or through the round window membrane , has been shown to protect from cisplatin - induced ototoxicity [ 13,2330 ] . d - met is a sulphur - containing nucleophile and antioxidant with multiple protective mechanisms . d - met may protect against cisplatin ototoxicity by reversing cellular platinum - thiol complexes , protecting the essential amino acid l - methionine , and also functioning as an antioxidant . campbell reported d - met pre - treatment provided complete protection in vivo against cisplatin - induced hearing loss and outer hair cell loss in the rat at 72 hours post - administration . gabaizadeh demonstrated that in combination with brain - derived neurotrophic factor , d - met also protects against cisplatin - induced loss of auditory neurons . ekborn found that pre - treatment with d - met in guinea pigs affects the concentration of free cisplatin in the systemic circulation . ekborn assumed that a cisplatin - methionine complex would not be cytotoxic for cancer cells , but deegan documented that a cisplatin - methionine complex is significantly cytotoxic for cancer cells in vitro but lacks the associated renal toxicity . n - l - acetylcysteine ( nac ) , a precursor of glutathione , is an antioxidant that limits the extent of the oxidative stress damage to the cell and is able to improve the oxidant / antioxidant cellular balance . pre - treatment with nac was shown by dickey to protect against cisplatin ototoxicity in the long - evans rat model . ebselen , an anti - inflammatory agent , has been shown to reduce cisplatin ototoxicity in wistar rats after high doses ( 16 mg / kg ) . it has also been demonstrated in fisher 344 rats that ebselen alone ( 16 mg / kg ) or in combination with allopurinol ( each compound was given at 8 mg / kg ) can protect against cisplatin - induced ototoxicity . based on the experience in this laboratory that many pharmacological agents show a species dependence [ 4244 ] , the sprague - dawley rat was used as an acute cisplatin ototoxicity model [ 4547 ] to compare the otoprotective efficacy of 2 sulphur - containing antioxidants , d - methionine , n - l - acetylcysteine , and 1 seleno - organic compound , ebselen . each putative protective agent was tested at 3 different dosages in order to assess the influence of dose on auditory preservation . the dosages were derived from the literature or from data collected in this laboratory ( d - met , nac ) . forty male albino sprague - dawley rats ( charles river , italy ) were divided into 10 groups ; 3 groups for each protective agent and a cisplatin - treated control group . assessment of the auditory function , including auditory brainstem response ( abr ) and distortion product otoacoustic emissions ( dpoae ) recordings . injection of the tested protector ( d - met , nac , ebselen , and saline ) after 1 hour delay , cisplatin ( 14 mg / kg ) was administered to each animal by a slow i.v . abr and dpoae data ( ps96 recordings ) were acquired after 96 hours from the time cisplatin was administered . in previous papers the authors have shown that a cisplatin dosage of 16 mg / kg damages the rat cochlea . this dosage serves for an acute ototoxicity model , but it is not well related to the dosages administered in humans . in order to better evaluate the behavior of the tested otoprotectors in the sprague - dawley rat , a lower cisplatin dosage ( 14 mg / kg ) was administered to the tested animals . injection of each protective agent and the i.v . d - methionine ( d - met ; sigma chemical co. ) was dissolved in saline ( 50 mg / ml ) and administered as a bolus i.p . d - met treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 1 , 2 and 3 received 300 , 350 and 400 mg / kg d - met , respectively ( dosages derived from previous pilot studies in this laboratory ) . n - l - acetylcysteine ( nac ; sigma chemical co. ) was dissolved in saline ( 100 mg / ml ) adjusted to ph 7.0 and administered as a bolus i.p . nac - treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 4 , 5 and 6 received 275 , 375 and 475 mg / kg nac , respectively ( dosages derived from previous pilot studies in this laboratory ) . ebselen - treated animals were divided in 3 groups ( 4 animals per group ) according to dosage : groups 7 , 8 and 9 received 4 , 8 and 12 mg / kg ebselen , respectively , by i.p . for example , lynch reported that 16 mg / kg of ebselen can provide protection against 16 mg / kg of cisplatin in the fischer-344 rat . the same authors also suggested that 8 mg / kg of ebselen and 8 mg / kg of allopurinol can provide similar effects . considering this information , it was decided that it was safer for the objectives of the study ( ie , using cisplatin at a lower concentration ) to set 12 mg / kg as the maximum ebselen dose . the abrs were elicited by 8 , 12 and 16 khz tone pips ( 1 ms rise - fall time , 10 ms plateau ) , over the intensity range of 30110 db spl . as in previous studies , the threshold level of the sprague - dawley rat at frequencies up to 16 khz ear plugs were used to occlude the contralateral ear in order to avoid binaural stimulation . the recordings of the distortion product otoacoustic emissions ( dpoae ) were conducted by a starkey 2000 ( starkey labs , usa ) device . the dpoae amplitudes were analyzed in the frequencies from 6.0 to 17.0 khz ( referred to as f2 ) and 5 frequency points were sampled . a rectal probe was placed in order to assess the rat s body temperature changes , and a homoeothermic blanket under the rat s body regulated the heating to keep the body temperature constant for the time needed for the acquisition of recordings . each animal was weighed on the day before the pre recordings and 4 days after cisplatin administration before the post recordings . the abr and dpoae variables were evaluated for statistical significance and post - pre differences were evaluated . tukey intervals were used to maintain an overall confidence level for each variable , and a bonferroni adjustment was made to ensure an overall 0.05 level for all intervals per interval type ( mean difference or pairwise difference ) and protocol . for the analysis of body weight alterations , a paired t - test was performed between the body weight at pre - and post - cisplatin administration of each experimental group . forty male albino sprague - dawley rats ( charles river , italy ) were divided into 10 groups ; 3 groups for each protective agent and a cisplatin - treated control group . assessment of the auditory function , including auditory brainstem response ( abr ) and distortion product otoacoustic emissions ( dpoae ) recordings . injection of the tested protector ( d - met , nac , ebselen , and saline ) after 1 hour delay , cisplatin ( 14 mg / kg ) was administered to each animal by a slow i.v . abr and dpoae data ( ps96 recordings ) were acquired after 96 hours from the time cisplatin was administered . in previous papers the authors have shown that a cisplatin dosage of 16 mg / kg damages the rat cochlea . this dosage serves for an acute ototoxicity model , but it is not well related to the dosages administered in humans . in order to better evaluate the behavior of the tested otoprotectors in the sprague - dawley rat , a lower cisplatin dosage ( 14 mg / kg ) was administered to the tested animals . d - methionine ( d - met ; sigma chemical co. ) was dissolved in saline ( 50 mg / ml ) and administered as a bolus i.p . d - met treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 1 , 2 and 3 received 300 , 350 and 400 mg / kg d - met , respectively ( dosages derived from previous pilot studies in this laboratory ) . n - l - acetylcysteine ( nac ; sigma chemical co. ) was dissolved in saline ( 100 mg / ml ) adjusted to ph 7.0 and administered as a bolus i.p . nac - treated animals were divided into 3 groups ( 4 animals per group ) according to dosage : groups 4 , 5 and 6 received 275 , 375 and 475 mg / kg nac , respectively ( dosages derived from previous pilot studies in this laboratory ) . ebselen - treated animals were divided in 3 groups ( 4 animals per group ) according to dosage : groups 7 , 8 and 9 received 4 , 8 and 12 mg / kg ebselen , respectively , by i.p . for example , lynch reported that 16 mg / kg of ebselen can provide protection against 16 mg / kg of cisplatin in the fischer-344 rat . considering this information , it was decided that it was safer for the objectives of the study ( ie , using cisplatin at a lower concentration ) to set 12 mg / kg as the maximum ebselen dose . as in previous studies , the threshold level of the sprague - dawley rat at frequencies up to 16 khz was found to be approximately at 3540 db spl . ear plugs were used to occlude the contralateral ear in order to avoid binaural stimulation . the recordings of the distortion product otoacoustic emissions ( dpoae ) were conducted by a starkey 2000 ( starkey labs , usa ) device . the dpoae amplitudes were analyzed in the frequencies from 6.0 to 17.0 khz ( referred to as f2 ) and 5 frequency points were sampled . a rectal probe was placed in order to assess the rat s body temperature changes , and a homoeothermic blanket under the rat s body regulated the heating to keep the body temperature constant for the time needed for the acquisition of recordings . the levels of all the stimuli used in the present study were checked by the use of a bruel and kjaer impulse precision sound level meter type 2209 , coupled with an 1-inch condenser microphone bruel and kjaer type 4145 for free field use , which had a normal incidence - free field response linear from 1 to 2 hz ( 3 db ) to 18 khz ( 1.5 db ) and meets the requirement of the ansi ( american national standards institute ) for laboratory standard type l microphone . each animal was weighed on the day before the pre recordings and 4 days after cisplatin administration before the post recordings . the abr and dpoae variables were evaluated for statistical significance and post - pre differences were evaluated . tukey intervals were used to maintain an overall confidence level for each variable , and a bonferroni adjustment was made to ensure an overall 0.05 level for all intervals per interval type ( mean difference or pairwise difference ) and protocol . for the analysis of body weight alterations , a paired t - test was performed between the body weight at pre - and post - cisplatin administration of each experimental group . figure 1 shows the pre - treatment hearing levels of all animals , divided per group at 8 , 12 and 16 khz . the hearing levels at 12 and 16 khz were very similar across all groups with the exception of the 275 mg / kg nac group . at 8 khz no significant threshold change was found in the d - met- and nac - treated animals . in contrast , in the 3 groups pre - treated with ebselen significant hearing threshold elevations were observed . the animals treated with 12 mg / kg of ebselen presented the lowest threshold shifts among the ebselen groups , which were statistically significant in comparison to the pre - treatment data . at 12 and 16 khz , only 3 groups presented comparable thresholds to the pre - treatment data the animals that received 350 and 400 mg / kg of d - met and 475 mg / kg of nac . the ebselen - treated animals presented significant threshold shifts and showed the highest threshold elevations in the treated groups . at these frequencies the hearing thresholds across the 3 ebselen groups were similar , although the protector doses were increased 2- and 3-fold ( from 4 mg / kg to 12 mg / kg ) . the dpoae analysis showed that cisplatin causes an average negative shift in the dpoae amplitude in the order of 20 db in the frequency span from 6.5 to 17 khz . only the animals from the group treated with 350 mg / kg of d - met presented lack of statistical differences between the pre and post dpoae recordings . all other groups showed significant alterations in dpoae amplitude , including groups 3 and 6 ( 400 mg / kg d - met and 475 mg / kg nac ) . the 3 ebselen - treated groups showed statistical differences at all tested frequencies , and in a number of animals it was not possible to record a post - treatment dpoae response . we used the sprague dawley rat model to compare the efficacy of different dosages of 3 pharmacological agents that have been reported to exert a protective effect against cisplatin ototoxicity in other strains of rats . experimental studies have shown that cisplatin administration causes the formation of reactive oxygen species in the inner ear , leading to lipid peroxydation , triggering of apoptosis and a significant alteration of the auditory function . in the majority of studies the abr has been utilized to assess the hearing threshold of the tested animals . in this study the efficacy of the tested pharmacological agents was additionally evaluated with distortion product otoacoustic emissions . the abr data from the d - met group confirm the previous findings of campbell in the wister rat , suggesting that dosages of 300400 mg / kg have the potential to protect the inner ear . in terms of performance , the higher dosages ( 350 and 400 mg / kg ) showed better auditory preservation across all the tested abr frequencies . of the 3 tested protocols , only the moderate dose ( 350 mg / kg ) group generated responses with no significant pre - post differences , and in light of this only the moderate dose can be considered as a candidate for auditory preservation . the data from the d - met dpoae responses suggest that increasing the amount of otoprotector does not necessarily increase the index of auditory preservation . the abr data from the nac group showed a partial auditory preservation in the 275 and 375 mg / kg groups and a complete preservation in the 475 mg / kg group . these findings are similar to those of dickey in which a pre - treatment injection of 400 mg / kg nac was able to prevent ototoxicity at a considerably lower dose of cisplatin ( 6 mg / kg ) in the long - evans rat . the dpoae responses from the nac treated animals showed significant amplitude changes across many frequencies . in terms of performance , the best results were observed in the 375 mg / kg group . by integrating the information from the abr and dpoae data , we conclude that the nac protocols do not seem to offer complete auditory preservation in dosages of up to 400 mg / kg . an explanation for the observed discrepancy between the abr and dpoae findings in the d - met and nac groups can not be given only from the electrophysiological findings ; however , it is reasonable to speculate that the effect on the dpoae recordings could be a result of minor loss of outer hair cells in the treated animals . considering the time window of observation ( 96 hours ) the dpoae data might indicate cell death of the outer hair cell population . results of the present study clearly show that ebselen pre - treatment did not protect the cochlea in the sprague dawley albino male rat . this finding is contradictory to earlier reports , showing amelioration of cisplatin ototoxicity in the fischer 344 rat given a 16 mg / kg dose of cisplatin . moreover , rybak showed that the hearing of the wistar rat was protected by ebselen ( 16 mg / kg ) in connection to cisplatin treatment . have shown that in the fischer 344 rat , 8 mg / kg of ebselen administered orally with 8 mg / kg of allopurinol offers protection similar to a single 16 mg / kg ebselen dose . the abr and dpoae data from this study show that in the sprague dawley rat there is no significant otoprotection from an i.p . administration of ebselen in dosages of up to 12 mg / kg . the maximum ebselen dosage used in this study is lower than the dosages reported in the literature ( 16 mg / kg ) to compensate for the lower cisplatin dosage employed ( 14 vs. 16 mg / kg ) . in addition , the pattern of the abr / dpoae data from the ebselen - treated animals was different than the data from the other 2 protectors , even at dosages that did not offer protection at all frequencies . non - optimized dosages of d - met or nac presented some protection in 1 of the tested abr / dpoae frequencies , but this was not the case for ebselen . the reasons behind this objective are rooted in the clinical environment , where it is essential to obtain maximum chemotherapeutic effect while preserving the hearing of patients treated with cisplatin - based chemotherapy . in the sprague - dawley albino male rats . pre - administration of the thiol - containing antioxidant was used allow the protective agent to accumulate in the inner ear before cisplatin reached the target cells . no information can be found in the literature on the cochlear kinetics of the used otoprotectors after the i.v . thio - sulfate , another thiol - containing antioxidant , is readily transported to the inner ear and reaches its peak concentration in scala tympani perilymph within 10 minutes after an i.v . the terminal half - life of d - met in scala tympani perilymph has been estimated to 0.6 hour . the abr data show that animals given 350 and 400 mg / kg of d - met and 475 mg / kg of nac presented significantly better auditory preservation than the other groups . the dpoae data show that only animals receiving 350 mg / kg of d - met presented no significant differences between the pre and the post recordings . combining this information with the abr data , it can be concluded that only d - met shows a complete auditory preservation 96 hours after cisplatin administration . findings from ebselen pre - treated sprague - dawley albino male rats demonstrate that ebselen dosages up to 12 mg / kg given by i.p .

herpesviruses are frequently found in humans , although their prevalence significantly varies depending on ethnicity , sex , and geographical location of individuals , among others [ 15 ] . currently , eight herpesviridae family members are known to infect humans : herpes simplex viruses ( hsv ) -1 and -2 ( hsv-1 , hhv-1 and hsv-2 , hhv-2 , resp . ) , varicella zoster virus ( vzv , hhv-3 ) , epstein barr ( ebv , hhv-4 ) , cytomegalovirus ( cmv , hhv-5 ) , human herpesvirus 6 ( hhv-6 ) , human herpesvirus 7 ( hhv-7 ) , and kaposi sarcoma - associated virus ( ksv or hhv-8 ) . all herpesviruses harbor large genomes encoding > 70 genes and share the capacity to establish lifelong persistent infections in the host ( and ncbi ) . human infection with herpes simplex viruses ( hsvs ) traces far back , even before the intercontinental migration of our ancestors , as proposed by recent phylogenetic analyses . symptomatic manifestations of hsvs have been described as early as 400 bc and these viruses are often considered the oldest viruses to be studied in the history of science . while hsv-1 is estimated to infect up to one - third of the world population , hsv-2 infects nearly 500 million people around the globe with more than 20 million new cases occurring every year . importantly , hsv-1 is the foremost important cause of infectious blindness in developed countries and has gained importance in primary genital infection , surpassing in many cases hsv-2 [ 922 ] . nevertheless , because hsv-2 recurs significantly more often than hsv-1 in the genitalia , hsv-2 remains overall the most frequent cause of genital ulcers worldwide [ 2326 ] . it is important to bear in mind that hsv-1 and hsv-2 also produce several other pathological conditions , such as encephalitis , conjunctivitis , zosteriform skin lesions , pneumonia , and systemic infections that compromise vital organs . an important concern regarding genital infection with hsv is its association with increased hiv infection . indeed , genital infection with hsv has been suggested to increase up to 3 - 4 times the susceptibility of acquiring hiv [ 2729 ] , which has been proposed to be mediated , at least in part by soluble mediators at the infection site [ 30 , 31 ] . furthermore , individuals coinfected with hsv and hiv shed significantly more these viruses than individuals with single viral infections [ 3234 ] . important efforts have been invested in the past 20 years on the development of a vaccine against hsvs . however , potential vaccine formulations that have reached the clinic have proven ineffective at preventing infection or reducing virus shedding [ 35 , 36 ] . discouraging results derived from the latest hsv-2 vaccine clinical trial , which used a viral subunit formulation , have led to new debates in the field and rethinking on the role of neutralizing antibodies in protecting against hsv-2 , as well as the need for correlates of protection [ 3739 ] . indeed , somewhat unexpected results were obtained with a subunit vaccine consisting of hsv-2 glycoprotein d ( gd ) plus an adjuvant , which was found to be more efficacious against hsv-1 than against hsv-2-induced genital disease [ 40 , 41 ] . again , these data are leading to new paradigm shifts in the field that hopefully will translate into novel vaccine approaches that could eventually reach the clinic . the lack of an effective vaccine against hsvs has flourished onto the development of novel microbicides against these viruses . hsv establishes a lifelong infection in the host by infecting neurons and persisting latently inside these cells [ 42 , 43 ] . because sensorial nervous termini innervate the skin and mucosae , infections at these sites with hsvs can lead to neuron infection with a significantly high frequency [ 44 , 45 ] . indeed , hsvs can readily gain access to neurons somewhat early after infecting epithelial cells , because these cells interact closely . nevertheless , to restrict virus access to neurons and other tissues , the host has evolved an arsenal of antimicrobial determinants that aim at blocking infection , progression of infection , and microbe replication . however , as masters of immune evasion , hsvs encode molecular determinants that promote their stealth and overcome host defenses by overriding several of the antiviral elements of the host . herpes simplex viruses are enveloped viruses with numerous proteins and glycoproteins embedded on their exterior ; whether 11 of the viral glycoproteins encoded by the viral genome are present on the virion surface remains to be thoroughly defined [ 46 , 47 ] . nevertheless , at least five viral glycoproteins have been implicated in viral entry : glycoprotein b ( gb ) , gc , gd , gh , and gl [ 48 , 49 ] . gb acts both as a viral attachment protein and fusion protein by binding to heparan sulfates ( hs ) on the surface of susceptible host cells and also is known to bind to paired immunoglobulin - like type 2 receptor ( pilr ) alpha [ 51 , 52 ] . a similar function has been described for gc in virus attachment , although only for hsv-1 . after gb - mediated attachment , gd binds to either of its receptors : nectin-1 ( pvrl1 ; poliovirus receptor - related 1 ) expressed on the surface of most host cells or alternatively hvem ( herpesvirus entry mediator , tnfrsf14 ) , mainly expressed on immune cells [ 54 , 55 ] . furthermore , 3-o - sulfate hs has also been suggested as a potential receptor for gd , although its physiological relevance requires additional research . binding of gd to its receptors is thought to induce conformational changes leading to the functional activation of a complex formed by gh / gl . activated gh / gl complex would in turn then promote changes in gb that activate the fusogenic properties of this protein and mediate the fusion of viral and host cell membranes [ 58 , 59 ] . as an alternative pathway , hsvs can enter cells through endocytic vesicles [ 60 , 61 ] . in both cases , fusion of membranes promotes the entry of the capsid and accompanying viral proteins ( tegument ) into the cytoplasm . the tegument is a complex mesh of > 20 proteins beneath the envelope that wraps the viral capsid and contains molecular determinants that mediate , among others , the inhibition of cellular translation and apoptosis [ 62 , 63 ] . once released into the cytoplasm , the capsid associates with microtubules through two tegument proteins vp1 - 2 ( encoded by ul36 ) and ul37 and then travels to the outer nuclear membrane to bind to the host nuclear pore complex ( npc ) to release the viral dna into the nucleus [ 62 , 63 ] . nucleoporin nup358 has been associated with this process by docking vp1 - 2 onto the nuclear pore complex and facilitating the release of viral dna into the nucleus through this macromolecular complex . once released into the nucleus , the viral dna is transcribed by means of the host rna - polymerase ii activity [ 65 , 66 ] . however , not all hsv genes are expressed synchronously but instead in four consecutive rounds of transcription . first , immediate early genes ( alpha ) are transcribed , many of which encode for proteins contributing to immune evasion and work as factors controlling cell translation . then , follows the transcription of early genes ( beta ) that are required for dna replication . finally , early late and late genes ( gamma-1 and gamma-2 ) are transcribed , which mainly encode for structural components of virions , such as capsid , tegument , and surface proteins [ 69 , 70 ] . these proteins can work as well as important immune evasion determinants ( see below ) . to generate new virions , capsid proteins migrate from the cytoplasm into the nucleus to assemble with viral dna and acquire at this location a layer of tegument proteins . unlike other viruses , hsvs do not alter nuclear pores on exit but rather undergo envelopment in the inner nuclear membrane to form an enveloped capsid . the capsid then travels through the perinuclear space and immediately fuses with the outer nuclear membrane thanks to glycoproteins gb and gh , exposing a tegument - recovered capsid into the cytoplasm . once in the cytoplasm , the capsid is further coated with additional tegument proteins and is once again enveloped in the trans - golgi network . from here , virions are exported in vesicles to the cell surface and secreted . although host tetherin ( bst-2 or cd317 ) has been shown to block the release of certain enveloped viruses from the cell surface , the hsv protein vhs ( virion host shutoff protein , ul41 ) can counteract the antiviral function of this protein by depleting it . noteworthy , hsvs can also propagate directly onto adjacent cells through cell - cell interactions . in these circumstances , this type of infection is used by hsvs to infect t cells , which has been shown to occur through infected fibroblasts in vitro [ 75 , 76 ] . this type of infection is mediated through a process called virological synapse and provides the virus a safe haven from neutralization by antibodies or complement ( see below ) [ 77 , 78 ] . immune and nonimmune host cells express an array of surface and intracellular receptors intended to sense microbial elements and initiating local and systemic antimicrobial responses . such receptors , termed pathogen recognition receptors ( prrs ) , recognize pathogen associated molecular patterns ( pamps ) , which consist among others of microbe - derived molecules , such as lipids , proteins and nucleic acids . an important family of prrs is toll - like receptors ( tlrs ) , which upon binding with microbe elements lead to intracellular signaling cascades that promote early antiviral cellular responses and the secretion of soluble mediators that activate infected and noninfected neighboring cells , as well as the immune system [ 8084 ] . it has been shown that hsvs induce the activation of tlr2 in primary vaginal epithelial cells and also immune cells , such as dendritic cells ( dcs ) . interestingly , it has been suggested that in keratinocytes , neural cells , and epithelial cells tlr2-mediated effects after virus infection require the cooperation of 3-integrin , likely due to the binding of the hsv gh / gl complex to this integrin , leading to nf-b activation , interferon production , and il-10 secretion ( figure 1 ) [ 85 , 86 ] . in dcs , the activation of tlr2 induces a cell response that leads to the downstream activation of nf-b and the transcription of immunomodulatory cytokines , such as il-6 , il-8 , il-10 , il-12 , and tnf- . another study with dcs also showed that hsv recognition by tlr2 promoted il-6 and il-12 secretion and proposed that this cytokine outcome was mediated , at least in part , by tlr9 modulation , suggesting a previously uncharacterized mechanism for sequential recognition of viruses via tlr2 through tlr9 . consistent with this notion , tlr2 knockout mice secrete low levels of mcp-1 , a chemokine generally induced after tlr9 engagement . importantly , tlr2 knockout mice display prolonged survival after hsv infection , as compared to wild - type and tlr4 knockout mice . despite reduced mortality , viral loads remained similar in tlr2-knockout and wild - type animals . on the other hand , microglia from tlr2 mice display delayed and attenuated production of reactive oxygen species ( ros ) following viral infection and suffer lesser neuronal oxidative damage in mixed neural cell cultures , as compared to hsv - infected cells from wild - type animals . tlr3 has also been described to play a role in hsv infection , especially for neurons and during viral brain infection . for instance , it has been shown that tlr3 deficiencies ( tlr3 ) render astrocytes permissive to hsv infection , facilitating the establishment of cns infection in animals . consistently , it was shown that tlr3 expressed in astrocytes provided early control of hsv infection after viral entry into the central nervous system and induced type - i ifn responses in these cells . other studies have shown that astrocyte infection with hsv leads to tlr3 engagement and nf-b activation , upregulating the expression of tnf- and il-6 with antiviral functions attributed to these two molecules ( figure 1 ) . on the other hand , studies performed in humans carrying mutations that negatively modulate tlr3-mediated immunity have shown that these individuals are more prone to hsv encephalitis [ 9496 ] . consistent with these findings , a study with ex vivo differentiated neurons , astrocytes and oligodendrocytes derived from pluripotent stem cells from individuals with tlr3 deficiencies were shown to be more susceptible to hsv infection in vitro than control cells and displayed compromised interferon secretion . interestingly , these effects depended significantly on the cell types analyzed . on the other hand , mice pretreated either intravaginally or intraperitoneally with agonists for tlr3 , such as polyi : c , suffer significantly less virus burden upon intravaginal viral challenge than nontreated animals , suggesting that activating this pathway would play favorable roles against hsv infection ( figure 1 ) . as with tlr3 , pretreating animals with tlr7 agonists , such as imiquimod , has also been shown to significantly reduce hsv burden in the genital tract after viral infection ( figure 1 ) . because of these results , imiquimod has been tested in humans , particularly against hsv strains that are resistant to acyclovir in immunocompromised patients , with favorable results [ 100102 ] . however , it is important to note that another study found that imiquimod produced ifn - independent anti - hsv effects in nonimmune cells , which was independent of tlr signaling and ifn production , suggesting that tlr7 is likely not the only activation pathway involved in the favorable results observed against hsv in other studies . tlr9 has also been shown to play roles in hsv infection , although similar to tlr3 and tlr7 , because tlr9 agonists can positively influence the antiviral response against this pathogen . indeed , mice pretreated intranasally with tlr9 agonists , such as cpg - oligodeoxynucleotides ( cpg - odns ) , show reduced secretion of inflammatory cytokines , such as ccl2 , il-6 , and ccl5 , and reduced viral loads in the brain , resulting in mild encephalitis and increased survival rates , as compared to nontreated mice ( figure 1 ) . additionally , treatment with cpg - odn containing unmethylated cpg provides protection against lethal vaginal challenge with hsv that was probably mediated by an intricate crosstalk between plasmacytoid dcs ( pdcs ) and vaginal stromal cells , as well as type - i ifns [ 104106 ] . similar to the findings described with tlr3 and tlr7 , these results suggest that modulating tlr9 signaling could be a promising strategy for limiting hsv infection in the host , although results from another group suggest that the antiviral effects mediated by tlr9 antagonists might not necessarily be mediated uniquely by intracellular events linked to tlr9 signaling . nevertheless , the results obtained with tlr9 agonists suggest that activating this tlr might be a useful strategy for controlling pathological responses induced by hsvs . when combined with antivirals , such as acyclovir or anti - inflammatory molecules , this strategy could improve current therapies against these viruses [ 103 , 109 ] . importantly , hsvs also induce the activation of non - tlr sensors in target cells . namely , primary vaginal epithelial cells display increased activation of dna sensors , such as dai ( dna - dependent activator of interferon ) and ifi16 ( interferon - inducible 16 ) , which trigger the secretion of il-6 ( figure 1 ) . another non - tlr host sensor includes v3-integrin , mentioned above with tlr2 , which was also recently described to function as a major sensor of hsvs per se and activator of innate immunity by relocating the viruses ' nectin-1 receptor to cholesterol - rich microdomains , thus , enabling virus uptake into dynamin 2-dependent acidic endosomes . v3-integrin interacts with hsvs gh / gl complexes and is thought to signal at least through two pathways , one mediated by tlr2 with the activation of nf-b and consequently induction of type - i interferons and another involving sarcoma- ( src- ) spleen tyrosine kinase- ( syk- ) caspase recruitment domain - containing protein 9- ( card9- ) trif ( tir - domain - containing adapter - inducing interferon- ) , which affects interferon regulatory factor 3 ( irf3 ) and irf7 ( figure 1 ) [ 85 , 86 ] . importantly , the hsv viral protein icp0 can counteract these v3-integrin signaling pathways to impair sensing of hsvs by infected cells . a recent study suggests that tlr signaling via myd88 and trif is expendable for controlling hsv infection and spread . indeed , myd88 , trif , and myd88-trif double knockout mice displayed similar levels of hsv replication , when compared to wild - type mice , although this particular study was focused on hsv corneal infection . importantly , the dna sensor ifi-16/p204 was identified here to be key for the activation of irf3 and ifn- production for viral containment . consistently , silencing the genes that encode for ifi16/p204 inhibits the activation of irf3 and nf-b in response to hsv dna . furthermore , a recent study showed that ifi16 depletion was associated with increased hsv yield , while its overexpression reduced the amount of virus obtained in cell cultures . chip assays found that ifi16 binds to hsv promoters and that cells devoid of this protein display increased amounts of host proteins that promote viral gene transcription at these locations . these findings suggest that ifi16 possesses antiviral functions and negatively modulates hsv transcription after binding to viral dna . another intracellular , non - tlr receptor involved in the detection of hsv determinants is cyclic guanosine monophosphate - adenosine monophosphate ( cgamp ) synthase ( cgas ) , a novel cytosolic dna sensor ( figure 1 ) . this protein has been shown to detect hsv dna leading to type - i ifn ( ifn - i ) production in fibroblasts , macrophages , and dendritic cells and mice deficient for cgas succumb to death after infection with this virus . intracellular nucleic acid sensors rig - i and mda5 ( retinoic acid - inducible gene 1 and melanoma differentiation - associated protein 5 ) also play antiviral roles in infected cells , yet vhs can selectively inhibit the expression of these molecules and prevent downstream irf3 dimerization , as well as the translocation of this complex into the nucleus ( figure 1 ) . by doing so , vhs can block signaling mediated through non - tlr pathways . another mechanism by which host cells can sense and initiate antiviral responses is through the activation of the inflammasome . the inflammasome is a multiprotein complex involved in translating pathogen recognition events into the secretion of inflammatory molecules , such as il-1. relevant inflammasome sensors include nlrp3 and aim2 in the cytoplasm of cells and the nuclear sensor ifi16 , discussed above . recent studies have shown that hsv can induce early activation of the inflammasome and then , later on , inhibit its function during active infection . indeed , fibroblasts infected with hsv display activated ifi16 and nlrp3 and secrete il-1 early after infection , although later on ifi16 is targeted to the proteasome by icp0 , likely releasing the break that ifi16 imposes on the transcription of hsv genes [ 114 , 117 ] . subsequently , nlrp3 and aim2 remain unaltered in cells with an inhibited inflammasome and little secretion of mature il-1 . sensing of microbial components by immune and nonimmune cells can lead to cell apoptosis , as a host strategy to block virus replication and spread within cells . importantly , hsvs encode viral determinants that block or delay the onset of apoptosis in infected cells , likely as a mechanism to extend the viability of its substrate for replication . this process has been proposed to be mediated by viral glycoproteins such as gj and gd , as virus mutants lacking each one of these proteins initiated apoptotic cascades in epithelial cells ( figure 2(a ) ) . furthermore , the viral proteins icp10pk and ul14 have also been shown to prevent apoptotic processes triggered in neurons and epithelial cells after viral infection ( figure 2(a ) ) [ 119121 ] . finally , us3 an hsv protein kinase conserved throughout alphaherpesviruses has also been shown to play a key role in blocking apoptosis induced by viral gene products and exogenous agents in epithelial cells . the antiapoptotic effects of us3 would be mediated through its interaction with programmed cell death protein 4 ( pdcd4 ) , which is retained in the nucleus of infected cells ( figure 2(a ) ) . nevertheless , other hsv proteins have been proposed to induce apoptosis and necrosis in host cells . for instance , hsv has been shown to induce necrosis in mouse fibroblast cells ( l929 cells ) mediated by the interaction between the viral ribonucleotide reductase large subunit icp6 and rip3 ( receptor - interacting kinase 3 ) through rhim domains , which activate mlkl ( mixed lineage kinase domain - like protein ) . consistently , an hsv icp6 deletion mutant failed to cause effective necrosis of hsv - infected cells and mice lacking rip3 exhibited severely impaired control of hsv replication and pathogenesis , highlighting the importance of the latter in limiting virus pathology . noteworthily , another study showed that early after hsv infection , natural killer cells ( nk cells ) suffer apoptosis through fas / fasl when these cells interact with hsv - infected macrophages ( figure 2(b ) ) . similarly , hsv infection of dendritic cells induces apoptosis early after virus entry , particularly after the release of immunomodulatory cytokines [ 125 , 126 ] , and the viral protein 34.5 can interfere with dc autophagosome maturation , which is thought to play antiviral functions in these cells by degrading virus determinants ( figure 2(b ) ) [ 127 , 128 ] . a similar role for autophagy has been proposed in the context of hsv infection in neurons as an alternative to ifn responses , which would likely result in the death of these cells or detrimental outcomes for the host . indeed , neurons from dorsal root ganglia require autophagy to limit hsv replication in vivo and in vitro . antiviral functions in host cells are also mediated by protein kinase r ( pkr ) , which phosphorylates the translation initiation factor eif2a as a mechanism to inhibit the translation of rna messengers upon viral infections . importantly , this host protein has been shown to play a key role in controlling hsv replication in vitro and in vivo [ 129 , 130 ] . for instance , viral 34.5 and us11 have been proposed to inhibit the activity of pkr to promote the translation of viral proteins ( figure 3 ) [ 131 , 132 ] . furthermore , hsvs have evolved determinants that preferentially block the translation of host molecules over viral genes , in such a way to impair their antiviral activity . indeed , hsvs vhs protein can mediate the degradation of host messenger rna through their ribonuclease activity ( figure 3 ) . the spatial - temporal delivery of vhs has evolved in such a way to display optimal activity early after infection for hampering host mrna transcription and not to alter viral mrnas transcribed later on . early sensing of viral determinants by host prrs will generally lead to the activation of interferon pathways that aim at impairing virus replication and its shedding within the host . although cells in the genital tract infected with hsv-2 produce interferons in response to this virus , the magnitude of this response is generally hampered in the infected tissue , suggesting that these molecules likely play favorable antiviral roles . indeed , biopsies obtained from individuals infected with hsv show extremely low levels of type - i ifns ( ifn- and ifn- ) , despite the presence of a large number of cells capable of synthesizing these mediators , which suggests alterations in the host interferon response during hsv infection . consistent with this notion , type - i ifn receptor ( ifnar ) knockout mice inoculated in the footpads with hsv manifest systemic viral infections that affect the lungs , liver , and spleens , although disease is nonlethal . interference with host interferon pathways would be mediated , at least in part by the early viral protein icp0 , which can impair irf3 function and block the transcription of genes regulated by this transcription factor . additionally , hsv icp27 also inhibits type - i ifn signaling and interferes with nuclear accumulation of stat-1 ( figure 3 ) . furthermore , the hsv ser / thr kinase us3 can hamper ifn- production by hyperphosphorylating irf3 and by blocking the dimerization and nuclear translocation of this factor ( figure 3 ) . similarly , the tegument protein vp16 can also abrogate ifn- expression by inhibiting nf-b and irf3 activation by impairing the recruitment of the coactivator cbp , without interfering with irf3 dimerization , nuclear translocation , or its dna binding activity ( figure 3 ) . yet , another mechanism by which hsv inhibits ifn- expression is through the deubiquitination of traf3 by the viral ubiquitin - specific protease ul36 , which inhibits stimuli - induced irf3 dimerization , promoter activation , and the transcription of ifn- ( figure 3 ) . as noted , hsvs have evolved redundant and nonredundant mechanisms to specifically impair the function of host molecules that are key for the expression of antiviral molecules , namely , interferons . the importance of ifns in controlling infection by hsvs is highlighted by the fact that the frequency of genital herpetic recurrences can be reduced in patients by applying topical ifn- , which also reduces viral dissemination . moreover , the recently described interferon ifn- , characterized as a type - i ifn constitutively expressed by epithelial cells in the female and male reproductive tract , is proposed to be a potent antiviral host mediator that likely contributes to control of hsv infection [ 144 , 145 ] . however , the exact mechanism by which ifn- exerts its anti - hsv effects remains to be determined . importantly , expression of ifn- varies with the female hormonal cycle and seems to be limited to cells belonging to reproductive organs [ 145 , 146 ] . after hsv has blocked immediate host antiviral responses , which rely on the sensing of microbe elements and early interferon responses , cell damage resulting from virus replication likely spreads virus - elicited danger signals and damage - associated molecular patterns ( damps ) onto other noninfected cells . these neighboring cells , as well as patrolling immune cells , may sense these danger elements and initiate cytokine and chemokine responses that will modulate the milieu and other immune components [ 126 , 147 , 148 ] . whether the soluble mediators produced in response to these danger signals or hsv itself promote the clearance of the virus or favor its persistence and spread in the host is largely unclear . because cytokine secretion is generally dependent on the canonical activation of nf-b , hsvs have evolved several molecular mechanisms to modulate the activity of this transcription factor . a recent report showed that the viral dna polymerase processivity factor ul42 interacts with p65/rela and p50/nf-b1 to block the translocation of nf-b to the nucleus in response to stimuli , such as tnf- . consistently , another study found that hsv icp0 inhibits tnf--induced nf-b activation , interacting similarly with p65/rela and p50/nf-b1 . us3 has also been shown to significantly inhibit nf-b activation and decrease the expression of inflammatory chemokines , such as il-8 . however , other hsv proteins , such as tegument protein ul37 , have been shown to promote nf-b activation and il-8 secretion in keratinocytes . activation of nf-b after cell infection has also been reported to facilitate viral replication [ 153 , 154 ] . taken together , hsvs have evolved strategies to both block and promote the activation of nf-b within infected cells . whether these opposing effects depend on the cell types targeted by these viruses or different stages of the infectious cycle requires further study . nevertheless , these findings highlight the importance of nf-b modulation by hsvs after infection . despite interference with nf-b activity , cells infected with hsv nonetheless secrete numerous modulatory cytokines and chemokines after infection or at the site of inflammation . for instance , hsv has been shown to induce the secretion of ccl2 , il-8 , il-6 , and tnf- in primary endometrial genital epithelial cells . in vivo importantly , this chemokine and cxcl10 have been shown to play important roles against hsv in cns infection in the mouse model , likely by recruiting nk and cytotoxic t cells to the infected tissue . similarly , a recent study proposed that cxcl10 is needed for establishing protective immunity against hsv-2 genital infection after vaccination with an attenuated hsv strain . ccl2 induced upon hsv infection has been attributed a favorable role in corneal infection in the mouse model . indeed , ccl2 mice were unable to contain the virus and failed to recruit inflammatory monocytes to the infection site . furthermore , ccl2 expression driven by ifi16 recognition of hsv has been described to facilitate the recruitment of inflammatory monocytes to the infection site , as silencing of p204/ifi-16 resulted in the loss of ccl2 production and significantly more hsv shedding . as indicated above , another cytokine induced after noteworthily , a protective role has been attributed to this cytokine in microglia , likely through downstream signaling of signal transducer and activator of transcription 3 ( stat3 ) , although the precise mechanism leading to its protective role is unclear [ 160 , 161 ] . mast cells have also been shown to secrete il-6 early after hsv infection , as well as tnf- , yet these cytokines were not induced directly by hsv in this study but depended on supernatants from hsv - infected keratinocytes and the il-33 receptor on the former cells . importantly , mice lacking tnf- or il-6 succumbed to death , consistent with a protective role for these cytokines . contrarily , a recent study suggested that treating mice with anti - tnf- in combination with the antiviral valacyclovir could significantly improve the prognosis of encephalitis caused by hsv . one aspect that has brought important attention onto cytokines and chemokines produced after hsv infection is that some of the molecules secreted in the genital tract can favor host infection by other sexually transmitted pathogens , such as the human immunodeficiency virus ( hiv ) . indeed , infection with hsv-2 increases 3 - 4 times host susceptibility of acquiring hiv and , furthermore , coinfection increases the shedding of both viruses [ 2729 , 163 ] . the increased susceptibility to acquire hiv after hsv-2 infection has been suggested to result , within others , by an increased recruitment of target cells for hiv , such as dendritic cells and t cells to the site of infection [ 164 , 165 ] , increased expression of hiv - receptor molecules at the surface or specific cell populations [ 166 , 167 ] , and reduced expression of cell surface molecules that actually promote the capture and degradation of hiv . furthermore , immune cells such as dendritic cells infected with hsv have been shown to produce soluble mediators that promote the reactivation of hiv from cells latently infected with the latter virus . regretfully , the identities of the soluble molecules that account for the observed effect have not been identified so far . besides upregulating the expression of certain cytokines and chemokines , hsvs can also reduce the expression of certain antiviral molecules , such as the secreted leucocyte protease inhibitor ( slpi ) . indeed , hsv - infected cells secrete less slpi , which reduces the infectivity of hsv in vitro . furthermore , a decrease in the expression of slpi would likely promote an increase in the secretion of proinflammatory cytokines , which are associated with exacerbated damage to the infected tissues . nevertheless , virus - mediated inhibition of other chemokines could favor the host , such as cxcl2 which is secreted by monocytes in response to hsv which is known to recruit neutrophils that elicit damaging inflammatory immune responses to host cells and tissues , namely , neurons . despite the fact that hsvs have been extensively studied , it is surprising to note how little we know about the contribution of cytokines and chemokines induced upon infection by this virus to infection and pathology . cytokines and chemokines induced by hsv infection will likely play different roles for the host , either favorable or antagonizing , depending on the tissue infected , whether it is skin , genitalia , eyes , or the central nervous system . furthermore , differences in the nature and amounts of the cytokines and chemokines secreted upon viral infection , as well as the roles of these molecules on virus clearance and disease , will likely depend on whether infection is mediated by hsv-1 or hsv-2 . high throughput techniques such as multiplex cytokine arrays and the availability of numerous knockout mice for these molecules should provide new insights and valuable information on the role of these soluble mediators in hsv infection in the near future . innate immunity has evolved soluble components and specialized cells to block microbe infection , replication , and shedding . the complement is composed of serum proteins that , once triggered by microbial determinants or antibody bound to ligands , interact with each other in a cascade of events that lead to the damaging of the surfaces of the pathogen or cells infected with the microbe . however , hsvs have evolved molecular determinants to interfere with the function of complement protein c5 and block its downstream activating properties ; this process is mediated by glycoprotein c [ 78 , 171 ] . by doing so , the virus likely extends its lifespan in the serum and that of the cells it infects . nk cells play important roles against several viral pathogens ; however their role in hsv infection is frequently debated . although some studies propose key roles for these cells , others have underestimated their importance at controlling hsv infection [ 172 , 173 ] . hsv can directly activate nk cells through tlr2 ; whether this interaction promotes viral clearance or not in vivo is still unclear . hsv can also decrease the expression of nk - activating ligands such as mica ( mhc class i polypeptide - related sequence a ) on the surface of infected cells , thus interfering with the effector activity of these cells . this process would be mediated by a late hsv gene product , which would mask , internalize , or retain mica intracellularly [ 175 , 176 ] . natural killer t ( inkt ) cells are cd1d - restricted t cells that express invariant tcr chains , as well as nk surface markers , and are specialized in recognizing polar lipids presented on the surface of cd1d molecules . although variations can be observed in the amount of inkt cells present after hsv infection , changes in the number of cells and expression of markers on their surface are somewhat discrete when compared to patients in the steady state , suggesting potential modulation of these cells by hsvs . furthermore , infection with hsv has been described to negatively modulate the activity of nkt cells by simply directing cd1d molecules from the cell surface of infected cells into intracellular compartments , thus blocking antigen presentation [ 179 , 180 ] . world prevalence for hsvs is a truthful testimony of the success of these viruses in establishing latent infection in the host . successful infection with hsvs is likely the result of a wide array of viral determinants encoded by these viruses with the capacity to interfere with multiple host factors intended to control early infection by pathogenic microbes . indeed , hsvs effectively block early cellular antiviral mechanisms by extending the survival of cells that serve as substrates , hence favoring virus production and killing cells that initiate and modulate effective antiviral immune responses , such as dcs . additionally , these viruses promote their stealth by interfering with their sensing by infected cells and by mounting somewhat modest interferon and cytokine responses that favor their replication and shedding . these phenomena will ultimately allow these viruses to reach cells needed for establishing latency : neurons . noteworthily , important progress has been made in the last years in identifying early antiviral components elicited and blocked by hsvs . these studies will hopefully lead to the identification and development of drugs that specifically interfere with viral processes . noteworthily , findings , such as those related to the activation of particular tlrs that favor host responses against these viruses , will undoubtedly contribute to the development of novel antiviral therapies . indeed , potentiating early antiviral functions in the host before exposure could be as effective as novel anti - hsv microbicides currently under development , while an effective vaccine against these viruses reaches the clinic .

besides overcoming physical constraints , such as extreme temperatures , reduced humidity , elevated pressure , and natural predators , human pathogens further need to overcome an arsenal of antimicrobial components evolved by the host to limit infection , replication and optimally , reinfection . herpes simplex virus-1 ( hsv-1 ) and herpes simplex virus-2 ( hsv-2 ) infect humans at a high frequency and persist within the host for life by establishing latency in neurons . to gain access to these cells , herpes simplex viruses ( hsvs ) must replicate and block immediate host antiviral responses elicited by epithelial cells and innate immune components early after infection . during these processes , infected and noninfected neighboring cells , as well as tissue - resident and patrolling immune cells , will sense viral components and cell - associated danger signals and secrete soluble mediators . while type - i interferons aim at limiting virus spread , cytokines and chemokines will modulate resident and incoming immune cells . in this paper , we discuss recent findings relative to the early steps taking place during hsv infection and replication . further , we discuss how hsvs evade detection by host cells and the molecular mechanisms evolved by these viruses to circumvent early antiviral mechanisms , ultimately leading to neuron infection and the establishment of latency .

1. Introduction 2. HSV Infectious Cycle 3. Evasion of HSV Sensing by Host Receptors 4. Modulation of Cell Viability and Early Antiviral Response 5. Secretion of Immunomodulatory Mediators Early after HSV Infection 6. HSV Interferes with Innate Immune Functions 7. Concluding Remarks

currently , eight herpesviridae family members are known to infect humans : herpes simplex viruses ( hsv ) -1 and -2 ( hsv-1 , hhv-1 and hsv-2 , hhv-2 , resp . ) , varicella zoster virus ( vzv , hhv-3 ) , epstein barr ( ebv , hhv-4 ) , cytomegalovirus ( cmv , hhv-5 ) , human herpesvirus 6 ( hhv-6 ) , human herpesvirus 7 ( hhv-7 ) , and kaposi sarcoma - associated virus ( ksv or hhv-8 ) . human infection with herpes simplex viruses ( hsvs ) traces far back , even before the intercontinental migration of our ancestors , as proposed by recent phylogenetic analyses . symptomatic manifestations of hsvs have been described as early as 400 bc and these viruses are often considered the oldest viruses to be studied in the history of science . it is important to bear in mind that hsv-1 and hsv-2 also produce several other pathological conditions , such as encephalitis , conjunctivitis , zosteriform skin lesions , pneumonia , and systemic infections that compromise vital organs . discouraging results derived from the latest hsv-2 vaccine clinical trial , which used a viral subunit formulation , have led to new debates in the field and rethinking on the role of neutralizing antibodies in protecting against hsv-2 , as well as the need for correlates of protection [ 3739 ] . hsv establishes a lifelong infection in the host by infecting neurons and persisting latently inside these cells [ 42 , 43 ] . because sensorial nervous termini innervate the skin and mucosae , infections at these sites with hsvs can lead to neuron infection with a significantly high frequency [ 44 , 45 ] . indeed , hsvs can readily gain access to neurons somewhat early after infecting epithelial cells , because these cells interact closely . nevertheless , to restrict virus access to neurons and other tissues , the host has evolved an arsenal of antimicrobial determinants that aim at blocking infection , progression of infection , and microbe replication . herpes simplex viruses are enveloped viruses with numerous proteins and glycoproteins embedded on their exterior ; whether 11 of the viral glycoproteins encoded by the viral genome are present on the virion surface remains to be thoroughly defined [ 46 , 47 ] . gb acts both as a viral attachment protein and fusion protein by binding to heparan sulfates ( hs ) on the surface of susceptible host cells and also is known to bind to paired immunoglobulin - like type 2 receptor ( pilr ) alpha [ 51 , 52 ] . after gb - mediated attachment , gd binds to either of its receptors : nectin-1 ( pvrl1 ; poliovirus receptor - related 1 ) expressed on the surface of most host cells or alternatively hvem ( herpesvirus entry mediator , tnfrsf14 ) , mainly expressed on immune cells [ 54 , 55 ] . binding of gd to its receptors is thought to induce conformational changes leading to the functional activation of a complex formed by gh / gl . once released into the cytoplasm , the capsid associates with microtubules through two tegument proteins vp1 - 2 ( encoded by ul36 ) and ul37 and then travels to the outer nuclear membrane to bind to the host nuclear pore complex ( npc ) to release the viral dna into the nucleus [ 62 , 63 ] . finally , early late and late genes ( gamma-1 and gamma-2 ) are transcribed , which mainly encode for structural components of virions , such as capsid , tegument , and surface proteins [ 69 , 70 ] . these proteins can work as well as important immune evasion determinants ( see below ) . such receptors , termed pathogen recognition receptors ( prrs ) , recognize pathogen associated molecular patterns ( pamps ) , which consist among others of microbe - derived molecules , such as lipids , proteins and nucleic acids . an important family of prrs is toll - like receptors ( tlrs ) , which upon binding with microbe elements lead to intracellular signaling cascades that promote early antiviral cellular responses and the secretion of soluble mediators that activate infected and noninfected neighboring cells , as well as the immune system [ 8084 ] . it has been shown that hsvs induce the activation of tlr2 in primary vaginal epithelial cells and also immune cells , such as dendritic cells ( dcs ) . interestingly , it has been suggested that in keratinocytes , neural cells , and epithelial cells tlr2-mediated effects after virus infection require the cooperation of 3-integrin , likely due to the binding of the hsv gh / gl complex to this integrin , leading to nf-b activation , interferon production , and il-10 secretion ( figure 1 ) [ 85 , 86 ] . in dcs , the activation of tlr2 induces a cell response that leads to the downstream activation of nf-b and the transcription of immunomodulatory cytokines , such as il-6 , il-8 , il-10 , il-12 , and tnf- . importantly , tlr2 knockout mice display prolonged survival after hsv infection , as compared to wild - type and tlr4 knockout mice . tlr3 has also been described to play a role in hsv infection , especially for neurons and during viral brain infection . for instance , it has been shown that tlr3 deficiencies ( tlr3 ) render astrocytes permissive to hsv infection , facilitating the establishment of cns infection in animals . consistently , it was shown that tlr3 expressed in astrocytes provided early control of hsv infection after viral entry into the central nervous system and induced type - i ifn responses in these cells . on the other hand , mice pretreated either intravaginally or intraperitoneally with agonists for tlr3 , such as polyi : c , suffer significantly less virus burden upon intravaginal viral challenge than nontreated animals , suggesting that activating this pathway would play favorable roles against hsv infection ( figure 1 ) . tlr9 has also been shown to play roles in hsv infection , although similar to tlr3 and tlr7 , because tlr9 agonists can positively influence the antiviral response against this pathogen . indeed , mice pretreated intranasally with tlr9 agonists , such as cpg - oligodeoxynucleotides ( cpg - odns ) , show reduced secretion of inflammatory cytokines , such as ccl2 , il-6 , and ccl5 , and reduced viral loads in the brain , resulting in mild encephalitis and increased survival rates , as compared to nontreated mice ( figure 1 ) . additionally , treatment with cpg - odn containing unmethylated cpg provides protection against lethal vaginal challenge with hsv that was probably mediated by an intricate crosstalk between plasmacytoid dcs ( pdcs ) and vaginal stromal cells , as well as type - i ifns [ 104106 ] . similar to the findings described with tlr3 and tlr7 , these results suggest that modulating tlr9 signaling could be a promising strategy for limiting hsv infection in the host , although results from another group suggest that the antiviral effects mediated by tlr9 antagonists might not necessarily be mediated uniquely by intracellular events linked to tlr9 signaling . when combined with antivirals , such as acyclovir or anti - inflammatory molecules , this strategy could improve current therapies against these viruses [ 103 , 109 ] . namely , primary vaginal epithelial cells display increased activation of dna sensors , such as dai ( dna - dependent activator of interferon ) and ifi16 ( interferon - inducible 16 ) , which trigger the secretion of il-6 ( figure 1 ) . v3-integrin interacts with hsvs gh / gl complexes and is thought to signal at least through two pathways , one mediated by tlr2 with the activation of nf-b and consequently induction of type - i interferons and another involving sarcoma- ( src- ) spleen tyrosine kinase- ( syk- ) caspase recruitment domain - containing protein 9- ( card9- ) trif ( tir - domain - containing adapter - inducing interferon- ) , which affects interferon regulatory factor 3 ( irf3 ) and irf7 ( figure 1 ) [ 85 , 86 ] . this protein has been shown to detect hsv dna leading to type - i ifn ( ifn - i ) production in fibroblasts , macrophages , and dendritic cells and mice deficient for cgas succumb to death after infection with this virus . intracellular nucleic acid sensors rig - i and mda5 ( retinoic acid - inducible gene 1 and melanoma differentiation - associated protein 5 ) also play antiviral roles in infected cells , yet vhs can selectively inhibit the expression of these molecules and prevent downstream irf3 dimerization , as well as the translocation of this complex into the nucleus ( figure 1 ) . another mechanism by which host cells can sense and initiate antiviral responses is through the activation of the inflammasome . the inflammasome is a multiprotein complex involved in translating pathogen recognition events into the secretion of inflammatory molecules , such as il-1. indeed , fibroblasts infected with hsv display activated ifi16 and nlrp3 and secrete il-1 early after infection , although later on ifi16 is targeted to the proteasome by icp0 , likely releasing the break that ifi16 imposes on the transcription of hsv genes [ 114 , 117 ] . this process has been proposed to be mediated by viral glycoproteins such as gj and gd , as virus mutants lacking each one of these proteins initiated apoptotic cascades in epithelial cells ( figure 2(a ) ) . furthermore , the viral proteins icp10pk and ul14 have also been shown to prevent apoptotic processes triggered in neurons and epithelial cells after viral infection ( figure 2(a ) ) [ 119121 ] . consistently , an hsv icp6 deletion mutant failed to cause effective necrosis of hsv - infected cells and mice lacking rip3 exhibited severely impaired control of hsv replication and pathogenesis , highlighting the importance of the latter in limiting virus pathology . noteworthily , another study showed that early after hsv infection , natural killer cells ( nk cells ) suffer apoptosis through fas / fasl when these cells interact with hsv - infected macrophages ( figure 2(b ) ) . similarly , hsv infection of dendritic cells induces apoptosis early after virus entry , particularly after the release of immunomodulatory cytokines [ 125 , 126 ] , and the viral protein 34.5 can interfere with dc autophagosome maturation , which is thought to play antiviral functions in these cells by degrading virus determinants ( figure 2(b ) ) [ 127 , 128 ] . a similar role for autophagy has been proposed in the context of hsv infection in neurons as an alternative to ifn responses , which would likely result in the death of these cells or detrimental outcomes for the host . the spatial - temporal delivery of vhs has evolved in such a way to display optimal activity early after infection for hampering host mrna transcription and not to alter viral mrnas transcribed later on . early sensing of viral determinants by host prrs will generally lead to the activation of interferon pathways that aim at impairing virus replication and its shedding within the host . indeed , biopsies obtained from individuals infected with hsv show extremely low levels of type - i ifns ( ifn- and ifn- ) , despite the presence of a large number of cells capable of synthesizing these mediators , which suggests alterations in the host interferon response during hsv infection . consistent with this notion , type - i ifn receptor ( ifnar ) knockout mice inoculated in the footpads with hsv manifest systemic viral infections that affect the lungs , liver , and spleens , although disease is nonlethal . interference with host interferon pathways would be mediated , at least in part by the early viral protein icp0 , which can impair irf3 function and block the transcription of genes regulated by this transcription factor . additionally , hsv icp27 also inhibits type - i ifn signaling and interferes with nuclear accumulation of stat-1 ( figure 3 ) . yet , another mechanism by which hsv inhibits ifn- expression is through the deubiquitination of traf3 by the viral ubiquitin - specific protease ul36 , which inhibits stimuli - induced irf3 dimerization , promoter activation , and the transcription of ifn- ( figure 3 ) . moreover , the recently described interferon ifn- , characterized as a type - i ifn constitutively expressed by epithelial cells in the female and male reproductive tract , is proposed to be a potent antiviral host mediator that likely contributes to control of hsv infection [ 144 , 145 ] . after hsv has blocked immediate host antiviral responses , which rely on the sensing of microbe elements and early interferon responses , cell damage resulting from virus replication likely spreads virus - elicited danger signals and damage - associated molecular patterns ( damps ) onto other noninfected cells . these neighboring cells , as well as patrolling immune cells , may sense these danger elements and initiate cytokine and chemokine responses that will modulate the milieu and other immune components [ 126 , 147 , 148 ] . whether the soluble mediators produced in response to these danger signals or hsv itself promote the clearance of the virus or favor its persistence and spread in the host is largely unclear . a recent report showed that the viral dna polymerase processivity factor ul42 interacts with p65/rela and p50/nf-b1 to block the translocation of nf-b to the nucleus in response to stimuli , such as tnf- . whether these opposing effects depend on the cell types targeted by these viruses or different stages of the infectious cycle requires further study . despite interference with nf-b activity , cells infected with hsv nonetheless secrete numerous modulatory cytokines and chemokines after infection or at the site of inflammation . furthermore , ccl2 expression driven by ifi16 recognition of hsv has been described to facilitate the recruitment of inflammatory monocytes to the infection site , as silencing of p204/ifi-16 resulted in the loss of ccl2 production and significantly more hsv shedding . mast cells have also been shown to secrete il-6 early after hsv infection , as well as tnf- , yet these cytokines were not induced directly by hsv in this study but depended on supernatants from hsv - infected keratinocytes and the il-33 receptor on the former cells . one aspect that has brought important attention onto cytokines and chemokines produced after hsv infection is that some of the molecules secreted in the genital tract can favor host infection by other sexually transmitted pathogens , such as the human immunodeficiency virus ( hiv ) . the increased susceptibility to acquire hiv after hsv-2 infection has been suggested to result , within others , by an increased recruitment of target cells for hiv , such as dendritic cells and t cells to the site of infection [ 164 , 165 ] , increased expression of hiv - receptor molecules at the surface or specific cell populations [ 166 , 167 ] , and reduced expression of cell surface molecules that actually promote the capture and degradation of hiv . furthermore , immune cells such as dendritic cells infected with hsv have been shown to produce soluble mediators that promote the reactivation of hiv from cells latently infected with the latter virus . besides upregulating the expression of certain cytokines and chemokines , hsvs can also reduce the expression of certain antiviral molecules , such as the secreted leucocyte protease inhibitor ( slpi ) . nevertheless , virus - mediated inhibition of other chemokines could favor the host , such as cxcl2 which is secreted by monocytes in response to hsv which is known to recruit neutrophils that elicit damaging inflammatory immune responses to host cells and tissues , namely , neurons . despite the fact that hsvs have been extensively studied , it is surprising to note how little we know about the contribution of cytokines and chemokines induced upon infection by this virus to infection and pathology . cytokines and chemokines induced by hsv infection will likely play different roles for the host , either favorable or antagonizing , depending on the tissue infected , whether it is skin , genitalia , eyes , or the central nervous system . furthermore , differences in the nature and amounts of the cytokines and chemokines secreted upon viral infection , as well as the roles of these molecules on virus clearance and disease , will likely depend on whether infection is mediated by hsv-1 or hsv-2 . high throughput techniques such as multiplex cytokine arrays and the availability of numerous knockout mice for these molecules should provide new insights and valuable information on the role of these soluble mediators in hsv infection in the near future . innate immunity has evolved soluble components and specialized cells to block microbe infection , replication , and shedding . although some studies propose key roles for these cells , others have underestimated their importance at controlling hsv infection [ 172 , 173 ] . hsv can also decrease the expression of nk - activating ligands such as mica ( mhc class i polypeptide - related sequence a ) on the surface of infected cells , thus interfering with the effector activity of these cells . natural killer t ( inkt ) cells are cd1d - restricted t cells that express invariant tcr chains , as well as nk surface markers , and are specialized in recognizing polar lipids presented on the surface of cd1d molecules . although variations can be observed in the amount of inkt cells present after hsv infection , changes in the number of cells and expression of markers on their surface are somewhat discrete when compared to patients in the steady state , suggesting potential modulation of these cells by hsvs . world prevalence for hsvs is a truthful testimony of the success of these viruses in establishing latent infection in the host . successful infection with hsvs is likely the result of a wide array of viral determinants encoded by these viruses with the capacity to interfere with multiple host factors intended to control early infection by pathogenic microbes . indeed , hsvs effectively block early cellular antiviral mechanisms by extending the survival of cells that serve as substrates , hence favoring virus production and killing cells that initiate and modulate effective antiviral immune responses , such as dcs . additionally , these viruses promote their stealth by interfering with their sensing by infected cells and by mounting somewhat modest interferon and cytokine responses that favor their replication and shedding . these phenomena will ultimately allow these viruses to reach cells needed for establishing latency : neurons . noteworthily , findings , such as those related to the activation of particular tlrs that favor host responses against these viruses , will undoubtedly contribute to the development of novel antiviral therapies . indeed , potentiating early antiviral functions in the host before exposure could be as effective as novel anti - hsv microbicides currently under development , while an effective vaccine against these viruses reaches the clinic .

several lines of evidence confirm that the adaptive immune response plays a critical role in the effective control and clearance of various kinds of viruses [ 1 , 2 ] . in addition , recent studies have demonstrated that innate immune responses are also important for viral clearance [ 3 , 4 ] . viral infection triggers various innate immune receptors known as pattern recognition receptors ( prrs ) , including toll - like receptor ( tlr ) , nod - like receptor ( nlr ) , and rig - i - like receptor ( rlr ) . the tlr family of prrs was the first to be identified and to date is the most extensively studied . both tlr7 and tlr8 are located in endosomes ; they recognize the genomes of single - stranded rna ( ssrna ) viruses such as influenza virus and human immunodeficiency virus ( hiv ) . recognition by these receptors results in the activation of intracellular signaling by nuclear factor-b ( nf-b ) and mitogen activated protein kinases ( mapks ) through myd88 activation , which in turn leads to the production of proinflammatory cytokines and chemokines and initiates various antiviral responses [ 5 , 6 ] . in the lungs , macrophages as well as dendritic cells ( dcs ) constitute the first line of innate host defenses against viral infection by contributing to the inhibition of viral replication [ 4 , 7 ] . for example , in influenza virus infection , the highly pathogenic h5n1 avian influenza virus and the h1n1 virus identified as the cause of the 2009 pandemic tend to directly infect alveolar macrophages in addition to epithelial cells . pharmacological depletion of macrophages reportedly reduces the rate of survival in animal models of influenza virus pneumonia , suggesting that macrophages are essential for effective immune responses to influenza virus infection . during viral infection and associated tlr7/8 stimulation , macrophages produce various proinflammatory cytokines such as tnf- and il-6 , which leads to an enhanced inflammatory response [ 1012 ] . the production of another major immunomodulatory cytokine , il-10 , is also upregulated in tlr7/8-stimulated macrophages and ssrna virus - infected macrophages . mesenchymal stem cells ( mscs ) are multipotent mesenchymal stromal cells that can be isolated from various tissues . they are capable of differentiating into mesodermal lineage cells such as bone , cartilage , and fat cells . a growing body of evidence indicates that mscs display unique immunomodulatory properties during inflammation in innate immune systems and that these cells are thus promising candidates for use in cell - mediated therapies for inflammatory diseases . for example , msc administration protects against experimental sepsis in mice as a result of enhanced production of the anti - inflammatory cytokine il-10 by macrophages in response to msc - secreted prostaglandin e2 ( pge2 ) . animal model studies have shown that the protective actions of mscs also occur in response to acute lung injury induced by the tlr4 ligand lipopolysaccharide ( lps ) , chronic obstructive pulmonary disease , pulmonary fibrosis , and bacterial pneumonia . in addition , treatment with mscs has been shown to improve lung function in a human ex vivo - perfused tlr4-mediated acute lung injury ( ali ) model . although little is known about the role that mscs may play in the immune response to viruses such as influenza viruses , the mechanism of msc - induced immunomodulation has been studied . both cell - cell contact and soluble factors secreted from mscs , such as pge2 , indoleamine 2,3 dioxygenase ( ido ) , transforming growth factor beta-1 ( tgf-1 ) , hepatocyte growth factor ( hgf ) , inducible nitric - oxide synthase ( inos ) , and heme oxygenase-1 ( ho1 ) , contribute to the modulation of immune responses . in the present study , we hypothesized that mscs and their soluble factors may modulate the cytokine expression induced by the activation of tlr7/8-mediated signaling , a major signaling pathway in innate immunity during ssrna virus infections . to investigate this hypothesis , we focused on macrophages , which are critical components of the innate immune response to viral infection . here , we investigated the effect of mscs and msc - conditioned medium on bone marrow - derived macrophages ( bmdms ) during tlr7/8 stimulation . we found that tlr7/8-mediated expression of tnf- , il-6 , and il-10 is modulated by enhancement of extracellular signal - related kinase ( erk)-mediated signaling and suppression of nf-b - mediated signaling in response to pge2 secreted by mscs . r848 ( tlr7/8 ligand ) and loxoribine ( tlr7 ligand ) were purchased from invivogen ( san diego , ca , usa ) . the prostaglandin receptor ep2 antagonist ah6809 and ep4 antagonist gw 627368x were purchased from cayman ( ann arbor , mi , usa ) . the mek / erk inhibitor u0126 , antibodies against the phosphorylated form of erk , p38 , c - jun n - terminal kinase ( jnk ) , and total inhibitor of kappa b ( ib)- , as well as horseradish peroxidase - conjugated rabbit or mouse igg secondary antibodies were purchased from cell signaling technology ( danvers , ma , usa ) . anti--actin antibody was purchased from sigma - aldrich ( st . louis , mo , usa ) . human mscs were obtained from lonza ( allendale , nj , usa ) , cultured in mesenchymal stem cell basal medium ( mscbm ) supplemented with mscgm singlequots ( lonza ) , and subcultured every 3 - 4 days by using fresh media . the culture medium was collected and centrifuged at 600 g for 10 min . the supernatant was used in further experiments as msc - conditioned medium , while cell - free medium incubated under the same conditions as the msc - conditioned medium served as a control . bone marrow cells were harvested from 8- to 10-week - old male c57/b6 mice ( charles river laboratories japan , inc . , yokohama , japan ) by flushing the femur and tibia with rpmi 1640 medium . recovered cells were then cultured in bone marrow cell medium ( 20% fcs , 30% l - cell supernatant , 2 mm l - glutamine , 1% penicillin / streptomycin , and 0.25 g / ml amphotericin b in rpmi 1640 ) , the adherent cells were replated in rpmi 1640 medium supplemented with 10% fcs , 2 mm l - glutamine , and 1% penicillin / streptomycin for use as bmdms . on day 7 , the medium was removed and replaced with either msc - conditioned medium , or control medium and the cells were incubated for 1 h , after which they were stimulated with either r848 or loxoribine . levels of tnf- , il-6 , gm - csf , il-12 p70 , and il-10 were determined using a duoset elisa kit ( r&d systems , minneapolis , mn , usa ) , and pge2 levels were measured using an elisa kit ( cayman ) , according to the manufacturer 's instructions . total rna was isolated using rneasy mini kits ( qiagen , valencia , ca , usa ) and reverse transcribed using high capacity cdna reverse transcription kits ( applied biosystems , foster city , ca , usa ) , according to the manufacturers ' instructions . quantitative real - time pcr ( qrt - pcr ) analysis with sybr green was performed on a 7500 fast real - time pcr system ( applied biosystems ) . the sequences of the primers were as follows : 5-ggtcaaaggtttggaagcag-3 ( forward ) and 5-tgtgaaatgccaccttttga-3 ( reverse ) for il1b ; 5-tctccgttacttggggacac-3 ( forward ) and 5-ccacactcaagaatggtcgc-3 ( reverse ) for cxcl1 ; 5-gtggaatcttccggctgtag-3 ( forward ) and 5-accatgacactctgcaacca-3 ( reverse ) for ccl3 ; 5-ccacttcttctctgggttgg-3 ( forward ) and 5-gtgcccacgtcaaggagtat-3 ( reverse ) for ccl5 ; and 5-ttgatggcaacaatctccac-3 ( forward ) and 5-cgtcccgtagacaaaatggt-3 ( reverse ) for gapdh , which was used as a loading control . following r848 stimulation , bmdms were lysed in ripa buffer ( thermo fisher scientific , waltham , ma , usa ) supplemented with protease inhibitor cocktail ( sigma - aldrich ) and phosphatase inhibitor ( thermo fisher scientific ) at various time points . lysed bmdms were kept on ice for 30 min and then centrifuged at 15,000 g for 15 min . the total protein concentration of each sample was determined using the bicinchoninic acid protein assay ( thermo fisher scientific ) . equal amounts ( 1030 g ) of cell lysate were separated by sds - page ( bio - rad , hercules , ca , usa ) , and the proteins were then transferred onto polyvinyl difluoride membranes ( invitrogen , carlsbad , ca , usa ) . after overnight incubation with each primary antibody , the membrane was washed , stained with horseradish peroxidase - conjugated rabbit or mouse igg secondary antibody , and visualized using enhanced chemiluminescence detection reagents ( ecl ; ge healthcare , piscataway , nj , usa ) . bmdms were transfected with the rela cflag pcdna3 plasmid ( gift from professor stephen smale ) or pcdna3.1 plasmid ( gift from professor stephen smale ) using the fugene6 transfection reagent ( promega , madison , wi , usa ) , according to the manufacturer 's instructions . at 24 h after transfection , the cells were cultured for 1 h in either msc - conditioned medium or control medium , after which they were incubated with either tlr7/8 ligand ( r848 ) or vehicle , without changing the medium . differences were analyzed for statistical significance using an unpaired t - test or anova , followed by tukey 's test for multiple comparisons . to investigate the effect of mscs on cytokine production by bmdms following tlr7/8 ligand stimulation , we cocultured mscs and bmdms and examined cytokine production following stimulation with either the tlr7/8 ( r848 ) or tlr7 ( loxoribine ) ligand . mscs inhibited the production of tnf- , il-6 , and gm - csf by bmdms after r848 and loxoribine stimulation . the level of il-12 p70 was lower in cocultured bmdms after r848 stimulation , but not after loxoribine stimulation . the level of il-10 was higher in cocultured bmdms after r848 stimulation , but not after loxoribine stimulation ( figure 1 ) . next , we examined whether mscs produce soluble factors that modulate cytokine production by macrophages stimulated with tlr7/8 ligands . bmdms were preincubated in either msc - conditioned or control medium and then stimulated with the tlr7/8 ( r848 ) or tlr7 ( loxoribine ) ligand . the production of tnf- , il-6 , and gm - csf following r848 or loxoribine stimulation was significantly lower in bmdms preincubated in msc - conditioned medium than in bmdms preincubated in control medium ( figures 2(a)2(c ) ) . in contrast , r848- and loxoribine - stimulated bmdms preincubated in msc - conditioned medium produced higher levels of il-10 than did r848- and loxoribine - stimulated bmdms preincubated in control medium ( figure 2(e ) ) . these results indicate that mscs secrete factors that suppress the production of tnf- , il-6 , and gm - csf and enhance the production of il-10 by tlr7/8 ligand - stimulated bmdms . the production of mrna encoding il1b , cxc ligand ( cxcl)1 , cc ligand ( ccl)3 , and ccl5 was suppressed in both r848- and loxoribine - stimulated bmdms preincubated in msc - conditioned medium ( figures 2(f)2(i ) ) . pge2 is a major immunomodulator secreted by mscs , and a previous study demonstrated that pge2 secreted from mscs contributes to enhanced il-10 production by macrophages . therefore , we examined whether pge2 contributes to the immunomodulatory effect of msc - conditioned medium on tlr7/8 ligand ( r848)-stimulated bmdms . we first confirmed that pge2 was present in msc - conditioned medium , but not in control medium ( figure 3(a ) ) . we then measured the level of pge2 in the supernatant of bmdms before and after tlr7/8 ligand ( r848 ) stimulation . the level was below the detection limit , suggesting that pge2 is not secreted by macrophages after tlr7/8 ligand stimulation ( data not shown ) . next , bmdms were preincubated for 1 h with the prostaglandin e2 receptor ep2 or ep4 antagonist in msc - conditioned or control medium , after which the cells were stimulated with r848 . the level of tnf- produced by cells preincubated with the ep2 or ep4 receptor antagonist was significantly higher than that produced by cells stimulated with r848 alone ( figure 3(b ) ) . the level of il-6 increased significantly in bmdms preincubated with the ep4 antagonist in msc - conditioned medium , but not in cells preincubated with the ep2 antagonist ( figure 3(c ) ) . in addition , the observed enhancement in il-10 production was partly abrogated by the ep4 antagonist , but not by the ep2 antagonist ( figure 3(d ) ) . these results indicate that the observed decreases in the expression of tnf- and il-6 and the observed increase in the expression of il-10 were due , at least in part , to pge2 secreted by mscs . because msc - conditioned medium modulated cytokine expression , especially after r848 stimulation , we examined the effect of msc - conditioned medium on signaling mediated by the mapk and nf-b pathways , both of which play important roles in tlr - mediated cytokine expression . the level of phosphorylated ( p)-erk was elevated 0.5 h after r848 stimulation in bmdms preincubated in msc - conditioned medium compared to the level in cells preincubated in control medium ( figures 4(a ) and 4(b ) ) . at 1 h after r848 stimulation , the level of total ib- , the inhibitory protein of nf-b , was higher in bmdms preincubated in msc - conditioned medium than in cells preincubated in control medium ( figures 4(c ) and 4(d ) ) . these results indicate that msc - conditioned medium enhances signaling mediated by erk and suppresses signaling mediated by nf-b in bmdms stimulated with r848 . to determine whether the pge2 present in msc - conditioned medium is responsible for the observed enhancement of erk signaling in r848-stimulated bmdms , we measured the level of p - erk after r848 stimulation in bmdms cultured in msc - conditioned medium or control medium with or without a cocktail composed of the ep2 and ep4 antagonists . treatment with the ep2/ep4 antagonist cocktail abrogated the increase in the level of p - erk in r848-stimulated bmdms cultured in msc - conditioned medium ( figure 5(a ) ) , suggesting that pge2/ep plays a role in increasing the level of p - erk in bmdms incubated in msc - conditioned medium . we then examined whether enhanced p - erk expression contributes to the observed suppression of tnf- and il-6 expression and enhancement of il-10 expression in bmdms incubated in msc - conditioned medium . for this experiment , bmdms were preincubated in msc - conditioned or control medium containing the mek / erk inhibitor u0126 or dimethyl sulfoxide ( dmso , as a vehicle control ) , after which the cells were stimulated with r848 . in cells incubated in the control medium , addition of u0126 resulted in a decrease in the levels of tnf- , il-6 , and il-10 after r848 stimulation ( figures 5(b)5(d ) ) , suggesting that mek / erk activation is essential for the production of these cytokines in r848-stimulated bmdms . as shown previously in figure 2(a ) , we confirmed that incubation in msc - conditioned medium results in a significant decrease in the levels of tnf- and il-6 and a significant increase in the level of il-10 after r848 stimulation ( figures 5(b)5(d ) ) . in cells incubated in msc - conditioned medium , addition of u0126 did not increase the production of tnf- and il-6 after r848 stimulation ( figures 5(b ) and 5(c ) ) , suggesting that the stimulation of p - erk expression induced by msc - conditioned medium does not contribute to the decreased expression of tnf- or il-6 . on the other hand , treatment with u0126 resulted in a significant decrease in il-10 production in r848-stimulated bmdms incubated in msc - conditioned medium . the il-10 level in these cells was comparable to that in r848-stimulated / u0126-treated bmdms cultured in control medium ( figure 5(d ) ) . these results suggest that enhanced p - erk expression contributes at least partially to the enhancement of il-10 production in bmdms cultured in msc - conditioned medium . to determine whether the suppression of nf-b signaling observed in r848-stimulated bmdms incubated in msc - conditioned medium is pge2-dependent , we measured the total ib- level in bmdms cultured in msc - conditioned or control medium with or without a cocktail composed of the ep2 and ep4 antagonists . the level of total ib- was significantly lower in bmdms cultured in msc - conditioned medium containing the ep2/ep4 antagonist cocktail than in cells cultured in msc - conditioned medium without the antagonist cocktail , suggesting that pge2/ep plays a role in the inhibition of total ib- degradation ( i.e. , suppression of nf-b signaling ) in cells cultured in msc - conditioned medium ( figure 6(a ) ) . we also investigated whether suppression of nf-b signaling contributes to the suppression of tnf- and il-6 expression and enhancement of il-10 expression by transfecting bmdms with either the rela cflag pcdna3 plasmid or a control c - flag pcdna3 plasmid . transfected cells were then incubated in either msc - conditioned medium or control medium and stimulated with r848.as shown in figure 6(b ) , rela mrna production was strongly induced in cells transfected with the rela cflag pcdna3 plasmid . in cells incubated in the control medium , rela overexpression resulted in no significant increase in the levels of tnf- , il-6 , or il-10 after r848 stimulation ( figures 6(c)6(e ) ) . in contrast , rela overexpression enhanced the expression of tnf- ( but not il-6 or il-10 ) in cells incubated in msc - conditioned medium ( figures 6(c)6(e ) ) . the production of tnf- in rela - transfected cells cultured in msc - conditioned medium and control medium was comparable ( figure 6(c ) ) . these results indicate that the expression of tnf- in bmdms incubated in msc - conditioned medium is mediated at least in part by nf-b signaling . in this study , we investigated the effects of mscs and msc - conditioned medium on macrophages during tlr7/8-mediated immune responses and demonstrated the following salient findings : ( 1 ) tlr7/8-mediated cytokine expression , including that of tnf- , il-6 , and il-10 , in macrophages is modulated by coincubation with mscs ; ( 2 ) soluble factors secreted by mscs modulate tlr7/8-mediated cytokine expression in macrophages ; ( 3 ) pge2 secreted by mscs is involved in this modulation ; ( 4 ) erk signaling is enhanced and nf-b signaling is suppressed after tlr7/8 ligand stimulation when macrophages are cultured in msc - conditioned medium , and these effects are primarily due to pge2 ; ( 5 ) erk signaling is involved in enhanced il-10 production in macrophages cultured in msc - conditioned medium ; and ( 6 ) nf-b signaling is involved in suppressed tnf- ( but not il-6 ) expression in macrophages cultured in msc - conditioned medium . a growing body of evidence indicates that mscs have immunomodulatory roles in innate immune responses ; therefore , msc - based therapies are increasingly viewed as promising means of treating various inflammatory diseases , such as sepsis , acute lung injury , and bacterial pneumonia . in the present study , we hypothesized that mscs may modulate tlr7/8-mediated signaling , a major signaling pathway activated by infection with ssrna viruses such as influenza virus and hiv . macrophages are one of the most important cellular components of the inflammatory and innate immune responses that occur in the lung following infection with microorganisms such as bacteria and viruses [ 4 , 28 ] . recent reports indicate that macrophages play a pivotal role in the immunomodulation of mscs during tlr4 ( lps)-mediated innate immune responses [ 17 , 20 ] ; however , the effect of mscs on innate immune responses to ligands for tlr7/8 , the receptors that recognize viral single - stranded rna , is largely unknown . therefore , we focused our investigation on the effect of mscs on tlr7/8-mediated cytokine expression in macrophages . our results indicate that tlr7/8-mediated induction of proinflammatory cytokine expression ( e.g. , tnf- and il-6 ) was suppressed in bmdms cultured in msc - conditioned medium , suggesting that msc - conditioned medium played an anti - inflammatory role in the present study . the significance of the induction of tnf- and il-6 expression following the tlr7/8-mediated response to ssrna virus infection is not fully known . infection with influenza virus , a major ssrna virus , induces a significant increase in the production of tnf- and il-6 by macrophages , leading to enhanced inflammatory responses [ 11 , 29 ] . in addition , increased tnf- and il-6 production can potentiate the severity of combined influenza a virus and bacterial infections [ 30 , 31 ] . another study demonstrated that the level of il-6 expression correlates with disease severity in pediatric h1n1 infection . based upon these data , we speculate that the suppression of tlr7/8-mediated tnf- and il-6 expression by msc - conditioned medium modulates the pathogenesis of ssrna virus infection , including infection by influenza virus . the expression of il-10 was highly upregulated in bmdms after tlr7/8 ligand stimulation in the present study , consistent with previous reports demonstrating that il-10 expression is highly upregulated in macrophages following tlr7/8 ligand stimulation and influenza virus infection . interestingly , we found that culturing bmdms in msc - conditioned medium increased the level of il-10 after tlr7/8 ligand stimulation . however , given that il-10 suppresses the induction of tnf- and il-6 expression in tlr ligand - stimulated macrophages , it is possible that il-10 also inhibits the induction of tnf- and il-6 expression by macrophages , leading to the suppression of inflammatory responses observed in the present study . in the presence of loxoribine ( tlr7 ligand ) , however , in the presence of loxoribine , the il-10 level was higher in bmdms cultured in msc - conditioned medium than in cells cultured in control medium . a possible reason for the discrepancy is the effect of cell - cell contact between bmdms and mscs , which may modulate cell surface markers , change intracellular signaling , and modulate loxoribine - induced il-10 production in the present study . soluble factors produced by mscs as well as cell - cell contact are thought to be important for the immunomodulatory effects . various immunosuppressive factors secreted by mscs mediate the immunomodulatory effects , including pge2 , ido , tgf-1 , hgf , inos , and ho1 . among these factors , we focused on the role of pge2 because pge2 , is a major secretory product of mscs , and it can modulate cytokine expression , especially in macrophages . four pge2 receptors have been identified : ep1 , ep2 , ep3 , and ep4 . the camp / pka / creb signaling pathway is activated through ep2 and ep4 , both of which are responsible for the anti - inflammatory function of pge2 . the results of the present study indicate that the pge2-ep2 and pge2-ep4 pathways play important roles in the modulation of cytokine expression . the role of pge2 in tnr7/8-mediated responses , including ssrna virus infection , is not fully known ; however , recent reports suggest that pge2 protects against ssrna virus infection in vitro [ 37 , 38 ] . these results led us to speculate that pge2 secreted by mscs following ssrna virus infection may play an immunoregulatory role in vivo by modulating cytokine expression . msc - based therapy against ssrna virus infection may be useful for patients with severe viral infection , such as those infected by the pandemic influenza 2009 h1n1 virus . in these cases , a cytokine storm can be lethal , and msc - mediated suppression of proinflammatory cytokines ( e.g. , tnf- and il-6 ) protects against severe influenza virus infection . in vivo animal studies and clinical trial activation of tlr7/8 signaling through myd88 results in the activation of mapks and the nf-b signaling pathway , leading to the expression of various cytokines . previous studies have demonstrated that pge2 inhibits nf-b - mediated transcription [ 40 , 41 ] . therefore , we examined the expression of signaling molecules associated with the mapk and nf-b pathways . interestingly , the expression of p - erk , an activated form of erk , was higher and the expression of total ib- , an inhibitory protein of nf-b , was lower in bmdms cultured in msc - conditioned medium than in cells cultured in control medium , indicating that erk signaling was activated and nf-b signaling was suppressed by the msc - conditioned medium . in the present study , the ep2/ep4 antagonist cocktail decreased the level of p - erk and total - ib- , confirming that pge2 contributes to enhanced erk and suppressed nf-b signaling . we also found that enhanced erk signaling was associated with enhanced il-10 expression , consistent with a previous report indicating that erk signaling is one of the primary pathways leading to il-10 production in macrophages . macrophages can be phenotypically polarized into 2 main groups depending on the microenvironment : m1 ( classical ) or m2 ( alternative ) activated macrophages . tnf- and il-6 , proinflammatory cytokines , are m1 macrophage markers , and il-10 is an m2 macrophage marker . therefore , our results indicate that msc or msc - conditioned medium can polarize macrophage population toward an m2 phenotype . a recent report indicates that mscs and msc - conditioned medium protect against lps - induced acute lung injury by polarizing the macrophage population toward an m2 phenotype . these cells were used because results obtained using human mscs may be more relevant to clinical settings and because little is known about the behavior of human mscs during inflammatory responses , although the beneficial effects of human mscs have been reported in studies of a mouse model of gram - negative sepsis , lps - induced ali , and escherichia coli pneumonia . suppression of tnf- and il-6 expression and enhancement of il-10 expression were observed in the present study when r848-stimulated bmdms were cultured in murine msc - conditioned medium ( data not shown ) , confirming that these immunomodulatory effects were not specific to human mscs . first , the pathway responsible for the suppression of il-6 expression in cells incubated in msc - conditioned medium is unknown . neither the erk nor nf-b signaling pathways were responsible for the suppression of il-6 expression . the janus kinase / signal transducer and activator of transcription / suppressor of cytokine signaling and phosphoinositol-3-kinase pathways , both of which are important for il-6 production [ 49 , 50 ] , could be responsible . second , mscs secrete soluble factors aside from pge2 that can modulate tlr7/8-mediated signaling , such as tgf- , which has been shown to suppress the production of proinflammatory cytokines in macrophages . further studies are required in order to obtain a complete understanding of the modulatory roles of mscs . in summary , we have demonstrated that msc - conditioned medium has an immunomodulatory effect on macrophages . in macrophages incubated in msc - conditioned medium , tlr7/8-mediated expression of the proinflammatory cytokines tnf- and il-6 was suppressed , and the expression of il-10 was enhanced . suppression of nf-b signaling contributed to the suppression of tnf- expression , and activation of erk signaling contributed to enhanced il-10 expression . these results indicate that msc - conditioned medium modulates the cytokine expression profile of cells stimulated with tlr7/8 . our results also suggest that mscs may play anti - inflammatory roles by modulating cytokine expression during infection by ssrna viruses , such as influenza virus .

increasing evidence suggests that mesenchymal stem cells ( mscs ) play anti - inflammatory roles during innate immune responses . however , little is known about the effect of mscs or their secretions on the ligand response of toll - like receptor ( tlr ) 7 and tlr8 , receptors that recognize viral single - stranded rna ( ssrna ) . macrophages play a critical role in the innate immune response to ssrna virus infection ; therefore , we investigated the effect of msc - conditioned medium on cytokine expression in macrophages following stimulation with tlr7/8 ligands . after stimulation with tlr7/8 ligand , bone marrow - derived macrophages cultured with mscs or in msc - conditioned medium expressed lower levels of tumor necrosis factor ( tnf ) and interleukin ( il ) 6 and higher levels of il-10 compared to macrophages cultured without mscs or in control medium , respectively . the modulations of cytokine expression were associated with prostaglandin e2 ( pge2 ) secreted by the mscs . pge2 enhanced extracellular signal - related kinase ( erk ) signaling and suppressed nuclear factor-b ( nf-b ) signaling . enhanced erk signaling contributed to enhanced il-10 production , and suppression of nf-b signaling contributed to the low production of tnf-. collectively , these results indicate that mscs and msc - conditioned medium modulate the cytokine expression profile in macrophages following tlr7/8-mediated stimulation , which suggests that mscs play an immunomodulatory role during ssrna virus infection .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

several lines of evidence confirm that the adaptive immune response plays a critical role in the effective control and clearance of various kinds of viruses [ 1 , 2 ] . viral infection triggers various innate immune receptors known as pattern recognition receptors ( prrs ) , including toll - like receptor ( tlr ) , nod - like receptor ( nlr ) , and rig - i - like receptor ( rlr ) . both tlr7 and tlr8 are located in endosomes ; they recognize the genomes of single - stranded rna ( ssrna ) viruses such as influenza virus and human immunodeficiency virus ( hiv ) . recognition by these receptors results in the activation of intracellular signaling by nuclear factor-b ( nf-b ) and mitogen activated protein kinases ( mapks ) through myd88 activation , which in turn leads to the production of proinflammatory cytokines and chemokines and initiates various antiviral responses [ 5 , 6 ] . mesenchymal stem cells ( mscs ) are multipotent mesenchymal stromal cells that can be isolated from various tissues . for example , msc administration protects against experimental sepsis in mice as a result of enhanced production of the anti - inflammatory cytokine il-10 by macrophages in response to msc - secreted prostaglandin e2 ( pge2 ) . animal model studies have shown that the protective actions of mscs also occur in response to acute lung injury induced by the tlr4 ligand lipopolysaccharide ( lps ) , chronic obstructive pulmonary disease , pulmonary fibrosis , and bacterial pneumonia . although little is known about the role that mscs may play in the immune response to viruses such as influenza viruses , the mechanism of msc - induced immunomodulation has been studied . in the present study , we hypothesized that mscs and their soluble factors may modulate the cytokine expression induced by the activation of tlr7/8-mediated signaling , a major signaling pathway in innate immunity during ssrna virus infections . to investigate this hypothesis , we focused on macrophages , which are critical components of the innate immune response to viral infection . here , we investigated the effect of mscs and msc - conditioned medium on bone marrow - derived macrophages ( bmdms ) during tlr7/8 stimulation . we found that tlr7/8-mediated expression of tnf- , il-6 , and il-10 is modulated by enhancement of extracellular signal - related kinase ( erk)-mediated signaling and suppression of nf-b - mediated signaling in response to pge2 secreted by mscs . the supernatant was used in further experiments as msc - conditioned medium , while cell - free medium incubated under the same conditions as the msc - conditioned medium served as a control . on day 7 , the medium was removed and replaced with either msc - conditioned medium , or control medium and the cells were incubated for 1 h , after which they were stimulated with either r848 or loxoribine . at 24 h after transfection , the cells were cultured for 1 h in either msc - conditioned medium or control medium , after which they were incubated with either tlr7/8 ligand ( r848 ) or vehicle , without changing the medium . to investigate the effect of mscs on cytokine production by bmdms following tlr7/8 ligand stimulation , we cocultured mscs and bmdms and examined cytokine production following stimulation with either the tlr7/8 ( r848 ) or tlr7 ( loxoribine ) ligand . the production of tnf- , il-6 , and gm - csf following r848 or loxoribine stimulation was significantly lower in bmdms preincubated in msc - conditioned medium than in bmdms preincubated in control medium ( figures 2(a)2(c ) ) . in contrast , r848- and loxoribine - stimulated bmdms preincubated in msc - conditioned medium produced higher levels of il-10 than did r848- and loxoribine - stimulated bmdms preincubated in control medium ( figure 2(e ) ) . these results indicate that mscs secrete factors that suppress the production of tnf- , il-6 , and gm - csf and enhance the production of il-10 by tlr7/8 ligand - stimulated bmdms . the production of mrna encoding il1b , cxc ligand ( cxcl)1 , cc ligand ( ccl)3 , and ccl5 was suppressed in both r848- and loxoribine - stimulated bmdms preincubated in msc - conditioned medium ( figures 2(f)2(i ) ) . pge2 is a major immunomodulator secreted by mscs , and a previous study demonstrated that pge2 secreted from mscs contributes to enhanced il-10 production by macrophages . therefore , we examined whether pge2 contributes to the immunomodulatory effect of msc - conditioned medium on tlr7/8 ligand ( r848)-stimulated bmdms . we first confirmed that pge2 was present in msc - conditioned medium , but not in control medium ( figure 3(a ) ) . next , bmdms were preincubated for 1 h with the prostaglandin e2 receptor ep2 or ep4 antagonist in msc - conditioned or control medium , after which the cells were stimulated with r848 . the level of il-6 increased significantly in bmdms preincubated with the ep4 antagonist in msc - conditioned medium , but not in cells preincubated with the ep2 antagonist ( figure 3(c ) ) . these results indicate that the observed decreases in the expression of tnf- and il-6 and the observed increase in the expression of il-10 were due , at least in part , to pge2 secreted by mscs . because msc - conditioned medium modulated cytokine expression , especially after r848 stimulation , we examined the effect of msc - conditioned medium on signaling mediated by the mapk and nf-b pathways , both of which play important roles in tlr - mediated cytokine expression . the level of phosphorylated ( p)-erk was elevated 0.5 h after r848 stimulation in bmdms preincubated in msc - conditioned medium compared to the level in cells preincubated in control medium ( figures 4(a ) and 4(b ) ) . at 1 h after r848 stimulation , the level of total ib- , the inhibitory protein of nf-b , was higher in bmdms preincubated in msc - conditioned medium than in cells preincubated in control medium ( figures 4(c ) and 4(d ) ) . these results indicate that msc - conditioned medium enhances signaling mediated by erk and suppresses signaling mediated by nf-b in bmdms stimulated with r848 . to determine whether the pge2 present in msc - conditioned medium is responsible for the observed enhancement of erk signaling in r848-stimulated bmdms , we measured the level of p - erk after r848 stimulation in bmdms cultured in msc - conditioned medium or control medium with or without a cocktail composed of the ep2 and ep4 antagonists . treatment with the ep2/ep4 antagonist cocktail abrogated the increase in the level of p - erk in r848-stimulated bmdms cultured in msc - conditioned medium ( figure 5(a ) ) , suggesting that pge2/ep plays a role in increasing the level of p - erk in bmdms incubated in msc - conditioned medium . we then examined whether enhanced p - erk expression contributes to the observed suppression of tnf- and il-6 expression and enhancement of il-10 expression in bmdms incubated in msc - conditioned medium . for this experiment , bmdms were preincubated in msc - conditioned or control medium containing the mek / erk inhibitor u0126 or dimethyl sulfoxide ( dmso , as a vehicle control ) , after which the cells were stimulated with r848 . in cells incubated in the control medium , addition of u0126 resulted in a decrease in the levels of tnf- , il-6 , and il-10 after r848 stimulation ( figures 5(b)5(d ) ) , suggesting that mek / erk activation is essential for the production of these cytokines in r848-stimulated bmdms . as shown previously in figure 2(a ) , we confirmed that incubation in msc - conditioned medium results in a significant decrease in the levels of tnf- and il-6 and a significant increase in the level of il-10 after r848 stimulation ( figures 5(b)5(d ) ) . in cells incubated in msc - conditioned medium , addition of u0126 did not increase the production of tnf- and il-6 after r848 stimulation ( figures 5(b ) and 5(c ) ) , suggesting that the stimulation of p - erk expression induced by msc - conditioned medium does not contribute to the decreased expression of tnf- or il-6 . on the other hand , treatment with u0126 resulted in a significant decrease in il-10 production in r848-stimulated bmdms incubated in msc - conditioned medium . these results suggest that enhanced p - erk expression contributes at least partially to the enhancement of il-10 production in bmdms cultured in msc - conditioned medium . to determine whether the suppression of nf-b signaling observed in r848-stimulated bmdms incubated in msc - conditioned medium is pge2-dependent , we measured the total ib- level in bmdms cultured in msc - conditioned or control medium with or without a cocktail composed of the ep2 and ep4 antagonists . the level of total ib- was significantly lower in bmdms cultured in msc - conditioned medium containing the ep2/ep4 antagonist cocktail than in cells cultured in msc - conditioned medium without the antagonist cocktail , suggesting that pge2/ep plays a role in the inhibition of total ib- degradation ( i.e. , suppression of nf-b signaling ) in cells cultured in msc - conditioned medium ( figure 6(a ) ) . we also investigated whether suppression of nf-b signaling contributes to the suppression of tnf- and il-6 expression and enhancement of il-10 expression by transfecting bmdms with either the rela cflag pcdna3 plasmid or a control c - flag pcdna3 plasmid . transfected cells were then incubated in either msc - conditioned medium or control medium and stimulated with r848.as shown in figure 6(b ) , rela mrna production was strongly induced in cells transfected with the rela cflag pcdna3 plasmid . in cells incubated in the control medium , rela overexpression resulted in no significant increase in the levels of tnf- , il-6 , or il-10 after r848 stimulation ( figures 6(c)6(e ) ) . in contrast , rela overexpression enhanced the expression of tnf- ( but not il-6 or il-10 ) in cells incubated in msc - conditioned medium ( figures 6(c)6(e ) ) . the production of tnf- in rela - transfected cells cultured in msc - conditioned medium and control medium was comparable ( figure 6(c ) ) . these results indicate that the expression of tnf- in bmdms incubated in msc - conditioned medium is mediated at least in part by nf-b signaling . in this study , we investigated the effects of mscs and msc - conditioned medium on macrophages during tlr7/8-mediated immune responses and demonstrated the following salient findings : ( 1 ) tlr7/8-mediated cytokine expression , including that of tnf- , il-6 , and il-10 , in macrophages is modulated by coincubation with mscs ; ( 2 ) soluble factors secreted by mscs modulate tlr7/8-mediated cytokine expression in macrophages ; ( 3 ) pge2 secreted by mscs is involved in this modulation ; ( 4 ) erk signaling is enhanced and nf-b signaling is suppressed after tlr7/8 ligand stimulation when macrophages are cultured in msc - conditioned medium , and these effects are primarily due to pge2 ; ( 5 ) erk signaling is involved in enhanced il-10 production in macrophages cultured in msc - conditioned medium ; and ( 6 ) nf-b signaling is involved in suppressed tnf- ( but not il-6 ) expression in macrophages cultured in msc - conditioned medium . a growing body of evidence indicates that mscs have immunomodulatory roles in innate immune responses ; therefore , msc - based therapies are increasingly viewed as promising means of treating various inflammatory diseases , such as sepsis , acute lung injury , and bacterial pneumonia . recent reports indicate that macrophages play a pivotal role in the immunomodulation of mscs during tlr4 ( lps)-mediated innate immune responses [ 17 , 20 ] ; however , the effect of mscs on innate immune responses to ligands for tlr7/8 , the receptors that recognize viral single - stranded rna , is largely unknown . therefore , we focused our investigation on the effect of mscs on tlr7/8-mediated cytokine expression in macrophages . , tnf- and il-6 ) was suppressed in bmdms cultured in msc - conditioned medium , suggesting that msc - conditioned medium played an anti - inflammatory role in the present study . the significance of the induction of tnf- and il-6 expression following the tlr7/8-mediated response to ssrna virus infection is not fully known . infection with influenza virus , a major ssrna virus , induces a significant increase in the production of tnf- and il-6 by macrophages , leading to enhanced inflammatory responses [ 11 , 29 ] . based upon these data , we speculate that the suppression of tlr7/8-mediated tnf- and il-6 expression by msc - conditioned medium modulates the pathogenesis of ssrna virus infection , including infection by influenza virus . the expression of il-10 was highly upregulated in bmdms after tlr7/8 ligand stimulation in the present study , consistent with previous reports demonstrating that il-10 expression is highly upregulated in macrophages following tlr7/8 ligand stimulation and influenza virus infection . interestingly , we found that culturing bmdms in msc - conditioned medium increased the level of il-10 after tlr7/8 ligand stimulation . however , given that il-10 suppresses the induction of tnf- and il-6 expression in tlr ligand - stimulated macrophages , it is possible that il-10 also inhibits the induction of tnf- and il-6 expression by macrophages , leading to the suppression of inflammatory responses observed in the present study . in the presence of loxoribine ( tlr7 ligand ) , however , in the presence of loxoribine , the il-10 level was higher in bmdms cultured in msc - conditioned medium than in cells cultured in control medium . a possible reason for the discrepancy is the effect of cell - cell contact between bmdms and mscs , which may modulate cell surface markers , change intracellular signaling , and modulate loxoribine - induced il-10 production in the present study . among these factors , we focused on the role of pge2 because pge2 , is a major secretory product of mscs , and it can modulate cytokine expression , especially in macrophages . these results led us to speculate that pge2 secreted by mscs following ssrna virus infection may play an immunoregulatory role in vivo by modulating cytokine expression . msc - based therapy against ssrna virus infection may be useful for patients with severe viral infection , such as those infected by the pandemic influenza 2009 h1n1 virus . in these cases , a cytokine storm can be lethal , and msc - mediated suppression of proinflammatory cytokines ( e.g. interestingly , the expression of p - erk , an activated form of erk , was higher and the expression of total ib- , an inhibitory protein of nf-b , was lower in bmdms cultured in msc - conditioned medium than in cells cultured in control medium , indicating that erk signaling was activated and nf-b signaling was suppressed by the msc - conditioned medium . in the present study , the ep2/ep4 antagonist cocktail decreased the level of p - erk and total - ib- , confirming that pge2 contributes to enhanced erk and suppressed nf-b signaling . we also found that enhanced erk signaling was associated with enhanced il-10 expression , consistent with a previous report indicating that erk signaling is one of the primary pathways leading to il-10 production in macrophages . therefore , our results indicate that msc or msc - conditioned medium can polarize macrophage population toward an m2 phenotype . a recent report indicates that mscs and msc - conditioned medium protect against lps - induced acute lung injury by polarizing the macrophage population toward an m2 phenotype . these cells were used because results obtained using human mscs may be more relevant to clinical settings and because little is known about the behavior of human mscs during inflammatory responses , although the beneficial effects of human mscs have been reported in studies of a mouse model of gram - negative sepsis , lps - induced ali , and escherichia coli pneumonia . suppression of tnf- and il-6 expression and enhancement of il-10 expression were observed in the present study when r848-stimulated bmdms were cultured in murine msc - conditioned medium ( data not shown ) , confirming that these immunomodulatory effects were not specific to human mscs . first , the pathway responsible for the suppression of il-6 expression in cells incubated in msc - conditioned medium is unknown . in summary , we have demonstrated that msc - conditioned medium has an immunomodulatory effect on macrophages . in macrophages incubated in msc - conditioned medium , tlr7/8-mediated expression of the proinflammatory cytokines tnf- and il-6 was suppressed , and the expression of il-10 was enhanced . suppression of nf-b signaling contributed to the suppression of tnf- expression , and activation of erk signaling contributed to enhanced il-10 expression . these results indicate that msc - conditioned medium modulates the cytokine expression profile of cells stimulated with tlr7/8 . our results also suggest that mscs may play anti - inflammatory roles by modulating cytokine expression during infection by ssrna viruses , such as influenza virus .

some primary alcohols , ethers and alkyl carbonates are used for gasoline additives as octane boosters . diethyl carbonate ( dec ) compared with the tradition gasoline additive methyl tert - butyl ether ( mtbe ) , dec has a higher oxygen content , and it is both low in toxicity and biodegrades quickly . adding dec to gasoline can not only enhance the octane rating , but it also reduces the content of alkenes and aromatic hydrocarbons which can give rise to environmental pollution . in recent years these is considerable interest in using dimethyl carbonate or dec to replace mtbe for meeting the oxygenate specifications on gasoline . the components of the gasoline are mainly c4c12 alkanes , cyclanes , olefins and aromatic hydrocarbon . considering the pollution from gasoline in the aqueous environment , such as the migration of gasoline additives from engines , the leakage of oil tanks in gasoline stations and in the process of transportation and storage , we considered the effect of adding water in these systems to simulate the real environment . liquid liquid equilibrium ( lle ) data are necessary and important basic data , which play an important role in understanding phase behavior for the multicomponent systems . to provide accurate solubility data and check thermodynamic models , people have studied lle and physical properties of mixtures containing alkyl carbonates [ 26 ] . here , we report lle for three ternary systems ( water + dec + propan-1-ol or benzene or cyclohexane ) and three quaternary systems ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) at t = 298.15 k and ambient pressure . the measured lle results were correlated by using the modified and extended uniquac models [ 7 , 8 ] having binary , ternary , and quaternary parameters . the binary energy parameters for partially miscible binary mixtures were obtained from mutual solubility data [ 1517 ] . the binary , ternary and quaternary parameters are required to represent accurately the quaternary lle data . the chemicals used in these experiments were dec , benzene , propan-1-ol , cyclohexane , water and heptane . the specifications of the chemicals used in this work are given in table 1.table 1purities and suppliers of the chemicalschemicalmass fraction puritysupplierbenzene ( ar ) 0.9920guangzhou - reagent co. ( guangzhou , china)cyclohexane ( ar)0.9910tianjin - reagent co. ( tianjin , china)dec ( ar)0.9945aladdin - reagent co.(shanghai , china)heptanes ( ar)0.9965fuyu - reagent co. ( tianjin , china)propan-1-ol ( ar)0.9950tianjin - reagent co. ( tianjin , china ) ar means analytical reagent purities and suppliers of the chemicals ar means analytical reagent measurements were carried out on a binary test system ( dmc + water ) at 298.15 k to validate the experimental technique by determining the mole fraction of dmc in water solution and comparing it with literature value . the estimated error in mole fraction is less than 5 10 . for maintaining temperature , about 70 cm of each mixture was loaded into the equilibrium glass cell placed in a thermostatted water bath . the temperature of the water bath was controlled at t = 298.15 k , and the temperature uncertainty was 0.05 k. contents were stirred well by a magnetic stirrer for about 3 h and were then allowed to settle for more than 8 h at constant temperature , which was long enough to reach thermodynamic equilibrium . for analysis , compositional analysis of samples was carried out in a gas chromatograph ( shimadzu analyses apparatus co. , shuzhou , china , gc-14c ) with a thermal conductivity detector . the analysis was performed with a porapak qs packed column ( 3 mm 2.5 m ) . the detector and injector temperatures were kept at 510.15 and 490.15 k , respectively . hydrogen was used as carrier gas at a rate of 60 mlmin throughout the column , and the column inlet pressure was 0.1 mpa . a chromatopac ( n2000 ) was used to detect the peak areas of the components . the accuracy in these experimental measurements was found to be ( 6 10 ) for the mole fraction.fig . the chemicals used in these experiments were dec , benzene , propan-1-ol , cyclohexane , water and heptane . the specifications of the chemicals used in this work are given in table 1.table 1purities and suppliers of the chemicalschemicalmass fraction puritysupplierbenzene ( ar ) 0.9920guangzhou - reagent co. ( guangzhou , china)cyclohexane ( ar)0.9910tianjin - reagent co. ( tianjin , china)dec ( ar)0.9945aladdin - reagent co.(shanghai , china)heptanes ( ar)0.9965fuyu - reagent co. ( tianjin , china)propan-1-ol ( ar)0.9950tianjin - reagent co. ( tianjin , china ) ar means analytical reagent purities and suppliers of the chemicals ar means analytical reagent measurements were carried out on a binary test system ( dmc + water ) at 298.15 k to validate the experimental technique by determining the mole fraction of dmc in water solution and comparing it with literature value . the estimated error in mole fraction is less than 5 10 . for maintaining temperature , about 70 cm of each mixture was loaded into the equilibrium glass cell placed in a thermostatted water bath . the temperature of the water bath was controlled at t = 298.15 k , and the temperature uncertainty was 0.05 k. contents were stirred well by a magnetic stirrer for about 3 h and were then allowed to settle for more than 8 h at constant temperature , which was long enough to reach thermodynamic equilibrium . for analysis , compositional analysis of samples was carried out in a gas chromatograph ( shimadzu analyses apparatus co. , shuzhou , china , gc-14c ) with a thermal conductivity detector . the analysis was performed with a porapak qs packed column ( 3 mm 2.5 m ) . the detector and injector temperatures were kept at 510.15 and 490.15 k , respectively . hydrogen was used as carrier gas at a rate of 60 mlmin throughout the column , and the column inlet pressure was 0.1 mpa . a chromatopac ( n2000 ) was used to detect the peak areas of the components . the accuracy in these experimental measurements was found to be ( 6 10 ) for the mole fraction.fig . the experimental lle results for the three ternary systems ( water + propan-1-ol + dec ) , ( water + dec + benzene ) and ( water + dec + cyclohexane ) measured at t = 298.15 k are reported in tables 2 , 3 and 4 . tables 5 , 6 and 7 list the experimental lle results for the three quaternary systems ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) at t = 298.15 k. figure 2 shows a tetrahedron to depict three planes of the quaternary lle for the ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) systems . for example , the quaternary system ( water + propan-1-ol + dec + benzene ) is comprised of three ternary subsystems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + dec + benzene).table 2experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) for mole fractions x at the temperature t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04750.00000.95250.99780.00000.00220.01780.10730.87490.97670.01760.00570.09690.16420.73890.92800.06410.00790.13910.23900.62190.92940.06600.00460.20990.28790.50220.90860.08680.00460.26360.32120.41520.94270.05190.00540.32310.35820.31870.93200.06140.0066table 3experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + benzene ( 3 ) for mole fractions x at t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04250.12340.83410.99330.00480.00190.03360.22850.73790.99260.00520.00220.03730.30030.66240.99100.00660.00240.04120.38520.57360.99060.00660.00280.04230.46660.49110.99220.00540.00240.04050.50970.44980.99340.00390.00270.03830.56340.39830.99510.00290.0020table 4experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + cyclohexane ( 3 ) for mole fractions x at t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04630.09480.85890.97100.00190.02710.03470.17700.78830.97650.00140.02210.05280.25920.68800.98030.00120.01850.05870.31980.62150.98790.00130.01080.04720.38800.56480.98170.00140.01690.04980.40840.54180.98010.00150.01840.04400.44620.50980.97640.00180.02180.04750.48500.46750.97630.00160.02210.05020.56640.38340.97960.00200.01840.04450.62200.33350.97950.00190.0186table 5experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + benzene ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3diethyl carbonate + ( 1 x 1 x 2 x 3)benzene } x 3 = 0.20 0.96420.03580.00000.03200.09130.2290 0.95230.04770.00000.08230.20970.1960 0.95180.04820.00000.07320.26530.1721 0.93870.06130.00000.10710.32930.1630 0.92220.07780.00000.12110.37060.1429 0.93200.06800.00000.13850.41850.1320 0.92350.07650.00000.16890.44320.1164 0.92900.07100.00000.19330.47340.1064 0.92070.07930.00000.24990.45350.0817 0.92440.07560.00000.28830.43940.0706 0.92430.07570.00000.32710.43340.0541 x 3 = 0.40 0.93060.06940.00000.03590.12740.4270 0.92040.07960.00000.06740.21290.3407 0.90300.09700.00000.07280.28410.3150 0.92780.07220.00000.10490.30290.3050 0.89950.10050.00000.14460.38300.2328 0.90090.09910.00000.16110.40890.2209 0.87560.12440.00000.20800.44540.1701 0.91050.08950.00000.27530.45710.1216 x 3 = 0.60 0.91750.08010.00230.05980.08820.5652 0.90730.09070.00200.10360.16660.4888 0.91120.08720.00160.10270.22790.4832 0.89240.10590.00170.15480.28700.3883 0.90750.09090.00150.20890.33310.3070 0.90220.09590.00190.25240.37840.2222 0.87360.12430.00220.30480.38750.1723 0.87310.12400.00290.33320.40330.1521 0.88810.10950.00240.45050.37140.0968 x 3 = 0.80 0.87650.11960.00390.46190.36930.1195 0.87710.11920.00370.52000.35700.0837 0.86970.12680.00350.51480.35040.0910 0.88230.11400.00370.56830.32710.0656 0.87370.12230.00400.57940.32060.0661 0.89410.10020.00570.18350.27440.4418 0.89480.10190.00330.25200.33960.3213 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) benzene } mole fraction ratio of dec and benzene in the binary mixturestable 6experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + cyclohexane ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3 diethyl carbonate + ( 1 x 1 x 2 x 3 ) cyclohexane } x 3 = 0.20 0.94710.04860.00430.01520.01370.1937 0.94270.05310.00420.01740.04690.1758 0.92680.06910.00410.02860.09010.1642 0.92510.07290.00210.03870.15230.1300 0.92040.07760.00190.05890.20940.1271 0.91510.08280.00210.10970.24400.1069 0.91650.08180.00170.17410.25570.0889 0.91770.08070.00160.19840.24990.0795 x 3 = 0.40 0.95250.04300.00450.02830.03610.3716 0.94180.05420.00400.03900.06360.3458 0.93380.06330.00290.03470.12710.3111 0.94110.05670.00210.08100.18740.2750 0.92900.06910.00190.14370.22050.2246 0.92840.06980.00190.17710.24440.1925 0.92590.07220.00190.21780.27780.1669 0.92570.07220.00210.24840.28270.1422 x 3 = 0.60 0.96020.03740.00240.04720.04550.5430 0.94630.05170.00190.05930.08200.5452 0.93810.05950.00240.06120.14710.4862 0.93590.06160.00250.14440.17890.3642 0.93690.06120.00190.19740.24020.3246 0.94010.05790.00200.22840.25470.2710 0.93110.06710.00180.25120.27170.2316 0.92800.06920.00280.28740.28360.1957 x 3 = 0.80 0.96430.03250.00320.08860.04910.7099 0.95520.04190.00290.11900.09570.6311 0.94580.05120.00300.15380.15020.5490 0.93990.05720.00290.20870.19650.4566 0.93640.05620.00740.24130.24050.3934 0.93630.05850.00510.28260.25460.3359 0.93080.06430.00490.32340.27110.2878 0.92130.07500.00370.36730.27870.2441 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) cyclohexane } mole fraction ratio of dec and cyclohexane in the binary mixturestable 7experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + heptane ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3diethyl carbonate + ( 1 x 1 x 2 x 3 ) heptane } x 3 = 0.20 0.95140.04600.00000.03080.09500.2079 0.92860.07140.00000.04110.25870.1657 0.84360.15640.00000.06020.38290.1323 0.90980.09020.00000.07050.41480.1247 0.89610.10390.00000.12260.48160.0854 0.83830.16170.00000.16860.48420.1047 0.81600.18400.00000.20930.50070.0729 0.83370.16630.00000.21950.50720.0642 0.86510.13490.00000.30010.50060.0439 x 3 = 0.40 0.91810.07240.00460.03120.11020.5550 0.94740.04400.00400.03870.20090.4438 0.90370.08790.00210.05260.29570.3276 0.90190.09010.00200.09500.38690.2509 0.90420.08920.00190.14250.46410.1865 0.89890.09420.00200.21220.47790.1498 0.89330.09930.00300.23260.50010.1183 0.88340.10950.00260.27660.50090.0983 x 3 = 0.60 0.95490.03300.00540.04540.05900.5137 0.94360.04650.00390.04510.12320.4845 0.93570.05470.00400.06760.19690.4218 0.92560.06460.00380.10790.25500.3637 0.92620.06230.00620.15860.32200.2947 0.91880.06830.00540.22120.34120.2482 0.91400.07420.00510.25790.36030.2126 0.91660.07400.00500.28590.37930.1837 x 3 = 0.80 0.95240.03740.00400.06120.08680.6616 0.94490.04570.00400.07630.13680.6154 0.93630.05330.00370.13360.20680.5077 0.93410.05770.00340.17610.25990.4263 0.92760.06300.00300.24230.30410.3459 0.92440.06520.00370.27890.32570.2948 0.92100.06790.00410.30330.34760.2612 0.88990.09700.00520.33980.35240.2267 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) heptane } mole fraction ratio of dec and heptane in the binary mixturesfig . 2phase equilibria of ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) ; x 3 denotes a quaternary section plane experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) for mole fractions x at the temperature t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + benzene ( 3 ) for mole fractions x at t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + cyclohexane ( 3 ) for mole fractions x at t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + benzene ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) benzene } mole fraction ratio of dec and benzene in the binary mixtures experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + cyclohexane ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) cyclohexane } mole fraction ratio of dec and cyclohexane in the binary mixtures experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + heptane ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) heptane } mole fraction ratio of dec and heptane in the binary mixtures phase equilibria of ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) ; x 3 denotes a quaternary section plane the modified uniquac and extended uniquac models were employed to correlate the experimental lle data . for most multicomponent systems , especially for type 1 systems having a plait point , the original uniquac model with only two binary parameters did not always give accurate results . so , in order to accurately correlate ternary and quaternary lle , it is necessary to use ternary and quaternary parameters in addition to the binary ones . the ternary and quaternary parameters were determined from the experimental lle data using a simplex method by minimizing the function f:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f = 100 \cdot \left\ { { \sum\limits_{k } { \sum\limits_{i } { \sum\limits_{j } { \left ( { x_{ijk}^ { \exp } - x_{ijk}^{\text{cal } } } \right)^{2 } /2ni } } } \}^{0.5 } } \right\ } $ $ \end{document } where x denotes the mole fraction in the liquid phase , i = 1 to 3 for a ternary system or i = 1 to 4 for a quaternary system , and j = 1 , 2 ( phases ) , k = 1 , 2 , , n , where n stands for the number of tie lines as shown in tables 10 and 11 . here table 8 shows the molecular structural volume and area parameters , where r and q are taken from the literature [ 5 , 19 ] . the interaction correction factors q , for nonassociating components such as dec , benzene , cyclohexane and heptanes , were set to q = q in the modified uniquac model and q = q in the extended uniquac model , while those for associating components such as water and propan-1-ol were taken from the literature [ 7 , 8].table 8structural parameters for pure componentscomponent r q q q benzene3.192.40q q cyclohexane3.973.01q q dec4.413.90 q q heptane5.174.40q q propan-1-ol2.782.511.320.89water0.921.401.280.96 from references [ 5 , 19 ] modified uniquac model extended uniquac model structural parameters for pure components from references [ 5 , 19 ] modified uniquac model extended uniquac model table 9 lists the binary parameters aij of the modified uniquac and extended uniquac models for the constituent binary mixtures , along with the standard deviations between experimental and calculated values : (p ) for pressure , (t ) for temperature , (x ) for liquid phase mole fraction , and (y ) for vapor phase mole fraction . good agreement was obtained between experimental results and those calculated by both models.table 9calculated results from binary phase equilibrium data reductionsystem(1 + 2 ) a 12/k a 21/k (p)/kpa (t)/k10 (x ) 10 (y ) lit.propan-1-ol + water159.06 138.20 262.46244.511.491.520.110.112.802.906.706.80propan-1-ol + dec75.71 97.62 175.10176.761.901.900.120.122.502.503.003.00benzene + propan-1-ol606.64 586.62 92.9891.701.451.460.050.050.600.605.405.40dec + benzene193.28 160.28 191.39157.751.761.900.010.010.600.708.609.40cyclohexane + propan-1-ol1018.29 965.40 110.86122.690.870.920.030.030.300.404.304.50cyclohexane + dec52.53 47.09 211.09230.092.552.540.140.142.902.808.108.10heptane + dec144.20 181.84 31.2269.081.301.300.070.070.800.808.007.90heptane + propan-1-ol757.15 738.64 117.88164.474.304.320.190.194.404.4017.0016.90water + cyclohexane1157.80 1315.60 2429.901942.50water + dec248.21 273.66 1177.60961.41water + heptane1022.10 1839.60 1884.202135.50water + benzene762.26 750.12 1663.801365.30 modified uniquac model extended uniquac model root - mean - square deviation calculated results from binary phase equilibrium data reduction modified uniquac model extended uniquac model root - mean - square deviation table 10 presents the ternary mixture parameters , 231 , 132 and 123 , together with the root - mean - square deviation ( rmsd ) values between the experimental and calculated tie lines for the ternary lle . the comparison is shown on the phase diagram in fig . 3 by means of the experimental and calculated tie lines for the three ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) . as shown in this figure , excellent correlation is seen by the extended and modified uniquac models . for the three ternary systems investigated in this work , the average rmsd values of correlated results are 0.88 and 1.03 % for the modified and extended uniquac models , respectively . good agreement between the experimental and correlated tie line data of the two models is indicated by the low rmsd values.table 10calculated results of ternary liquid liquid equilibriasystem ( 1 + 2 + 3 ) n 231 132 123 rms rms lit.water + propan-1-ol + dec70.0342 0.0063 0.62150.31341.54950.80053.611.200.761.07this workwater + dec + benzene70.1465 0.0914 0.18340.11650.15750.04831.311.030.600.67this workwater + dec + cyclohexane100.1018 0.1554 4.39060.14481.24520.00942.001.821.271.34this workwater + propan-1-ol + benzene120.0011 5.0259 0.00094.15700.90610.83812.793.602.122.57water + propan-1-ol + cyclohexane70.0488 0.0059 1.30820.61802.84340.20232.543.051.411.79water + propan-1-ol + heptane110.0217 0.1289 1.57641.11710.19410.203118.6116.652.902.42water + dec + heptane130.0333 0.0509 0.15760.18640.01750.83190.851.310.260.65 number of tie lines modified uniquac model extended uniquac model root - mean - square deviation ( mol-% ) predicted results using binary parameters taken from the table 9 correlated results using binary and ternary parametersfig . 3experimental and calculated lle of ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) at t = 298.15 k. filled circle , experimental tie - line data ; lines , predicted results by the modified uniquac model using binary parameters taken from table 9 ; dotted lines , correlated results by the modified uniquac model using binary and ternary parameters taken from tables 9 and 10 calculated results of ternary liquid liquid equilibria modified uniquac model extended uniquac model root - mean - square deviation ( mol-% ) predicted results using binary parameters taken from the table 9 correlated results using binary and ternary parameters experimental and calculated lle of ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) at t = 298.15 k. filled circle , experimental tie - line data ; lines , predicted results by the modified uniquac model using binary parameters taken from table 9 ; dotted lines , correlated results by the modified uniquac model using binary and ternary parameters taken from tables 9 and 10 table 11 summarizes the quaternary parameters , 2341 , 1342 , 1243 and 1234 , together with the correlated results obtained by fitting the modified and extended uniquac models with binary , ternary , and quaternary parameters to the experimental quaternary lle data , together with the predicted results by the models with only the binary and ternary parameters listed in tables 9 and 10 . for the three investigated quaternary systems , the average rmsd values of correlated results are 2.68 and 2.96 % for the extended and modified uniquac models , respectively . it can be seen that the correlated results obtained from both models are better than the predicted ones in representing the quaternary lle measured in this work . this is due to adding the quaternary parameters in the correlation.table 11calculated results of quaternary liquid liquid equilibriasystem ( 1 + 2 + 3 + 4 ) n 2341 1342 1243 1234 rms rms water + propan-1-ol + dec + benzene351.7213 0.1941 25.364120.50464.90442.97201.32720.08444.765.422.592.86water + propan-1-ol + dec + cyclohexane321.3683 4.9950 4.783419.86991.210515.69781.01802.51364.664.222.651.21water + propan-1-ol + dec + heptane310.1506 0.0319 0.64090.58041.35212.63060.42570.84804.247.342.792.80 number of tie lines modified uniquac model extended uniquac model root - mean - square deviation ( mol-% ) predicted results using binary and ternary parameters taken from the tables 9 and 10 correlated results using binary , ternary and quaternary parameters calculated results of quaternary liquid liquid equilibria modified uniquac model extended uniquac model root - mean - square deviation ( mol-% ) predicted results using binary and ternary parameters taken from the tables 9 and 10 correlated results using binary , ternary and quaternary parameters the equilibrium distribution coefficient of dec , calculated from the experimental lle data , is defined as : the ratio of the concentration of dec in the aqueous phase to the concentration in the organic phase:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d = x _ { 3}^{\rm{aqueous\;phase } } /x _ { 3}^{\rm{organic\;phase } } $ $ \end{document}where d is equilibrium distribution coefficient of dec and x3is the mole fraction of dec . figures 4 , 5 and 6 show the equilibrium distribution coefficient of dec for the quaternary systems ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) , at four different distribution ratio of x3 = 0.2 , 0.4 , 0.6 , and 0.8 . for the three measured quaternary systems , the equilibrium distribution coefficients of dec show low values as shown in figs . 4 , 5 and 6 . it can be concluded that adding dec does not result in an evident increase of solubility of dec in the aqueous phase . since dec has two ethyl groups , while alkyl is a hydrophobic group , dec is more soluble in the organic phase.fig . 4distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + benzene ( 4 ) as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectivelyfig . 5distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + cyclohexane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectivelyfig . 6distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + heptane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + benzene ( 4 ) as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + cyclohexane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + heptane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively under atmospheric pressure , the experimental lle data for the ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) , and quaternary systems of ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) were obtained at t = 298.15 k. the experimental lle results were successfully correlated using the extended and modified uniquac models . the correlated results obtained by using the quaternary parameters as well as the binary and ternary parameters , with a rmsd value of less than 3 mol- % , showed good agreement with the experimental lle results

in this study liquid phase equilibrium compositions were measured at 298.15 k under atmospheric pressure for ( water + propan-1-ol + diethyl carbonate ( dec ) + benzene or cyclohexane or heptane ) quaternary systems and ( water + dec + propan-1-ol or benzene or cyclohexane ) ternary systems . good correlation of the experimental lle data was seen for the measured systems by both modified and extended uniquac models . the solubility of dec in aqueous and organic phases is shown by equilibrium distribution coefficients calculated from the lle data .

Introduction Experimental Materials Procedures Calculation Procedure and Results Discussion Conclusions

here , we report lle for three ternary systems ( water + dec + propan-1-ol or benzene or cyclohexane ) and three quaternary systems ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) at t = 298.15 k and ambient pressure . the experimental lle results for the three ternary systems ( water + propan-1-ol + dec ) , ( water + dec + benzene ) and ( water + dec + cyclohexane ) measured at t = 298.15 k are reported in tables 2 , 3 and 4 . tables 5 , 6 and 7 list the experimental lle results for the three quaternary systems ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) at t = 298.15 k. figure 2 shows a tetrahedron to depict three planes of the quaternary lle for the ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) systems . for example , the quaternary system ( water + propan-1-ol + dec + benzene ) is comprised of three ternary subsystems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + dec + benzene).table 2experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) for mole fractions x at the temperature t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04750.00000.95250.99780.00000.00220.01780.10730.87490.97670.01760.00570.09690.16420.73890.92800.06410.00790.13910.23900.62190.92940.06600.00460.20990.28790.50220.90860.08680.00460.26360.32120.41520.94270.05190.00540.32310.35820.31870.93200.06140.0066table 3experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + benzene ( 3 ) for mole fractions x at t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04250.12340.83410.99330.00480.00190.03360.22850.73790.99260.00520.00220.03730.30030.66240.99100.00660.00240.04120.38520.57360.99060.00660.00280.04230.46660.49110.99220.00540.00240.04050.50970.44980.99340.00390.00270.03830.56340.39830.99510.00290.0020table 4experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + cyclohexane ( 3 ) for mole fractions x at t = 298.15 korganic phaseaqueous phase x 1 x 2 x 3 x 1 x 2 x 3 0.04630.09480.85890.97100.00190.02710.03470.17700.78830.97650.00140.02210.05280.25920.68800.98030.00120.01850.05870.31980.62150.98790.00130.01080.04720.38800.56480.98170.00140.01690.04980.40840.54180.98010.00150.01840.04400.44620.50980.97640.00180.02180.04750.48500.46750.97630.00160.02210.05020.56640.38340.97960.00200.01840.04450.62200.33350.97950.00190.0186table 5experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + benzene ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3diethyl carbonate + ( 1 x 1 x 2 x 3)benzene } x 3 = 0.20 0.96420.03580.00000.03200.09130.2290 0.95230.04770.00000.08230.20970.1960 0.95180.04820.00000.07320.26530.1721 0.93870.06130.00000.10710.32930.1630 0.92220.07780.00000.12110.37060.1429 0.93200.06800.00000.13850.41850.1320 0.92350.07650.00000.16890.44320.1164 0.92900.07100.00000.19330.47340.1064 0.92070.07930.00000.24990.45350.0817 0.92440.07560.00000.28830.43940.0706 0.92430.07570.00000.32710.43340.0541 x 3 = 0.40 0.93060.06940.00000.03590.12740.4270 0.92040.07960.00000.06740.21290.3407 0.90300.09700.00000.07280.28410.3150 0.92780.07220.00000.10490.30290.3050 0.89950.10050.00000.14460.38300.2328 0.90090.09910.00000.16110.40890.2209 0.87560.12440.00000.20800.44540.1701 0.91050.08950.00000.27530.45710.1216 x 3 = 0.60 0.91750.08010.00230.05980.08820.5652 0.90730.09070.00200.10360.16660.4888 0.91120.08720.00160.10270.22790.4832 0.89240.10590.00170.15480.28700.3883 0.90750.09090.00150.20890.33310.3070 0.90220.09590.00190.25240.37840.2222 0.87360.12430.00220.30480.38750.1723 0.87310.12400.00290.33320.40330.1521 0.88810.10950.00240.45050.37140.0968 x 3 = 0.80 0.87650.11960.00390.46190.36930.1195 0.87710.11920.00370.52000.35700.0837 0.86970.12680.00350.51480.35040.0910 0.88230.11400.00370.56830.32710.0656 0.87370.12230.00400.57940.32060.0661 0.89410.10020.00570.18350.27440.4418 0.89480.10190.00330.25200.33960.3213 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) benzene } mole fraction ratio of dec and benzene in the binary mixturestable 6experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + cyclohexane ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3 diethyl carbonate + ( 1 x 1 x 2 x 3 ) cyclohexane } x 3 = 0.20 0.94710.04860.00430.01520.01370.1937 0.94270.05310.00420.01740.04690.1758 0.92680.06910.00410.02860.09010.1642 0.92510.07290.00210.03870.15230.1300 0.92040.07760.00190.05890.20940.1271 0.91510.08280.00210.10970.24400.1069 0.91650.08180.00170.17410.25570.0889 0.91770.08070.00160.19840.24990.0795 x 3 = 0.40 0.95250.04300.00450.02830.03610.3716 0.94180.05420.00400.03900.06360.3458 0.93380.06330.00290.03470.12710.3111 0.94110.05670.00210.08100.18740.2750 0.92900.06910.00190.14370.22050.2246 0.92840.06980.00190.17710.24440.1925 0.92590.07220.00190.21780.27780.1669 0.92570.07220.00210.24840.28270.1422 x 3 = 0.60 0.96020.03740.00240.04720.04550.5430 0.94630.05170.00190.05930.08200.5452 0.93810.05950.00240.06120.14710.4862 0.93590.06160.00250.14440.17890.3642 0.93690.06120.00190.19740.24020.3246 0.94010.05790.00200.22840.25470.2710 0.93110.06710.00180.25120.27170.2316 0.92800.06920.00280.28740.28360.1957 x 3 = 0.80 0.96430.03250.00320.08860.04910.7099 0.95520.04190.00290.11900.09570.6311 0.94580.05120.00300.15380.15020.5490 0.93990.05720.00290.20870.19650.4566 0.93640.05620.00740.24130.24050.3934 0.93630.05850.00510.28260.25460.3359 0.93080.06430.00490.32340.27110.2878 0.92130.07500.00370.36730.27870.2441 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) cyclohexane } mole fraction ratio of dec and cyclohexane in the binary mixturestable 7experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + heptane ( 4 ) for mole fractions x at t = 298.15 kaqueous phaseorganic phase x 1 x 2 x 3 x 1 x 2 x 3 { x 1water + x 2propan-1-ol + x 3diethyl carbonate + ( 1 x 1 x 2 x 3 ) heptane } x 3 = 0.20 0.95140.04600.00000.03080.09500.2079 0.92860.07140.00000.04110.25870.1657 0.84360.15640.00000.06020.38290.1323 0.90980.09020.00000.07050.41480.1247 0.89610.10390.00000.12260.48160.0854 0.83830.16170.00000.16860.48420.1047 0.81600.18400.00000.20930.50070.0729 0.83370.16630.00000.21950.50720.0642 0.86510.13490.00000.30010.50060.0439 x 3 = 0.40 0.91810.07240.00460.03120.11020.5550 0.94740.04400.00400.03870.20090.4438 0.90370.08790.00210.05260.29570.3276 0.90190.09010.00200.09500.38690.2509 0.90420.08920.00190.14250.46410.1865 0.89890.09420.00200.21220.47790.1498 0.89330.09930.00300.23260.50010.1183 0.88340.10950.00260.27660.50090.0983 x 3 = 0.60 0.95490.03300.00540.04540.05900.5137 0.94360.04650.00390.04510.12320.4845 0.93570.05470.00400.06760.19690.4218 0.92560.06460.00380.10790.25500.3637 0.92620.06230.00620.15860.32200.2947 0.91880.06830.00540.22120.34120.2482 0.91400.07420.00510.25790.36030.2126 0.91660.07400.00500.28590.37930.1837 x 3 = 0.80 0.95240.03740.00400.06120.08680.6616 0.94490.04570.00400.07630.13680.6154 0.93630.05330.00370.13360.20680.5077 0.93410.05770.00340.17610.25990.4263 0.92760.06300.00300.24230.30410.3459 0.92440.06520.00370.27890.32570.2948 0.92100.06790.00410.30330.34760.2612 0.88990.09700.00520.33980.35240.2267 obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) heptane } mole fraction ratio of dec and heptane in the binary mixturesfig . 2phase equilibria of ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) ; x 3 denotes a quaternary section plane experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) for mole fractions x at the temperature t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + benzene ( 3 ) for mole fractions x at t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the ternary system of water ( 1 ) + diethyl carbonate ( 2 ) + cyclohexane ( 3 ) for mole fractions x at t = 298.15 k experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + benzene ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) benzene } mole fraction ratio of dec and benzene in the binary mixtures experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + cyclohexane ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) cyclohexane } mole fraction ratio of dec and cyclohexane in the binary mixtures experimental ( liquid + liquid ) equilibrium data for the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + diethyl carbonate ( 3 ) + heptane ( 4 ) for mole fractions x at t = 298.15 k obtained by mixing pure water and propan-1-ol with the binary mixtures of { x 3 dec + ( 1 x 3 ) heptane } mole fraction ratio of dec and heptane in the binary mixtures phase equilibria of ( water + propan-1-ol + dec + benzene or cyclohexane or heptane ) ; x 3 denotes a quaternary section plane the modified uniquac and extended uniquac models were employed to correlate the experimental lle data . 3experimental and calculated lle of ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) at t = 298.15 k. filled circle , experimental tie - line data ; lines , predicted results by the modified uniquac model using binary parameters taken from table 9 ; dotted lines , correlated results by the modified uniquac model using binary and ternary parameters taken from tables 9 and 10 calculated results of ternary liquid liquid equilibria modified uniquac model extended uniquac model root - mean - square deviation ( mol-% ) predicted results using binary parameters taken from the table 9 correlated results using binary and ternary parameters experimental and calculated lle of ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) at t = 298.15 k. filled circle , experimental tie - line data ; lines , predicted results by the modified uniquac model using binary parameters taken from table 9 ; dotted lines , correlated results by the modified uniquac model using binary and ternary parameters taken from tables 9 and 10 table 11 summarizes the quaternary parameters , 2341 , 1342 , 1243 and 1234 , together with the correlated results obtained by fitting the modified and extended uniquac models with binary , ternary , and quaternary parameters to the experimental quaternary lle data , together with the predicted results by the models with only the binary and ternary parameters listed in tables 9 and 10 . 6distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + heptane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + benzene ( 4 ) as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + cyclohexane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively distribution coefficient , d , of dec in the quaternary system of water ( 1 ) + propan-1-ol ( 2 ) + dec ( 3 ) + heptane ( 4 ) , as a function of mole fraction of dec in the organic - rich phase , x 3 : filled circle , white circle , filled square , white square , x 3 = 0.2 , 0.4 , 0.6 , and 0.8 , respectively under atmospheric pressure , the experimental lle data for the ternary systems ( water + propan-1-ol + dec ) , ( water + propan-1-ol + benzene ) and ( water + propan-1-ol + cyclohexane ) , and quaternary systems of ( water + propan-1-ol + dec + benzene ) , ( water + propan-1-ol + dec + cyclohexane ) and ( water + propan-1-ol + dec + heptane ) were obtained at t = 298.15 k. the experimental lle results were successfully correlated using the extended and modified uniquac models .

primary sjgren 's syndrome ( pss ) is an autoimmune disease with exocrine gland dysfunction and at least one - third of patients experience multiorgan involvement . furthermore , 5% of patients may develop lymphoma , mainly the mucosa - associated lymphoid tissue ( malt ) non - hodgkin lymphoma ( nhl ) , which represents the most severe complication of the disease . histologically , pss is characterized by extensive target tissue infiltration of lymphocytes , mainly represented in the salivary glands by t cells , predominantly cd4 t cells , but also cd8 t cells . although t cells predominate in mild lesions , b cells are the most represented cell subset in the advanced lesions , with a decreased percentage of macrophages and an increased percentage of dendritic cells [ 46 ] . infiltrating lymphocytes are often organized into tertiary ectopic lymphoid structures , showing a network including specific segregated t- and b - cell zones , associated with follicular dendritic cells , with a specific glandular cytokine profile . despite the presence , and sometimes predominance , of t cells in salivary gland infiltrates , cd4 t helper ( th ) lymphocytes have been long known to be distributed into th1 and th2 cells , based on distinct cytokine patterns . an imbalance between type 1 cytokine - producing th1 cells and type 2 cytokine - producing th2 cells has been considered as predisposing to autoimmunity . historically , pss was thought to be a th1 driven disease due to the predominance of cd4 t lymphocytes and their products , namely , interferon- ( ifn- ) , in target organs and peripheral blood ( pb ) of these patients . inevitably , on the basis of in vitro and in vivo observations , the role of th1 and th2 cells in pss has become contradictory . in the last decade , a number of th cell lineages , including th0 , th17 , regulatory t ( treg ) , and follicular helper t ( tfh ) cells , have been identified . this challenged the long - standing paradigm of a th1/th2 immune response and prompted to identify their role in the pathogenesis of autoimmune diseases including pss . in particular , th17 cells were described and il-17 was acknowledged as a prime representative of the new generation of proinflammatory cytokines . concomitantly , regulatory t ( treg ) cells were identified as a unique population of th cells that restrain excessive activation of effector lymphocytes . besides the role of different cell subsets in pss pathogenesis , the impact of abnormal cytokine production , such as il-6 , il-17 , and baff , has also attracted considerable attention . in particular , it is a challenge to understand how the interaction between several interconnected networks of cytokines impact so many different cell populations , on one hand , and how the interplay of cytokine - producing t and b cells shifts the balance towards autoreactive t and b lymphocytes , on the other . the ongoing progress in discovering lymphocyte subsets and the lengthening list of cytokines involved has further fuelled the debate on pss pathogenesis ( figure 1 ) . the main purpose of this review is to summarize and highlight the role of il-17-producing t cells and treg cells in pss pathogenesis , offering the rationale for new therapeutic approaches in this disease . treg cells were initially identified in mice and humans according to the high surface expression of the alpha chain of il-2 receptor ( il-2r , cd25 ) and the capability to prevent polyautoimmunity in an experimental animal model . this cellular subset exerts suppressive activity towards autoreactive lymphocytes via either cell - cell contact or the release of soluble mediators including il-10 and transforming growth factor ( tgf- ) . the commitment of a nave t lymphocyte towards a treg phenotype is dependent of a peculiar cytokine microenvironment and of the expression of the forkhead box protein p3 ( foxp3 ) transcriptional factor , which ensures treg suppressive function and represents to date the most specific treg marker [ 13 , 14 ] . intriguingly , soluble mediators required for treg commitment are also those which participate in the commitment of a pathogenic il-17-producing t helper ( th ) subset , the th17 cells . in fact , tgf- is required in both cases , but the concurrent presence or absence of il-6 leads to the generation of either th17 or treg cells , respectively . it is evident , therefore , that such a fine balance between these two cell subsets may be easily disturbed leading to a predominance of pathogenic cells and therefore to the development of autoimmunity . in this context , it has been demonstrated that a committed treg cell can be turned into a th17 cell in the presence of appropriate stimuli . an interesting study , employing a foxp3 reporter mouse , revealed that the blockade of indoleamine 2,3-dioxygenase , a master regulator of self - tolerance , in the presence of il-6 induced conversion of treg into th17-like cells in rodent tumor - draining lymph nodes . as far as the role of treg cells in pss pathogenesis is concerned , ten studies have been published and the results are often controversial [ 1726 ] . therefore , similar to systemic lupus erythematosus ( sle ) and rheumatoid arthritis ( ra ) , conclusive data are still lacking [ 27 , 28 ] ( table 1 ) . such discrepancies may be explained at least in part by the different strategies employed to assess treg cells over time . generally , the approach of earlier studies was to enumerate the proportion of circulating treg cells according to the high surface expression of cd25 . subsequently , however , the coexpression of foxp3 , the most specific marker of treg cells , was also evaluated . five studies reported an overall reduction of pb cd25treg cells [ 18 , 19 , 22 , 24 , 26 ] , but , of note , an association between this reduction and clinical or serological features was observed only in 2 studies . in detail , liu et al . reported an inverse correlation between the percentage of treg cells and c reactive protein , erythrocyte sedimentation rate , rheumatoid factor , and immunoglobulin ( ig ) g concentration . conversely , szodoray et al . described that treg cell reduction in the pb was more pronounced in patients with a milder clinical picture not presenting extraglandular manifestations . in striking contrast , two studies reported an increase of circulating treg cells in pss , not associated , however , with any clinical or serological features [ 17 , 23 ] and three described that pb cd4cd25 cell percentages were similar in pss and controls [ 20 , 21 , 25 ] . only two studies tried to correlate the disease activity with the percentage of peripheral treg cells . in particular , we subdivided patients into two groups according to the eular sjogren 's syndrome disease activity index ( essdai ) values : patients with essdai 2 , whose only manifestation of disease was a mild stable polyclonal hypergammaglobulinemia , were considered inactive , whereas patients with essdai > 2 were classified as active . we observed that the disease activity did not influence the number of circulating cd4cd25treg cells . similarly , another study defined clinical active disease as the presence of one or more glandular and extraglandular features recognized by the essdai and the sjgren syndrome disease activity index ( ssdai ) with the exception of fatigue and serologic activity as the presence of increased serum viscosity , elevated igg , igm , iga , or decreased levels of complements c3 and c4 . the apparently paradoxical increase as well as the normal values of circulating treg cell percentages described in some studies deserves some consideration . the surface expression of cd25 in not limited to treg cells but can be shared by recently activated lymphocytes . in this setting , han et al . observed that only a subgroup of cd25 t cells coexpresses foxp3 in ra patients . therefore , higher percentages of cd25 t cells in ra patients may be due more likely to a contamination of recently activated cells rather than to an increase of real treg cells . in line with this hypothesis , sarigul et al . reported that , besides an increase of circulating cd25 treg cells in pss , the proportion of foxp3 cells in the pb was comparable to that of normal subjects and patients with ra . in addition , unlike murine cd25 treg cells , those isolated from humans include different cell subsets that , although developmentally related , display different phenotype and function . on this basis , in 2011 , miyara and sakaguchi suggested that the assessment of cd45ra on the cell surface and of helios transcription factor may help to distinguish treg cell subsets with consistent suppressive activity . unfortunately such approach was not employed in the majority of studies investigating treg cell in pss ; hence several issues concerning the reproducibility and comparability of different studies remain open . finally , it has been recently put forward the hypothesis that cd25 expression is not mandatory to confer a regulatory phenotype . in this setting , recent studies identified a t lymphocyte subpopulation expressing foxp3 , but lacking cd25 surface molecule , that is expanded in the pb of patients with sle [ 33 , 34 ] . however , the lack of a consistent suppressive activity exerted by cd25foxp3 cells underscored the need to identify more specific treg cell markers to be combined with foxp3 . translating the knowledge on murine treg cells to humans , the glucocorticoid induced tumor necrosis factor receptor related protein ( gitr ) , a well - characterized marker of treg cells in mice , gained growing scientific interest . in fact , gitr and foxp3 coexpression allows identifying t cells with regulatory phenotype and function independently of cd25 expression in normal subjects . of interest , gitr blockade abrogates their suppressive activity suggesting that this molecule is involved in conferring regulatory properties . moreover , a functionally suppressive cd25gitr cell subset was found to be expanded in the pb of patients with sle or pss and this expansion was associated with a reduction of conventional cd25 treg cells [ 26 , 37 ] . intriguingly , the expansion of cd25gitr cell was more pronounced in patients with inactive disease suggesting a certain attempt to rebalance the reduction of conventional treg cells that is working , at least , in milder disease . moving to tissue level , the evaluation of treg cells in pss minor salivary glands ( msgs ) was performed in four studies [ 18 , 21 , 23 , 26 ] . observed a reduced number of cd25 cells in pss - msgs compared to those with nonautoimmune parotitis . however , taken the fact that also activated cells may express cd25 , these observations do not allow drawing any definitive conclusion on the presence of treg in pss - msgs . therefore , foxp3 has been subsequently analyzed with either immunohistochemistry [ 18 , 21 , 23 , 26 ] or polymerase chain reaction , showing a consistent expression of this transcription factor and supporting the presence of treg cells in pss - msg mononuclear cell infiltrate . of note , however , both cd25 and cd25 cells were included in the foxp3 area within the salivary gland infiltrate , and some of them coexpressed gitr , suggesting the presence of not only conventional cd25 , but also the aforementioned cd25gitr suppressive cell subset in msg during pss . of particular interest , an attempt to correlate foxp3 staining , namely the amount of infiltrating treg cells , and the extent of glandular involvement was also pursued in two studies [ 21 , 23 ] . these findings , concerning the correlation between the amount of infiltrating treg cells and the severity of tissue inflammation , are in line with those obtained in rheumatoid synovium and point out the role of treg cells in counteracting local tissue inflammation that , however , may be ineffective [ 38 , 39 ] . in fact , although the in vitro functional assays performed in four studies pointed out that the suppressive activity of pb cd25 cells seems preserved in pss [ 17 , 18 , 22 , 26 ] , it is not possible to ascertain whether treg cells are able to exert their suppressive activity in vivo or they are affected by local inflammatory microenvironment . il-17 is a family of cytokines including six members , from a to f , with a wide range of biological activities . besides physiological processes , such as host defense against microbial infections , il-17 is a leading actor in pathologic conditions , including cancer and autoimmune disorders , due to a strong proinflammatory potential . in fact , upon binding to its receptor , il-17 triggers downstream events culminating in the transcription of proinflammatory genes , such as cytokines , chemokines , and matrix degrading enzymes , by target cells . although the main cellular source of il-17 is represented by t lymphocytes , growing evidence suggests that also neutrophils and mast cells participate in the balance of this cytokine [ 42 , 43 ] . the commitment of a nave cd4 t lymphocyte towards a t helper ( th ) 17 cell occurs in the presence of a peculiar cytokine milieu . indeed , the upregulation of the retinoic acid orphan receptor ( ror ) t transcription , the expression of il-17 , and the stabilization of the th17 phenotype require the concurrent presence of il-6 , tgf- , il-21 , il-1 , and il-23 . besides il-17 , th17 cells are able to produce also il-21 and il-22 . il-21 collaborates with dendritic cell - derived tgf- to amplify the tendency to th17-cell differentiation and induces these lymphocytes to express receptors for il-23 . in addition , it has been recently reported that a subset of th17 cells , as identified by the coexpression of ccr6 and cxcr3 , is able to produce also ifn- . taken the well - established role of ifn- in the pathogenesis of autoimmune diseases , including pss , this intriguing finding further underscores the relevance of th17 cells in such scenario . it is interesting that , at least in mice , th17 lymphocytes can also function as b - cell helpers . they induce , indeed , a pronounced antibody response , with preferential immunoglobulin ( ig ) class switch to igg2a and igg3 for il-17 and to igg1 and igg2b for il-21 . these results establish that th17 cells are crucial in germinal center ( gc ) formation . as suggested above , the proinflammatory il-17 , normally considered a t - cell - associated factor , has been also reported to be a central driver of gc - derived autoantibodies . this was demonstrated by blocking il-17 signaling that disrupted the cd4t - cell and b - cell interactions required for gc formation . the evidence that il-17-knockout ( ko ) mice are less prone to develop autoimmune diseases such as type 1 diabetes , collagen - induced arthritis , and experimental autoimmune encephalomyelitis [ 41 , 47 ] raised the hypothesis that this cytokine , and therefore il-17-producing cells , may also be involved in the pathogenesis of pss . experimental ss in the il-17-ko mouse was only recently evaluated . despite being immunized with salivary gland peptides intriguingly , the adoptive transfer of th17 cells was able to induce a focal sialadenitis similar to that of wild type mice , underscoring the pathogenic role of il-17 also in pss . first reported that il-17 overexpression in salivary glands by adenovirus vectors was able to trigger a ss - like condition in nonsusceptible mice . in this setting , three studies described that il-17 is consistently expressed in the periductal infiltrates of all msgs from patients with pss [ 5052 ] and in two of them such expression was found to be associated with the severity of glandular inflammation [ 51 , 52 ] . in addition , most of the cytokines that support the th17 phenotype as well as other th17-cell products , including il-6 , il-21 , il-22 , and il-23 and their receptors , are consistently expressed in msgs of patients with pss [ 50 , 5257 ] . similar to il-17 , also il-21 expression in msgs appears to parallel the severity of glandular inflammation . the main cellular source of glandular il-17 in pss - msgs appears to be constituted by cd4 and , to a lesser extent , cd8 t lymphocytes [ 58 , 59 ] . of interest , however , two recent studies pointed out that also cd4cd8 ( double negative , dn ) t cells [ 60 , 61 ] and mast cells may participate in il-17 local balance in pss . dn t cells were initially identified as a source of il-17 in sle and their presence in the inflammatory infiltrate of kidney during lupus nephritis suggested a pathogenic role of this cell subset in sle . in pss , dn t cells were found to be associated not only with the extent of glandular involvement , as already demonstrated for il-17 [ 51 , 52 ] , but also with the presence of msg ectopic lymphoid structures , which have been associated with more severe clinical phenotype , including b - cell lymphoma [ 7 , 63 ] . this evidence appears to support the pathogenic role of il-17 and il-17-producing cells not only in the induction but also in the perpetuation of glandular lesions . il-17 and its related cytokines have also been evaluated in other biological samples from pss patients such as saliva , tears , and serum . to date , only one study assessed il-17 in pss saliva reporting higher levels of this cytokine compared to non - pss , but failing to identify any association between the concentration of this cytokine and the extent of msg lymphocytic infiltration . in addition , there is general agreement among available studies of increased il-17 concentration in the tears of pss patients compared to non - pss dry eye [ 6568 ] . as far as serum is concerned , the four studies that assessed il-17 pointed out that only a subgroup of pss patients display detectable levels of this cytokine [ 50 , 51 , 69 , 70 ] , but only two of these found an association between serum il-17 and clinical / histological features of pss [ 69 , 70 ] . in particular , it was shown that disease duration was significantly longer and parotid gland swelling was less prevalent in il-17-positive patients compared to those il-17-negative ones . however , multivariate analysis revealed that disease duration was associated with the presence of serum il-17 independently of concurrent parotid gland swelling . furthermore , il-17 serum concentration was higher in patients with ectopic gc - like structures compared to those without gcs . also il-21 has been found to be increased in pss serum , while il-6 and il-23 have been found to be increased in pss plasma in association with increased levels of il-17 . to shed additional light on peripheral il-17 balance , enumeration of il-17-producing cells in the pb has been performed in a series of studies . an overall increase of circulating cd4th17 cells [ 51 , 5961 ] and dn t cells [ 60 , 61 ] has been described in pss patients compared to controls . intriguingly , however , in a study performed by our group , differences in the percentage of these cell subsets according to disease duration were also highlighted . it appeared , indeed , that in early pss , with symptom duration less than 18 months , cd4th17 cells were expanded , while dn t cells were comparable to those of normal subjects . conversely , in patients with established disease and symptom duration over 5 years , cd4th17 cell percentage was comparable to that of controls and dn t cells were expanded . taken together , these findings in pss msg , serum / plasma , and pb suggest a highly dynamic scenario occurring in the course of the disease with a clear pathogenic role of il-17 in triggering and maintaining glandular inflammation and a recirculation of il-17-producing t - cell subsets from pb to msg and vice versa in different phases of the disease ( table 1 ) . however , despite their clear pathogenic role , no association between il-17/th17 cells and severity of clinical picture or specific extraglandular clinical manifestations has been reported . we believe that this last consideration does not diminish but rather reinforces the role of il-17/th17 system in pss pathogenesis . in fact , this biological system is crucial in the induction as well as in the maintenance of pss , independently of the clinical or serological features of the disease , thus representing an interesting and promising therapeutic targeting in this disease . in conclusion , although interesting , all the aforementioned studies evaluating treg and th17 cells are weighted by a number of intrinsic limitations that deserve some consideration . first , pss is a heterogeneous disease characterized by different genetic background ( e.g. , hla haplotypes ) , clinical manifestations ( glandular versus extraglandular ) , and serological status ( anti - ssa , anti - ssb , both , or none ) . all the above imply different therapeutic approaches ( lacrimal or salivary substitutes versus immune suppressants ) . since in most studies patients are not subgrouped according to disease features , including disease duration or therapy , results may be biased . furthermore , the majority of investigations are performed in peripheral blood rather than target organs providing a partial view on such scenario . therefore , the balance of different cytokines and soluble mediators as well as their effects on t cells should be investigated in vivo rather than in vitro . finally , it is still a matter of debate whether treg cell impairment and th17 cell predominance may be either a cause or a consequence of the ongoing inflammation . pss is an autoimmune disorder affecting exocrine glands and is characterized , in most cases , by a rather mild clinical picture . however , a subgroup of pss patients experience systemic extraglandular involvement leading to a worsening of disease prognosis . current therapeutic options for the treatment of pss are mainly empiric , often translated by other autoimmune diseases , and recent systematic reviews highlighted the lack of evidence - based recommendations for most of the immunosuppressive drugs commonly employed in the spectrum of extraglandular involvement . in this setting , therefore , pss may be still considered an orphan disease [ 7275 ] . in recent years , the effects of currently available therapies on treg and th17 cells have been extensively investigated in autoimmune diseases , in particular ra and psoriasis [ 40 , 76 ] . this is partially explained by the fact that only a subgroup of patients display extraglandular manifestations requiring immunosuppressive therapies . furthermore , most of the biologic therapies employed in ra are not currently used in pss for the lack of either proven clinical efficacy ( e.g. , tnf blockers ) , clinical trials ( e.g. , tocilizumab ) , or , although effective , specific licensing ( e.g. , abatacept ) . it is of note , however , that corticosteroids ( cs ) may affect the il-17 axis during pss . although a case report described infiltrate reduction in one patient with pss receiving cs at high doses , no further evidence supports the efficacy of cs in reducing glandular inflammation to date [ 77 , 78 ] . subsequently , it has been demonstrated that the subset of il-17-producing dn t lymphocytes , isolated from pss patients , is resistant to cs in vitro . conversely , dn t cells from normal controls promptly respond to cs in vitro by reducing il-17 production . these observations give rise to an intriguing line of investigation in the clinical scenario of cs - resistance of autoimmune / inflammatory diseases . as far as treg cell selective therapeutic targeting is concerned , approaches promoting the in vivo expansion of tregs or injection of in vitro expanded autologous / heterologous tregs are under intense investigation in different acute and chronic diseases to date with promising results . in particular , data from murine studies suggest that the transfer of autologous treg cells is able to prevent the development of collagen - induced arthritis and colitis , while ex vivo expanded cd4cd25 treg cells can attenuate the development of experimental autoimmune encephalomyelitis and ameliorate type i diabetes . in humans , treg cells transfer is able to prolong immune tolerance following hematopoietic stem cell transplantation and to prevent graft - versus - host disease . treg cell transfer is safe in patients with severe crohn 's disease and an open - label phase i trial to investigate safety and efficacy of intravenous infusion of ex vivo selected and expanded autologous polyclonal treg cells in patients with type 1 diabetes is currently ongoing ( trial nct01210664 ) . these encouraging results allow speculating that reprogramming treg cells or infusing in vitro expanded autologous / heterologous treg cells may affect the natural history of autoimmune diseases . first , taken the aforementioned debate about the specificity of cd25 as treg cell marker , additional molecules should be identified to isolate and expand the most suitable treg cell subset . in this setting , since under some conditions , the regulatory activity of cd4cd25gitr cells is superior to that of cd4cd25gitr cells , gitr may be a good candidate . second , stability and fate of transferred treg cells in vivo are not predictable and , according to recent findings , they may be converted into effector cells , namely , th17 cells , by proinflammatory cytokines [ 16 , 82 ] . on the other hand , the growing amount of data supporting the pathogenic role of il-17 axis in the pathogenesis of pss provided the rational for a number of clinical trials that are currently ongoing in pss . two monoclonal antibodies against il-17 ( the fully human igg1k secukinumab and the humanized igg4 ixekizumab ) are being investigated in inflammatory arthritides and a clinical trial evaluating secukinumab in dry eye patients is currently ongoing ( trial nct01250171 ) . concerning the molecules that are involved in the balance between treg and th17 cell commitment , the humanized anti - il-6 antibody tocilizumab is employed in clinical practice for the treatment of ra and a phase ii trial to assess its efficacy in pss is currently ongoing ( trial nct01782235 ) . since il-6 presence or absence in the local microenvironment drives the polarization of a nave t cell to a th17 or a treg phenotype , respectively , it would be of great interest to ascertain the effect of il-6 blockade in such a scenario . compounds targeting il-23 and il-21 have been or are under investigation in plaque psoriasis and chronic inflammatory arthritides , but not yet in pss [ 84 , 85 ] . another interesting aspect is the possible effect on il-17 axis exerted by non - t - cell selective compounds . in fact , previous studies that investigated the effects of anti - cd20 antibody rituximab on t lymphocytes in ra revealed that this compound is able to deplete cd4th17 cells in pb and synovium as they coexpress cd20 [ 8688 ] . taken that rituximab is currently the most employed biologic agent in pss , if these observations will be confirmed in the pb and msg of pss patients , an additional rational for therapeutic application of rituximab in pss may be defined . finally , another intriguing approach is represented by the interference with th17 commitment via the modulation of rort activity in the thymus . a study that employed a synthetic ligand binding to this transcription factor in a mouse model of multiple sclerosis reported impressive clinical efficacy . these data point out the need of randomized clinical trials to investigate the safety and efficacy of these drugs in pss in order to provide solid scientific evidence . taken together , the difficulty to build therapeutic recommendations in pss may be related to the heterogeneity of clinical picture , the frequent failure of first line treatments , the lack of scientific evidence for drugs licensed for other diseases , and , finally , the lack of innovative therapeutic compounds . pss encompasses several subsets of patients with different genetic background , pathophysiological pathways , demographic features , and different response to proposed therapies . despite the acknowledged role of t - cell subsets in pss , mechanisms leading to their abnormal activation and their contribution to pss pathogenesis are not fully elucidated . in particular , only few studies evaluated the role of treg cells in pss , and conclusive data are still lacking . similarly , although conventional cd4th17 and il-17-producing dn t cells are under active investigation , their origin , fate , and function are still a matter of debate . currently available data suggest a pivotal role of il-17 axis and il-17-producing cells in every step of pss pathogenesis , from the induction of autoimmune epithelitis to the maintenance and perpetuation of inflammation and ectopic gc formation . in this context , il-17-producing t cells seem to be a link between t- and b - cell compartments . in this setting , a therapeutic approach targeting il-17 axis may represent an intriguing issue worth to be investigated in pss . in the era of biologic agents , randomized double - blind controlled trials represent the most powerful tool to obtain comparable results and provide the rationale to build solid therapeutic recommendations also in pss .

historically , primary sjgren 's syndrome ( pss ) was thought to be a t helper ( h ) 1 driven disease due to the predominance of cd4+t lymphocytes and their products in target organs and peripheral blood of patients . in the last decades , the identification of a number of t cell subsets , including th17 , t regulatory ( treg ) , and follicular helper t cells , challenged this long - standing paradigm and prompted to identify their role in pss pathogenesis . in addition the impact of abnormal proinflammatory cytokine production , such as il-6 , il-17 , il-22 , and il-23 , has also attracted considerable attention . however , although several studies have been carried out in experimental models and patients with pss , many aspects concerning the role of treg cells and il-17/th17 cell system in pss pathogenesis are not fully elucidated . in particular , the role played by different il-17-producing t cell subsets as well as the effects of pharmacological therapies on treg / th17 cell balance represents an intriguing issue . the aim of this review article is to provide an overview of current knowledge on treg cells and il-17-producing t cells in pss pathogenesis . we believe that these insights into pss pathogenesis may provide the basis for successful therapeutic intervention in this disease .

1. Introduction 2. Regulatory T Cells in pSS 3. IL-17-Producing T Cells in pSS 4. Therapeutic Perspectives 5. Conclusion

primary sjgren 's syndrome ( pss ) is an autoimmune disease with exocrine gland dysfunction and at least one - third of patients experience multiorgan involvement . furthermore , 5% of patients may develop lymphoma , mainly the mucosa - associated lymphoid tissue ( malt ) non - hodgkin lymphoma ( nhl ) , which represents the most severe complication of the disease . histologically , pss is characterized by extensive target tissue infiltration of lymphocytes , mainly represented in the salivary glands by t cells , predominantly cd4 t cells , but also cd8 t cells . although t cells predominate in mild lesions , b cells are the most represented cell subset in the advanced lesions , with a decreased percentage of macrophages and an increased percentage of dendritic cells [ 46 ] . despite the presence , and sometimes predominance , of t cells in salivary gland infiltrates , cd4 t helper ( th ) lymphocytes have been long known to be distributed into th1 and th2 cells , based on distinct cytokine patterns . historically , pss was thought to be a th1 driven disease due to the predominance of cd4 t lymphocytes and their products , namely , interferon- ( ifn- ) , in target organs and peripheral blood ( pb ) of these patients . inevitably , on the basis of in vitro and in vivo observations , the role of th1 and th2 cells in pss has become contradictory . in the last decade , a number of th cell lineages , including th0 , th17 , regulatory t ( treg ) , and follicular helper t ( tfh ) cells , have been identified . this challenged the long - standing paradigm of a th1/th2 immune response and prompted to identify their role in the pathogenesis of autoimmune diseases including pss . besides the role of different cell subsets in pss pathogenesis , the impact of abnormal cytokine production , such as il-6 , il-17 , and baff , has also attracted considerable attention . in particular , it is a challenge to understand how the interaction between several interconnected networks of cytokines impact so many different cell populations , on one hand , and how the interplay of cytokine - producing t and b cells shifts the balance towards autoreactive t and b lymphocytes , on the other . the main purpose of this review is to summarize and highlight the role of il-17-producing t cells and treg cells in pss pathogenesis , offering the rationale for new therapeutic approaches in this disease . treg cells were initially identified in mice and humans according to the high surface expression of the alpha chain of il-2 receptor ( il-2r , cd25 ) and the capability to prevent polyautoimmunity in an experimental animal model . intriguingly , soluble mediators required for treg commitment are also those which participate in the commitment of a pathogenic il-17-producing t helper ( th ) subset , the th17 cells . in fact , tgf- is required in both cases , but the concurrent presence or absence of il-6 leads to the generation of either th17 or treg cells , respectively . it is evident , therefore , that such a fine balance between these two cell subsets may be easily disturbed leading to a predominance of pathogenic cells and therefore to the development of autoimmunity . in this context , it has been demonstrated that a committed treg cell can be turned into a th17 cell in the presence of appropriate stimuli . an interesting study , employing a foxp3 reporter mouse , revealed that the blockade of indoleamine 2,3-dioxygenase , a master regulator of self - tolerance , in the presence of il-6 induced conversion of treg into th17-like cells in rodent tumor - draining lymph nodes . as far as the role of treg cells in pss pathogenesis is concerned , ten studies have been published and the results are often controversial [ 1726 ] . generally , the approach of earlier studies was to enumerate the proportion of circulating treg cells according to the high surface expression of cd25 . subsequently , however , the coexpression of foxp3 , the most specific marker of treg cells , was also evaluated . reported an inverse correlation between the percentage of treg cells and c reactive protein , erythrocyte sedimentation rate , rheumatoid factor , and immunoglobulin ( ig ) g concentration . described that treg cell reduction in the pb was more pronounced in patients with a milder clinical picture not presenting extraglandular manifestations . in striking contrast , two studies reported an increase of circulating treg cells in pss , not associated , however , with any clinical or serological features [ 17 , 23 ] and three described that pb cd4cd25 cell percentages were similar in pss and controls [ 20 , 21 , 25 ] . in particular , we subdivided patients into two groups according to the eular sjogren 's syndrome disease activity index ( essdai ) values : patients with essdai 2 , whose only manifestation of disease was a mild stable polyclonal hypergammaglobulinemia , were considered inactive , whereas patients with essdai > 2 were classified as active . the apparently paradoxical increase as well as the normal values of circulating treg cell percentages described in some studies deserves some consideration . therefore , higher percentages of cd25 t cells in ra patients may be due more likely to a contamination of recently activated cells rather than to an increase of real treg cells . reported that , besides an increase of circulating cd25 treg cells in pss , the proportion of foxp3 cells in the pb was comparable to that of normal subjects and patients with ra . in addition , unlike murine cd25 treg cells , those isolated from humans include different cell subsets that , although developmentally related , display different phenotype and function . unfortunately such approach was not employed in the majority of studies investigating treg cell in pss ; hence several issues concerning the reproducibility and comparability of different studies remain open . in this setting , recent studies identified a t lymphocyte subpopulation expressing foxp3 , but lacking cd25 surface molecule , that is expanded in the pb of patients with sle [ 33 , 34 ] . however , the lack of a consistent suppressive activity exerted by cd25foxp3 cells underscored the need to identify more specific treg cell markers to be combined with foxp3 . translating the knowledge on murine treg cells to humans , the glucocorticoid induced tumor necrosis factor receptor related protein ( gitr ) , a well - characterized marker of treg cells in mice , gained growing scientific interest . moreover , a functionally suppressive cd25gitr cell subset was found to be expanded in the pb of patients with sle or pss and this expansion was associated with a reduction of conventional cd25 treg cells [ 26 , 37 ] . intriguingly , the expansion of cd25gitr cell was more pronounced in patients with inactive disease suggesting a certain attempt to rebalance the reduction of conventional treg cells that is working , at least , in milder disease . moving to tissue level , the evaluation of treg cells in pss minor salivary glands ( msgs ) was performed in four studies [ 18 , 21 , 23 , 26 ] . observed a reduced number of cd25 cells in pss - msgs compared to those with nonautoimmune parotitis . however , taken the fact that also activated cells may express cd25 , these observations do not allow drawing any definitive conclusion on the presence of treg in pss - msgs . therefore , foxp3 has been subsequently analyzed with either immunohistochemistry [ 18 , 21 , 23 , 26 ] or polymerase chain reaction , showing a consistent expression of this transcription factor and supporting the presence of treg cells in pss - msg mononuclear cell infiltrate . of note , however , both cd25 and cd25 cells were included in the foxp3 area within the salivary gland infiltrate , and some of them coexpressed gitr , suggesting the presence of not only conventional cd25 , but also the aforementioned cd25gitr suppressive cell subset in msg during pss . of particular interest , an attempt to correlate foxp3 staining , namely the amount of infiltrating treg cells , and the extent of glandular involvement was also pursued in two studies [ 21 , 23 ] . these findings , concerning the correlation between the amount of infiltrating treg cells and the severity of tissue inflammation , are in line with those obtained in rheumatoid synovium and point out the role of treg cells in counteracting local tissue inflammation that , however , may be ineffective [ 38 , 39 ] . in fact , although the in vitro functional assays performed in four studies pointed out that the suppressive activity of pb cd25 cells seems preserved in pss [ 17 , 18 , 22 , 26 ] , it is not possible to ascertain whether treg cells are able to exert their suppressive activity in vivo or they are affected by local inflammatory microenvironment . besides physiological processes , such as host defense against microbial infections , il-17 is a leading actor in pathologic conditions , including cancer and autoimmune disorders , due to a strong proinflammatory potential . in fact , upon binding to its receptor , il-17 triggers downstream events culminating in the transcription of proinflammatory genes , such as cytokines , chemokines , and matrix degrading enzymes , by target cells . although the main cellular source of il-17 is represented by t lymphocytes , growing evidence suggests that also neutrophils and mast cells participate in the balance of this cytokine [ 42 , 43 ] . the commitment of a nave cd4 t lymphocyte towards a t helper ( th ) 17 cell occurs in the presence of a peculiar cytokine milieu . indeed , the upregulation of the retinoic acid orphan receptor ( ror ) t transcription , the expression of il-17 , and the stabilization of the th17 phenotype require the concurrent presence of il-6 , tgf- , il-21 , il-1 , and il-23 . in addition , it has been recently reported that a subset of th17 cells , as identified by the coexpression of ccr6 and cxcr3 , is able to produce also ifn- . taken the well - established role of ifn- in the pathogenesis of autoimmune diseases , including pss , this intriguing finding further underscores the relevance of th17 cells in such scenario . as suggested above , the proinflammatory il-17 , normally considered a t - cell - associated factor , has been also reported to be a central driver of gc - derived autoantibodies . the evidence that il-17-knockout ( ko ) mice are less prone to develop autoimmune diseases such as type 1 diabetes , collagen - induced arthritis , and experimental autoimmune encephalomyelitis [ 41 , 47 ] raised the hypothesis that this cytokine , and therefore il-17-producing cells , may also be involved in the pathogenesis of pss . despite being immunized with salivary gland peptides intriguingly , the adoptive transfer of th17 cells was able to induce a focal sialadenitis similar to that of wild type mice , underscoring the pathogenic role of il-17 also in pss . in this setting , three studies described that il-17 is consistently expressed in the periductal infiltrates of all msgs from patients with pss [ 5052 ] and in two of them such expression was found to be associated with the severity of glandular inflammation [ 51 , 52 ] . in addition , most of the cytokines that support the th17 phenotype as well as other th17-cell products , including il-6 , il-21 , il-22 , and il-23 and their receptors , are consistently expressed in msgs of patients with pss [ 50 , 5257 ] . of interest , however , two recent studies pointed out that also cd4cd8 ( double negative , dn ) t cells [ 60 , 61 ] and mast cells may participate in il-17 local balance in pss . dn t cells were initially identified as a source of il-17 in sle and their presence in the inflammatory infiltrate of kidney during lupus nephritis suggested a pathogenic role of this cell subset in sle . in pss , dn t cells were found to be associated not only with the extent of glandular involvement , as already demonstrated for il-17 [ 51 , 52 ] , but also with the presence of msg ectopic lymphoid structures , which have been associated with more severe clinical phenotype , including b - cell lymphoma [ 7 , 63 ] . this evidence appears to support the pathogenic role of il-17 and il-17-producing cells not only in the induction but also in the perpetuation of glandular lesions . to date , only one study assessed il-17 in pss saliva reporting higher levels of this cytokine compared to non - pss , but failing to identify any association between the concentration of this cytokine and the extent of msg lymphocytic infiltration . also il-21 has been found to be increased in pss serum , while il-6 and il-23 have been found to be increased in pss plasma in association with increased levels of il-17 . intriguingly , however , in a study performed by our group , differences in the percentage of these cell subsets according to disease duration were also highlighted . taken together , these findings in pss msg , serum / plasma , and pb suggest a highly dynamic scenario occurring in the course of the disease with a clear pathogenic role of il-17 in triggering and maintaining glandular inflammation and a recirculation of il-17-producing t - cell subsets from pb to msg and vice versa in different phases of the disease ( table 1 ) . we believe that this last consideration does not diminish but rather reinforces the role of il-17/th17 system in pss pathogenesis . in fact , this biological system is crucial in the induction as well as in the maintenance of pss , independently of the clinical or serological features of the disease , thus representing an interesting and promising therapeutic targeting in this disease . in conclusion , although interesting , all the aforementioned studies evaluating treg and th17 cells are weighted by a number of intrinsic limitations that deserve some consideration . , hla haplotypes ) , clinical manifestations ( glandular versus extraglandular ) , and serological status ( anti - ssa , anti - ssb , both , or none ) . since in most studies patients are not subgrouped according to disease features , including disease duration or therapy , results may be biased . furthermore , the majority of investigations are performed in peripheral blood rather than target organs providing a partial view on such scenario . therefore , the balance of different cytokines and soluble mediators as well as their effects on t cells should be investigated in vivo rather than in vitro . current therapeutic options for the treatment of pss are mainly empiric , often translated by other autoimmune diseases , and recent systematic reviews highlighted the lack of evidence - based recommendations for most of the immunosuppressive drugs commonly employed in the spectrum of extraglandular involvement . in recent years , the effects of currently available therapies on treg and th17 cells have been extensively investigated in autoimmune diseases , in particular ra and psoriasis [ 40 , 76 ] . these observations give rise to an intriguing line of investigation in the clinical scenario of cs - resistance of autoimmune / inflammatory diseases . in particular , data from murine studies suggest that the transfer of autologous treg cells is able to prevent the development of collagen - induced arthritis and colitis , while ex vivo expanded cd4cd25 treg cells can attenuate the development of experimental autoimmune encephalomyelitis and ameliorate type i diabetes . treg cell transfer is safe in patients with severe crohn 's disease and an open - label phase i trial to investigate safety and efficacy of intravenous infusion of ex vivo selected and expanded autologous polyclonal treg cells in patients with type 1 diabetes is currently ongoing ( trial nct01210664 ) . in this setting , since under some conditions , the regulatory activity of cd4cd25gitr cells is superior to that of cd4cd25gitr cells , gitr may be a good candidate . second , stability and fate of transferred treg cells in vivo are not predictable and , according to recent findings , they may be converted into effector cells , namely , th17 cells , by proinflammatory cytokines [ 16 , 82 ] . on the other hand , the growing amount of data supporting the pathogenic role of il-17 axis in the pathogenesis of pss provided the rational for a number of clinical trials that are currently ongoing in pss . concerning the molecules that are involved in the balance between treg and th17 cell commitment , the humanized anti - il-6 antibody tocilizumab is employed in clinical practice for the treatment of ra and a phase ii trial to assess its efficacy in pss is currently ongoing ( trial nct01782235 ) . since il-6 presence or absence in the local microenvironment drives the polarization of a nave t cell to a th17 or a treg phenotype , respectively , it would be of great interest to ascertain the effect of il-6 blockade in such a scenario . compounds targeting il-23 and il-21 have been or are under investigation in plaque psoriasis and chronic inflammatory arthritides , but not yet in pss [ 84 , 85 ] . in fact , previous studies that investigated the effects of anti - cd20 antibody rituximab on t lymphocytes in ra revealed that this compound is able to deplete cd4th17 cells in pb and synovium as they coexpress cd20 [ 8688 ] . taken that rituximab is currently the most employed biologic agent in pss , if these observations will be confirmed in the pb and msg of pss patients , an additional rational for therapeutic application of rituximab in pss may be defined . taken together , the difficulty to build therapeutic recommendations in pss may be related to the heterogeneity of clinical picture , the frequent failure of first line treatments , the lack of scientific evidence for drugs licensed for other diseases , and , finally , the lack of innovative therapeutic compounds . pss encompasses several subsets of patients with different genetic background , pathophysiological pathways , demographic features , and different response to proposed therapies . despite the acknowledged role of t - cell subsets in pss , mechanisms leading to their abnormal activation and their contribution to pss pathogenesis are not fully elucidated . in particular , only few studies evaluated the role of treg cells in pss , and conclusive data are still lacking . similarly , although conventional cd4th17 and il-17-producing dn t cells are under active investigation , their origin , fate , and function are still a matter of debate . currently available data suggest a pivotal role of il-17 axis and il-17-producing cells in every step of pss pathogenesis , from the induction of autoimmune epithelitis to the maintenance and perpetuation of inflammation and ectopic gc formation . in this context , il-17-producing t cells seem to be a link between t- and b - cell compartments . in this setting , a therapeutic approach targeting il-17 axis may represent an intriguing issue worth to be investigated in pss . in the era of biologic agents , randomized double - blind controlled trials represent the most powerful tool to obtain comparable results and provide the rationale to build solid therapeutic recommendations also in pss .

the association between us military service and hearing loss continues to receive significant attention , especially in light of recently completed and ongoing combat deployments in iraq and afghanistan . nearly one - half million us veterans are currently receiving over $ 1 billion annually in department of veterans affairs ( va ) compensation for hearing loss . as a result , traditionally , most hearing loss associated with military service has been caused by high intensity and/or impulse noise . in recent years , an increasing number of us service members have hearing loss as a result of being in proximity to the detonation of explosive devices in the iraq and afghanistan operations . for example , deployment has been observed to increase the risk of hearing loss , with 71% of soldiers returning from iraq or afghanistan reporting exposure to loud noise , and more than 15% of returnees reporting ringing in their ears . hearing loss is a significant health and readiness issue for the us military since afflicted personnel exposed to hazardous noise are more likely to suffer additional hearing damage , and service members with hearing loss attrite at a higher rate from military service than those with normal hearing . yet , population - based studies to describe the association between deployment and hearing loss are limited . a study of us army soldiers who visited audiology clinics noted that hearing loss was identified in 68.6% of post - deployment diagnoses and 4.0% of non - deployment - related diagnoses . one population - based study that utilized a number of international classification of diseases , 9 revision , clinical modification diagnostic codes for hearing loss found annual incidence rates between 19.3 and 22.2/1000 ; however , the study did not include deployment as an exposure and was limited to active - duty service members . patient - based studies have reported hearing loss in 15% of patients with tympanic membrane perforation admitted to brooke army medical center , and 19% of patients admitted to a rehabilitation center with comorbid traumatic brain injury . the present study uses data from the millennium cohort study , the largest prospective study of military personnel to date , which follows participants to evaluate whether military service exposures are associated with long - term health outcomes . given the large sample of deployed participants in the millennium cohort , this study offers a unique opportunity to prospectively evaluate the association between military deployment and hearing loss among members of all service branches including active duty , reserve , and national guard members . the objective of this study was to examine the association between military deployment and subsequent hearing loss . the millennium cohort study , launched in 2001 , is a longitudinal cohort study designed to assess the effects of us military service on the health of participants over a follow - up period of at least 21 years . the study utilizes a comprehensive questionnaire , completed approximately every 3 years by participants via mail and a secure internet system . a random sample of us military members from all service branches and components who were on active rosters as of october 2000 was selected for the first enrollment panel . this panel was enrolled from 2001 to 2003 ( n = 77,047 , 36.0% response rate ) and was oversampled for women , reserve / guard personnel , and individuals deployed to southwest asia , bosnia , or kosovo from 1998 to 2000 . the eligible population for these analyses included participants from the first enrollment panel who completed at least one follow - up questionnaire ( first follow - up from 2004 to 2006 or second follow - up from 2007 to 2008 ) and did not self - report significant hearing loss at baseline ( n = 57,593 ) . participants were excluded from these analyses if they deployed before baseline or completed any of their assessments while deployed . additionally , participants were excluded if they were missing relevant outcome or covariate data . the final study population included 48,540 participants . this study was approved by the institutional review board at the naval health research center , and all millennium cohort members are voluntary participants . data for this study were obtained from the millennium cohort study questionnaire as well as electronic military records . the questionnaire collects self - reported demographic , health , behavioral , and exposure data , such as hearing loss , tobacco use , and combat - related experiences . electronic military records , including age , sex , service branch , service component , military occupation , education level , marital status , military separation status , and deployment dates , were provided by the defense manpower data center . when available , self - reported data were used to supplement electronic data from personnel records to minimize missing data . in addition , this study utilized audiometric data , maintained by the defense occupational and environmental health readiness system - hearing conservation data repository , when available , to validate self - reported hearing loss data . all military personnel have audiometric testing , at a minimum , at the time of entrance , at the time of discharge , and to assess readiness prior to any deployment or other hazardous duty . combat deployment , the exposure of interest for the primary analyses , was defined as having deployed to operation iraqi freedom or operation enduring freedom or in support of these conflicts and having personally : witnessed a death due to war , disaster , or tragic event;witnessed instances of physical abuse;been exposed to dead or decomposing bodies;been exposed to maimed soldiers or civilians ; orbeen exposed to prisoners of war or refugees . witnessed a death due to war , disaster , or tragic event ; witnessed instances of physical abuse ; been exposed to dead or decomposing bodies ; been exposed to maimed soldiers or civilians ; or been exposed to prisoners of war or refugees . those who had deployed with combat exposures were compared with those who had deployed without combat exposures and with those who had not deployed . secondary analyses examined a subset of the study population who had deployed and completed the 2007 - 2008 millennium cohort questionnaire that included questions regarding exposure to improvised explosive device ( ied ) blast- and combat - related head trauma . these secondary analyses assessed hearing loss in relation to blast and combat - related head trauma . additional variables , including self - reported smoking status , and exposure to pesticides , chemicals , and occupations requiring the use of personal protective equipment ( ppe ) , were included in all models . chemical exposure was defined as self - reported routine skin contact with paint and/or solvents and/or other hazardous substances during the past 3 years . pesticide exposure was defined as self - reported exposure to pesticides , including creams , sprays , or uniform treatments , or pesticides applied in the environment or around living facilities , during the past 3 years . occupations requiring ppe were defined as occupational hazards requiring protective equipment , such as respirators or hearing protection during the past 3 years . smokers were defined as those who reported smoking at least 100 cigarettes in their lifetime . past smokers were differentiated from current smokers if they reported to have successfully quit smoking . hearing loss was assessed using the millennium cohort questionnaire , which includes the baseline question : has your doctor or other health professional ever told you that you have any of the following conditions ? with the response being significant hearing loss . the question was modified in follow - up questionnaires to describe new hearing loss over the last 3 years , based on the study design of re - surveying participants at approximately 3-year intervals . participants who did not self - report hearing loss at baseline , but later positively endorsed self - reported hearing loss during a follow - up survey , were classified as having new - onset self - reported hearing loss . objective audiometric data were dichotomized using the va standards for impaired hearing . for va purposes , impaired hearing is considered a disability when the audiometric hearing threshold in any of the frequencies ( 500 , 1000 , 2000 , 3000 , or 4000 hz ) is 40 db or greater ; or when the auditory thresholds for at least three of these frequencies are 26 db or greater . note that the va definition also includes those who have speech recognition scores < 94% , but the audiometric database did not contain this additional information . initial descriptive analyses included frequencies , percentages , and chi - square tests to describe the variables within the population . multivariable logistic regression was used for the primary model to determine the odds of new - onset self - reported hearing loss in relation to combat deployment , while adjusting for all covariates noted previously . all variables were assessed at baseline except for military separation and deployment experiences , which were assessed throughout the study period . secondary analyses using multivariable logistic regression included an assessment of the relations between hearing loss and exposure to blasts and combat - related head trauma among a deployed subset of this study group who had completed a baseline survey in 2001 , deployed between 2004 and 2007 , and completed the 2007 survey . multicollinearity was assessed using a variance inflation factor of 4 or greater to identify potentially collinear variables . validation of self - reported hearing loss was evaluated by comparing millennium cohort survey data and objective audiometric data . va criteria for hearing impairment were applied as described previously , to define audiometrically normal or abnormal hearing . each self - report of hearing loss ( yes / no ) was evaluated based on the time stamp of the survey and the timing of available audiometric data . self - report of yes hearing loss was considered invalid if any subsequent audiogram was normal . in addition , those without a preceding validating test who also lacked a subsequent invalidating test and had all abnormal audiograms after self - report were considered to have valid hearing loss . self - report of no hearing loss was considered valid if any subsequent audiogram was normal . self - report of no hearing loss was considered invalid if any previous audiogram was abnormal . in addition , those without a subsequent validating test , who also lacked a previous invalidating test , and had all normal audiograms prior to self - report , were considered to have valid absence of hearing loss . all self - reports were independently assessed , and the degree of nonrandom agreement between the audiometric data and self - reported data was calculated using the kappa statistic . a kappa value between 0.6 and 0.8 was considered substantial agreement , and a kappa value between 0.4 and 0.6 was considered moderate agreement . in addition , a sensitivity analysis was conducted using multivariable logistic regression to examine the odds of self - reported new - onset hearing loss among only those subjects with a validated audiometric record . data management and statistical analyses were performed using sas software , version 9.3 ( sas institute , inc . , cary , north carolina ) . hearing loss was assessed using the millennium cohort questionnaire , which includes the baseline question : has your doctor or other health professional ever told you that you have any of the following conditions ? with the response being significant hearing loss . the question was modified in follow - up questionnaires to describe new hearing loss over the last 3 years , based on the study design of re - surveying participants at approximately 3-year intervals . participants who did not self - report hearing loss at baseline , but later positively endorsed self - reported hearing loss during a follow - up survey , were classified as having new - onset self - reported hearing loss . objective audiometric data were dichotomized using the va standards for impaired hearing . for va purposes , impaired hearing is considered a disability when the audiometric hearing threshold in any of the frequencies ( 500 , 1000 , 2000 , 3000 , or 4000 hz ) is 40 db or greater ; or when the auditory thresholds for at least three of these frequencies are 26 db or greater . note that the va definition also includes those who have speech recognition scores < 94% , but the audiometric database did not contain this additional information . initial descriptive analyses included frequencies , percentages , and chi - square tests to describe the variables within the population . multivariable logistic regression was used for the primary model to determine the odds of new - onset self - reported hearing loss in relation to combat deployment , while adjusting for all covariates noted previously . all variables were assessed at baseline except for military separation and deployment experiences , which were assessed throughout the study period . secondary analyses using multivariable logistic regression included an assessment of the relations between hearing loss and exposure to blasts and combat - related head trauma among a deployed subset of this study group who had completed a baseline survey in 2001 , deployed between 2004 and 2007 , and completed the 2007 survey . multicollinearity was assessed using a variance inflation factor of 4 or greater to identify potentially collinear variables . validation of self - reported hearing loss was evaluated by comparing millennium cohort survey data and objective audiometric data . va criteria for hearing impairment were applied as described previously , to define audiometrically normal or abnormal hearing . each self - report of hearing loss ( yes / no ) was evaluated based on the time stamp of the survey and the timing of available audiometric data . in addition , those without a preceding validating test who also lacked a subsequent invalidating test and had all abnormal audiograms after self - report were considered to have valid hearing loss . self - report of no hearing loss was considered valid if any subsequent audiogram was normal . self - report of no hearing loss was considered invalid if any previous audiogram was abnormal . in addition , those without a subsequent validating test , who also lacked a previous invalidating test , and had all normal audiograms prior to self - report , were considered to have valid absence of hearing loss . all self - reports were independently assessed , and the degree of nonrandom agreement between the audiometric data and self - reported data was calculated using the kappa statistic . a kappa value between 0.6 and 0.8 was considered substantial agreement , and a kappa value between 0.4 and 0.6 was considered moderate agreement . in addition , a sensitivity analysis was conducted using multivariable logistic regression to examine the odds of self - reported new - onset hearing loss among only those subjects with a validated audiometric record . data management and statistical analyses were performed using sas software , version 9.3 ( sas institute , inc . , cary , north carolina ) . this study included 48,540 millennium cohort study participants , of whom 3660 ( 7.5% ) self - reported new - onset hearing loss during follow - up . demographic , military , and behavioral characteristics for those who reported new - onset hearing loss , in comparison with those who reported no hearing loss during the study period , are shown in table 1 . in this comparison , all differences were statistically significant ( p < 0.001 ) with the exception of service component . distribution of demographic , military and behavioral characteristics of 48,540 millennium cohort participants in relation to hearing status * deployment occurred after baseline , self - report of close proximity to an ied blast as reported on 2007 questionnaire among those whose first deployment occurred between their 2004 and 2007 millennium cohort follow - up questionnaire ( n = 4245 ) , self - report of combat - related head trauma as reported on 2007 questionnaire among those whose first deployment occurred between their 2004 and 2007 millennium cohort follow - up questionnaire ( n = 4,245 ) , ied = improvised explosive device , ppe = personal protective equipment multivariable logistic regression was used to calculate adjusted odds ratios ( aors ) for new - onset hearing loss [ table 2 ] . in this analysis , millennium cohort study participants who were deployed with combat experience had increased odds ( aor = 1.63 , 95% confidence interval [ ci ] = 1.49 - 1.77 ) of reporting new - onset hearing loss compared with those who were not deployed . in this adjusted model , male sex , being born before 1970 ( compared with those born in 1980 or later ) , or being currently married were all demographic characteristics associated with increased odds of new - onset self - reported hearing loss . conversely , those of black non - hispanic race / ethnicity ( compared with other race / ethnicity ) were at decreased odds of new - onset self - reported hearing loss . military - specific characteristics with increased odds of hearing loss were serving in the army , navy / coast guard , or marines ( compared with serving in the air force ) , reporting exposures to occupational hazards that required ppe ( including hearing protection ) or routine contact with chemicals , or exposure to pesticides . conversely , officers , those serving in the reserve / national guard ( compared with active duty ) , those serving as health care specialists ( compared with functional support specialists ) , and those separated from the military had lower adjusted odds of reporting new - onset hearing loss . finally , past or current smokers ( compared with never smokers ) had increased adjusted odds of reporting new - onset hearing loss . aor of reporting new - onset hearing loss * all characteristics shown are included in the multivariable model , aor = adjusted odds ratio , ci = confidence interval , ppe = personal protective equipment results from the subanalysis indicated that among persons who deployed and completed the 2007 - 2008 questionnaire ( n = 4245 ) , 1069 reported proximity to a blast , of whom 144 ( 13.5% ) reported new - onset hearing loss . fifty - four deployers reported combat - related head trauma , of whom 21 ( 38.9% ) reported new - onset hearing loss . two separate logistic regression analyses that included all variables ( except for deployment / combat experience ) were performed on the data of the subset of participants who had been deployed during the study period , to assess the relations between combat - related head trauma or blast exposure and new - onset hearing loss [ table 3 ] . participants who reported combat - related head trauma were more than 6 times as likely to report new - onset hearing loss ( aor = 6.88 , 95% ci = 3.77 - 12.54 ) . similarly , participants who reported blast exposure were more than twice as likely to report new - onset hearing loss ( aor = 2.10 , 95% ci = 1.62 - 2.73 ) . aor of reporting new - onset hearing loss in relation to combat - related head trauma and exposure to ied blast among deployed service members adjusted for sex , birth year , education , marital status , race / ethnicity , smoking status , pay grade , service component , service branch , occupation , use of ppe , separation from the military and exposure to pesticides or chemicals , aor = adjusted odds ratio , ci = confidence interval , ppe = personal protective equipment , ied = improvised explosive device the audiometric validation procedure allowed for one audiometry record to correspond with one self - reported record from each survey time period . among the 48,540 individuals included in this study , there were 63,481 self - reports of hearing status during the study period validated among 25,987 millennium cohort participants . there was moderate to substantial agreement between self - reported hearing and audiometric data at each survey cycle , with kappa values of 0.69 ( 95% ci = 0.67 - 0.71 ) , 0.60 ( 95% ci = 0.58 - 0.62 ) , and 0.57 ( 95% ci = 0.56 - 0.59 ) for the self - reported data from 2001 , 2004 , and 2007 , respectively . the percent positive and percent negative agreement were also calculated for each year . the percent positive agreement for 2001 , 2004 , and 2007 were 83.0% , 56.9% , and 51.1% , respectively , while the percent negative agreement for 2001 , 2004 , and 2007 were 96.0% , 97.3% , and 97.7% , respectively . results from the sensitivity analysis examining new - onset hearing loss among only those subjects with a validated audiometric record indicated consistent findings for all measures of association and significance levels ( data not shown ) . to our knowledge , this is the first large - scale prospective study of a military cohort to describe self - reported hearing loss after military deployment that was validated with audiometric data . in this study , we observed moderate to substantial agreement between self - reported hearing loss and hearing loss defined by audiometric data . millennium cohort participants who were deployed with combat experience had a 1.6-fold increased odds for reporting new - onset hearing loss compared with non - deployers . furthermore , in analyses limited to deployed participants , being in close proximity to an explosive blast or experiencing head trauma were strongly associated with new - onset hearing loss . these findings quantify an important health risk faced by us service members who deploy to combat environments . individuals with the occupational code of combat specialists were 11% more likely to report hearing loss , but this association was not statistically significant . given that combat experience was associated with a statistically significant increased risk for self - reported hearing loss , it would seem logical to observe the same for combat specialists . the apparent lack of an association may be partially attributable inherent limitations in the dod occupational conversion index . for example , some combat specialists do not actively participate in ground combat that includes discharging weapons . using a large military audiometric database to validate self - reported hearing loss us military service represents one of the few occupations with a requirement for regular audiometric testing of its members , and the maintenance of clinical audiometric data in a large electronic data repository is a unique attribute of the military health care system . it might have been assumed that study participants would not self - report hearing loss very accurately or consistently , especially in response to a single question on the survey . hearing loss may be overreported when members learn they have to retake a hearing test for any reason ; hearing loss may also be underreported when members fail to notice or acknowledge subtle changes in their hearing . the results from this study suggest that negative reports of hearing loss have substantial accuracy , while positive reports of hearing loss have moderate accuracy , based on comparison with objective audiometric data that would meet va disability criteria for hearing impairment . finding that individuals who were deployed and had combat experiences were 1.6 times more likely to report new - onset hearing loss , compared with their nondeployed counterparts , appears to be a unique contribution to the epidemiologic study of hearing loss . one other study found an association between deployment and hearing loss , but the data were limited to an assessment of us army soldiers . another interesting and important contribution of the current study was the finding that deployed individuals without combat experiences were not at increased risk for new - onset hearing loss compared with nondeployed personnel . this implies that much of the hearing loss attributable to the deployment is related to specific combat experiences rather than to deployment itself . combat may include a significant amount of impulse noise , characterized as noise with a duration of < 1 second and with peak levels 15 db louder than background noise . sources of impulse noise include firing weapons or artillery , as well as detonation of explosive devices . a study conducted in finland reported that combat and shooting exercises can reach peak noise levels of 180 db , and researchers at the us national institute for occupational safety and health have stated that , firing a weapon poses a significant risk of noise - induced hearing loss , if hearing protection is not worn . impulse noise in addition to continuous noise exposure has been reported to be more damaging to hearing than continuous noise exposure alone . a study conducted among canadian armed forces found that ground combat troops were hesitant to use hearing protection because they felt it reduced detection of auditory warnings and reduced communication among the team members . although research is being conducted to develop appropriate hearing protection for combat , a 2009 study among us army cadets found that most devices are lacking in performance and acceptance . these findings suggest that additional research is needed to design hearing protection devices that will meet the needs of ground combat forces . as expected , subgroup analyses of the deployed study participants revealed that the likelihood of reporting new - onset hearing loss was increased with exposures to both combat - related head trauma and proximity to an explosive blast . participants reporting head trauma related to combat were over 6 times more likely to report new - onset hearing loss , whereas those reporting proximity to a blast were approximately twice as likely to report new - onset hearing loss . since the question on blast exposure relates to having an ied or booby trap explode near you , there could be some variability in how the word near is interpreted , and exposure at some distances may not have resulted in injury to the auditory system . we were not able to assess the nature of the blast , nor quantify proximity to the blast , use of hearing protection , or loss of consciousness . combat - related head trauma is likely to include those exposed to blasts , as well as exposure to small arms fire , artillery , grenades , and physical assault . besides the primary effects of blast overpressure , peripheral or central auditory system damage can occur from secondary effects ( shrapnel and other blast - accelerated debris ) , and tertiary effects ( body being thrown and impacting other objects ) . the most common types of blast - related injury involve middle and inner ear structures resulting in conductive , sensorineural , or mixed type of hearing loss . pure sensorineural hearing loss is the predominant type occurring in blast - related traumatic brain injury and was reported to be nearly 60% in a study of inpatients at a va rehabilitation unit . a recent study among us army soldiers reported low levels of referral to audiology clinics following indications of noise - induced hearing loss and head injury on post - deployment health assessments , highlighting the importance of attention to these issues after any suspected exposure . in addition to deployment , other key factors were associated with new - onset hearing loss in the multivariable analysis including male sex , increasing age , non - black race / ethnicity , tobacco use , exposure to other occupational hazards , contact with chemicals , and exposure to pesticides similar to previous reports . the consistency of findings observed here with other published studies lends further credibility to the use of new - onset self - reported hearing loss as a valid measure in this population . in possible contrast , one previous study found increased odds of hearing loss among adult hispanics who were unmarried , whereas in this study , married members were at increased odds for reporting new - onset hearing loss . no biologically plausible reason exists for the finding that married individuals were at increased risk for new - onset hearing loss in models that adjust for demographic variables . it may be that marriage was associated with other exposures that could not be assessed . it is also possible that this finding is due to correlation between marriage and age , and that birth - year cohorts incompletely adjusted for the age effect . this study , as well as a previous study , found an increased risk for hearing loss among active duty compared with reserve / guard members . serving in the military is associated with increased risk for hearing loss , and it is likely that serving in the reserve / guard means fewer hours of munitions - related noise exposure due to the non - continuous active - duty status of these us service members . officers were at decreased odds for reporting new - onset hearing loss , as were members of the air force . these differences may reflect less hazardous noise exposures and/or increased compliance with hearing protection programs in these groups . observing that those who had separated from service were at lower odds for reporting new - onset hearing loss may appear counterintuitive . first , all members of the us military receive an audiogram prior to leaving the military , which would likely increase hearing loss diagnoses among those separating . secondly , the literature suggests that those with hearing loss attrite from the military at a higher rate than those without hearing loss . given that the prevalence of hearing loss was found to be greater among military veterans compared with civilians , it may be possible that this observation reflects the reduced risk for new - onset hearing loss associated with being a civilian in comparison with the increased risk associated with continued military service in this adjusted model . the validation scheme , while imperfect , assumed that normal hearing has the potential to become abnormal and that established abnormal hearing can not return to normal , consistent with the physiology of noise - induced hearing loss . several previous studies conducted in australia , brazil , and the united states of the accuracy of a single self - reported question on hearing loss reported sensitivities ranging from 0.71 to 0.78 and specificities from 0.56 to 0.76 , compared with hearing impairment defined by audiometric testing . these studies evaluated older subjects , hence results of their validation testing may not apply to this younger population . one investigation recruited construction workers who face occupational noise exposure , and with an average age of 42.8 years , this study population is perhaps more comparable to the millennium cohort participants ; this study reported sensitivities of 0.87 - 0.88 and specificities of 0.68 - 0.74 for detection of lower frequency hearing loss using a question that elicited a rating of hearing ability on a 1 ( excellent ) to 5 ( poor ) scale , with fair or poor ratings defined as a positive self - report of hearing loss , and lower kappa values ( 0.25 - 0.45 ) were reported . in our study , validation of self - report of past diagnosis compared with audiometric testing was much stronger . the degree of misclassification may , therefore , be smaller in our study , but undoubtedly some nondifferential misclassification remains that reduced the magnitude of observed associations . however , a consistent result found from the sensitivity analysis performed on validated subjects is reassuring . there were a number of variables used in this analysis that could not be perfectly measured and may have introduced misclassification . we were not able to directly measure hazardous noise levels associated with combat , and had to rely on our imperfect measure of combat as described previously . this was done , in part , because such grouping provided consistent identification of participants who provided data across an 8 year span ( 2001 - 2008 ) . misclassification of true demographic characteristics may have occurred , most likely resulting in the reported association being weaker than the true association . as with all surveys , < 100% of those invited to participate opt to participate . as described in the first paragraph of the methods , the first enrollment panel had a participation rate of 36% . as a result , the cohort may not be representative of the entire military or those who deploy . however , previous investigations suggest that the cohort is a representative population of military personnel who report reliably with minimal health related tendency for enrollment , and showed little non - response bias at the first follow - up . we did not adjust for other important risk factors , including nonoccupational hazardous noise , such as recreational firearm use , and diabetes . although diabetes is another risk factor for hearing loss , individuals with diabetes are not able to join the us military . a recent study conducted using millennium cohort data reported the occurrence of diabetes during an approximate 3 year follow - up was 3/1,000 person - years . therefore , very few members of this cohort had diabetes and the likelihood that important confounding resulted from this condition is extremely low . loss to follow - up represents another limitation , with 71% of subjects enrolled in 2001 completing the survey at either the first or second follow - up . although loss to follow - up may result in bias , we have previously investigated this possibility using statistical techniques for missing data and have found that it did not bias risk estimates for several key outcomes of this study , including posttraumatic stress disorder ( ptsd ) , depression , and eating disorders . the potential remains though for residual confounding due to unmeasured variables or inaccurately measured variables meeting the criteria for confounding . this study has multiple strengths , including a large sample size permitting subgroup analyses and detection of smaller associations with excellent power , and a longitudinal design that permits assessment of new - onset outcomes in relation to previously measured exposures . in addition , the study included a large proportion of national guard / reservists and followed subjects even after separation from the military , thereby providing an advantage over analyses based on electronic data for active - duty service members until the time of separation from the military . we assessed multiple relevant exposures , including combat deployment , and specifically combat - related head trauma and proximity to an explosive blast , smoking status , occupation requiring ppe , or exposures that involve routine contact with chemicals , among other factors . in summary , these are the first analyses to our knowledge to define and quantify the substantial risk of new - onset hearing loss related to military combat - related exposures . we found that combat experience was associated with a 63% increased risk for hearing loss . in addition , we also identified that individuals who reported exposure to an explosive blast or had combat - related head trauma were much more likely to report hearing loss . this study also demonstrated the validity of self - reported hearing loss , when queried in the context of the millennium cohort study , in defining this important health outcome . from a clinical perspective , the 6-fold increase in risk of hearing loss after combat - related head trauma deserves further attention . a multidisciplinary approach to treatment of patients with combat - related head trauma should take into account possible overlapping symptoms with blast - related comorbidities including ptsd , dizziness and imbalance , and speech and language problems , in order to identify and properly manage auditory system outcomes . this may facilitate overall recovery , improve cognitive deficits , and result in better quality of life . preventive strategies should include early detection and monitoring of hearing loss , based on pre - deployment and post - deployment audiograms , to inform clinical practice guidelines , as well as development of improved and more acceptable hearing protection , protective head gear , and possible identification of effective otoprotectants .

the objective of this study was to define the risk of hearing loss among us military members in relation to their deployment experiences . data were drawn from the millennium cohort study . self - reported data and objective military service data were used to assess exposures and outcomes . among all 48,540 participants , 7.5% self - reported new - onset hearing loss . self - reported hearing loss showed moderate to substantial agreement ( k = 0.57 - 0.69 ) with objective audiometric measures . new - onset hearing loss was associated with combat deployment ( adjusted odds ratio [ aor ] = 1.63 , 95% confidence interval [ ci ] = 1.49 - 1.77 ) , as well as male sex and older age . among deployers , new - onset hearing loss was also associated with proximity to improvised explosive devices ( aor = 2.10 , 95% ci = 1.62 - 2.73 ) and with experiencing a combat - related head injury ( aor = 6.88 , 95% ci = 3.77 - 12.54 ) . these findings have implications for health care and disability planning , as well as for prevention programs .

Introduction Methods Exposures Outcomes Statistical analyses Results Discussion

the association between us military service and hearing loss continues to receive significant attention , especially in light of recently completed and ongoing combat deployments in iraq and afghanistan . as a result , traditionally , most hearing loss associated with military service has been caused by high intensity and/or impulse noise . in recent years , an increasing number of us service members have hearing loss as a result of being in proximity to the detonation of explosive devices in the iraq and afghanistan operations . for example , deployment has been observed to increase the risk of hearing loss , with 71% of soldiers returning from iraq or afghanistan reporting exposure to loud noise , and more than 15% of returnees reporting ringing in their ears . hearing loss is a significant health and readiness issue for the us military since afflicted personnel exposed to hazardous noise are more likely to suffer additional hearing damage , and service members with hearing loss attrite at a higher rate from military service than those with normal hearing . a study of us army soldiers who visited audiology clinics noted that hearing loss was identified in 68.6% of post - deployment diagnoses and 4.0% of non - deployment - related diagnoses . the present study uses data from the millennium cohort study , the largest prospective study of military personnel to date , which follows participants to evaluate whether military service exposures are associated with long - term health outcomes . given the large sample of deployed participants in the millennium cohort , this study offers a unique opportunity to prospectively evaluate the association between military deployment and hearing loss among members of all service branches including active duty , reserve , and national guard members . the objective of this study was to examine the association between military deployment and subsequent hearing loss . the millennium cohort study , launched in 2001 , is a longitudinal cohort study designed to assess the effects of us military service on the health of participants over a follow - up period of at least 21 years . the eligible population for these analyses included participants from the first enrollment panel who completed at least one follow - up questionnaire ( first follow - up from 2004 to 2006 or second follow - up from 2007 to 2008 ) and did not self - report significant hearing loss at baseline ( n = 57,593 ) . this study was approved by the institutional review board at the naval health research center , and all millennium cohort members are voluntary participants . data for this study were obtained from the millennium cohort study questionnaire as well as electronic military records . the questionnaire collects self - reported demographic , health , behavioral , and exposure data , such as hearing loss , tobacco use , and combat - related experiences . when available , self - reported data were used to supplement electronic data from personnel records to minimize missing data . in addition , this study utilized audiometric data , maintained by the defense occupational and environmental health readiness system - hearing conservation data repository , when available , to validate self - reported hearing loss data . those who had deployed with combat exposures were compared with those who had deployed without combat exposures and with those who had not deployed . secondary analyses examined a subset of the study population who had deployed and completed the 2007 - 2008 millennium cohort questionnaire that included questions regarding exposure to improvised explosive device ( ied ) blast- and combat - related head trauma . these secondary analyses assessed hearing loss in relation to blast and combat - related head trauma . additional variables , including self - reported smoking status , and exposure to pesticides , chemicals , and occupations requiring the use of personal protective equipment ( ppe ) , were included in all models . pesticide exposure was defined as self - reported exposure to pesticides , including creams , sprays , or uniform treatments , or pesticides applied in the environment or around living facilities , during the past 3 years . hearing loss was assessed using the millennium cohort questionnaire , which includes the baseline question : has your doctor or other health professional ever told you that you have any of the following conditions ? participants who did not self - report hearing loss at baseline , but later positively endorsed self - reported hearing loss during a follow - up survey , were classified as having new - onset self - reported hearing loss . multivariable logistic regression was used for the primary model to determine the odds of new - onset self - reported hearing loss in relation to combat deployment , while adjusting for all covariates noted previously . secondary analyses using multivariable logistic regression included an assessment of the relations between hearing loss and exposure to blasts and combat - related head trauma among a deployed subset of this study group who had completed a baseline survey in 2001 , deployed between 2004 and 2007 , and completed the 2007 survey . validation of self - reported hearing loss was evaluated by comparing millennium cohort survey data and objective audiometric data . each self - report of hearing loss ( yes / no ) was evaluated based on the time stamp of the survey and the timing of available audiometric data . self - report of yes hearing loss was considered invalid if any subsequent audiogram was normal . self - report of no hearing loss was considered valid if any subsequent audiogram was normal . self - report of no hearing loss was considered invalid if any previous audiogram was abnormal . in addition , those without a subsequent validating test , who also lacked a previous invalidating test , and had all normal audiograms prior to self - report , were considered to have valid absence of hearing loss . all self - reports were independently assessed , and the degree of nonrandom agreement between the audiometric data and self - reported data was calculated using the kappa statistic . in addition , a sensitivity analysis was conducted using multivariable logistic regression to examine the odds of self - reported new - onset hearing loss among only those subjects with a validated audiometric record . hearing loss was assessed using the millennium cohort questionnaire , which includes the baseline question : has your doctor or other health professional ever told you that you have any of the following conditions ? participants who did not self - report hearing loss at baseline , but later positively endorsed self - reported hearing loss during a follow - up survey , were classified as having new - onset self - reported hearing loss . multivariable logistic regression was used for the primary model to determine the odds of new - onset self - reported hearing loss in relation to combat deployment , while adjusting for all covariates noted previously . secondary analyses using multivariable logistic regression included an assessment of the relations between hearing loss and exposure to blasts and combat - related head trauma among a deployed subset of this study group who had completed a baseline survey in 2001 , deployed between 2004 and 2007 , and completed the 2007 survey . validation of self - reported hearing loss was evaluated by comparing millennium cohort survey data and objective audiometric data . each self - report of hearing loss ( yes / no ) was evaluated based on the time stamp of the survey and the timing of available audiometric data . in addition , those without a preceding validating test who also lacked a subsequent invalidating test and had all abnormal audiograms after self - report were considered to have valid hearing loss . self - report of no hearing loss was considered valid if any subsequent audiogram was normal . self - report of no hearing loss was considered invalid if any previous audiogram was abnormal . in addition , those without a subsequent validating test , who also lacked a previous invalidating test , and had all normal audiograms prior to self - report , were considered to have valid absence of hearing loss . all self - reports were independently assessed , and the degree of nonrandom agreement between the audiometric data and self - reported data was calculated using the kappa statistic . in addition , a sensitivity analysis was conducted using multivariable logistic regression to examine the odds of self - reported new - onset hearing loss among only those subjects with a validated audiometric record . this study included 48,540 millennium cohort study participants , of whom 3660 ( 7.5% ) self - reported new - onset hearing loss during follow - up . demographic , military , and behavioral characteristics for those who reported new - onset hearing loss , in comparison with those who reported no hearing loss during the study period , are shown in table 1 . distribution of demographic , military and behavioral characteristics of 48,540 millennium cohort participants in relation to hearing status * deployment occurred after baseline , self - report of close proximity to an ied blast as reported on 2007 questionnaire among those whose first deployment occurred between their 2004 and 2007 millennium cohort follow - up questionnaire ( n = 4245 ) , self - report of combat - related head trauma as reported on 2007 questionnaire among those whose first deployment occurred between their 2004 and 2007 millennium cohort follow - up questionnaire ( n = 4,245 ) , ied = improvised explosive device , ppe = personal protective equipment multivariable logistic regression was used to calculate adjusted odds ratios ( aors ) for new - onset hearing loss [ table 2 ] . in this analysis , millennium cohort study participants who were deployed with combat experience had increased odds ( aor = 1.63 , 95% confidence interval [ ci ] = 1.49 - 1.77 ) of reporting new - onset hearing loss compared with those who were not deployed . in this adjusted model , male sex , being born before 1970 ( compared with those born in 1980 or later ) , or being currently married were all demographic characteristics associated with increased odds of new - onset self - reported hearing loss . conversely , those of black non - hispanic race / ethnicity ( compared with other race / ethnicity ) were at decreased odds of new - onset self - reported hearing loss . military - specific characteristics with increased odds of hearing loss were serving in the army , navy / coast guard , or marines ( compared with serving in the air force ) , reporting exposures to occupational hazards that required ppe ( including hearing protection ) or routine contact with chemicals , or exposure to pesticides . conversely , officers , those serving in the reserve / national guard ( compared with active duty ) , those serving as health care specialists ( compared with functional support specialists ) , and those separated from the military had lower adjusted odds of reporting new - onset hearing loss . finally , past or current smokers ( compared with never smokers ) had increased adjusted odds of reporting new - onset hearing loss . aor of reporting new - onset hearing loss * all characteristics shown are included in the multivariable model , aor = adjusted odds ratio , ci = confidence interval , ppe = personal protective equipment results from the subanalysis indicated that among persons who deployed and completed the 2007 - 2008 questionnaire ( n = 4245 ) , 1069 reported proximity to a blast , of whom 144 ( 13.5% ) reported new - onset hearing loss . fifty - four deployers reported combat - related head trauma , of whom 21 ( 38.9% ) reported new - onset hearing loss . two separate logistic regression analyses that included all variables ( except for deployment / combat experience ) were performed on the data of the subset of participants who had been deployed during the study period , to assess the relations between combat - related head trauma or blast exposure and new - onset hearing loss [ table 3 ] . participants who reported combat - related head trauma were more than 6 times as likely to report new - onset hearing loss ( aor = 6.88 , 95% ci = 3.77 - 12.54 ) . similarly , participants who reported blast exposure were more than twice as likely to report new - onset hearing loss ( aor = 2.10 , 95% ci = 1.62 - 2.73 ) . aor of reporting new - onset hearing loss in relation to combat - related head trauma and exposure to ied blast among deployed service members adjusted for sex , birth year , education , marital status , race / ethnicity , smoking status , pay grade , service component , service branch , occupation , use of ppe , separation from the military and exposure to pesticides or chemicals , aor = adjusted odds ratio , ci = confidence interval , ppe = personal protective equipment , ied = improvised explosive device the audiometric validation procedure allowed for one audiometry record to correspond with one self - reported record from each survey time period . among the 48,540 individuals included in this study , there were 63,481 self - reports of hearing status during the study period validated among 25,987 millennium cohort participants . there was moderate to substantial agreement between self - reported hearing and audiometric data at each survey cycle , with kappa values of 0.69 ( 95% ci = 0.67 - 0.71 ) , 0.60 ( 95% ci = 0.58 - 0.62 ) , and 0.57 ( 95% ci = 0.56 - 0.59 ) for the self - reported data from 2001 , 2004 , and 2007 , respectively . results from the sensitivity analysis examining new - onset hearing loss among only those subjects with a validated audiometric record indicated consistent findings for all measures of association and significance levels ( data not shown ) . to our knowledge , this is the first large - scale prospective study of a military cohort to describe self - reported hearing loss after military deployment that was validated with audiometric data . in this study , we observed moderate to substantial agreement between self - reported hearing loss and hearing loss defined by audiometric data . millennium cohort participants who were deployed with combat experience had a 1.6-fold increased odds for reporting new - onset hearing loss compared with non - deployers . furthermore , in analyses limited to deployed participants , being in close proximity to an explosive blast or experiencing head trauma were strongly associated with new - onset hearing loss . given that combat experience was associated with a statistically significant increased risk for self - reported hearing loss , it would seem logical to observe the same for combat specialists . using a large military audiometric database to validate self - reported hearing loss us military service represents one of the few occupations with a requirement for regular audiometric testing of its members , and the maintenance of clinical audiometric data in a large electronic data repository is a unique attribute of the military health care system . the results from this study suggest that negative reports of hearing loss have substantial accuracy , while positive reports of hearing loss have moderate accuracy , based on comparison with objective audiometric data that would meet va disability criteria for hearing impairment . finding that individuals who were deployed and had combat experiences were 1.6 times more likely to report new - onset hearing loss , compared with their nondeployed counterparts , appears to be a unique contribution to the epidemiologic study of hearing loss . another interesting and important contribution of the current study was the finding that deployed individuals without combat experiences were not at increased risk for new - onset hearing loss compared with nondeployed personnel . sources of impulse noise include firing weapons or artillery , as well as detonation of explosive devices . as expected , subgroup analyses of the deployed study participants revealed that the likelihood of reporting new - onset hearing loss was increased with exposures to both combat - related head trauma and proximity to an explosive blast . participants reporting head trauma related to combat were over 6 times more likely to report new - onset hearing loss , whereas those reporting proximity to a blast were approximately twice as likely to report new - onset hearing loss . we were not able to assess the nature of the blast , nor quantify proximity to the blast , use of hearing protection , or loss of consciousness . combat - related head trauma is likely to include those exposed to blasts , as well as exposure to small arms fire , artillery , grenades , and physical assault . the most common types of blast - related injury involve middle and inner ear structures resulting in conductive , sensorineural , or mixed type of hearing loss . a recent study among us army soldiers reported low levels of referral to audiology clinics following indications of noise - induced hearing loss and head injury on post - deployment health assessments , highlighting the importance of attention to these issues after any suspected exposure . in addition to deployment , other key factors were associated with new - onset hearing loss in the multivariable analysis including male sex , increasing age , non - black race / ethnicity , tobacco use , exposure to other occupational hazards , contact with chemicals , and exposure to pesticides similar to previous reports . the consistency of findings observed here with other published studies lends further credibility to the use of new - onset self - reported hearing loss as a valid measure in this population . in possible contrast , one previous study found increased odds of hearing loss among adult hispanics who were unmarried , whereas in this study , married members were at increased odds for reporting new - onset hearing loss . no biologically plausible reason exists for the finding that married individuals were at increased risk for new - onset hearing loss in models that adjust for demographic variables . this study , as well as a previous study , found an increased risk for hearing loss among active duty compared with reserve / guard members . serving in the military is associated with increased risk for hearing loss , and it is likely that serving in the reserve / guard means fewer hours of munitions - related noise exposure due to the non - continuous active - duty status of these us service members . officers were at decreased odds for reporting new - onset hearing loss , as were members of the air force . observing that those who had separated from service were at lower odds for reporting new - onset hearing loss may appear counterintuitive . given that the prevalence of hearing loss was found to be greater among military veterans compared with civilians , it may be possible that this observation reflects the reduced risk for new - onset hearing loss associated with being a civilian in comparison with the increased risk associated with continued military service in this adjusted model . several previous studies conducted in australia , brazil , and the united states of the accuracy of a single self - reported question on hearing loss reported sensitivities ranging from 0.71 to 0.78 and specificities from 0.56 to 0.76 , compared with hearing impairment defined by audiometric testing . one investigation recruited construction workers who face occupational noise exposure , and with an average age of 42.8 years , this study population is perhaps more comparable to the millennium cohort participants ; this study reported sensitivities of 0.87 - 0.88 and specificities of 0.68 - 0.74 for detection of lower frequency hearing loss using a question that elicited a rating of hearing ability on a 1 ( excellent ) to 5 ( poor ) scale , with fair or poor ratings defined as a positive self - report of hearing loss , and lower kappa values ( 0.25 - 0.45 ) were reported . although loss to follow - up may result in bias , we have previously investigated this possibility using statistical techniques for missing data and have found that it did not bias risk estimates for several key outcomes of this study , including posttraumatic stress disorder ( ptsd ) , depression , and eating disorders . this study has multiple strengths , including a large sample size permitting subgroup analyses and detection of smaller associations with excellent power , and a longitudinal design that permits assessment of new - onset outcomes in relation to previously measured exposures . we assessed multiple relevant exposures , including combat deployment , and specifically combat - related head trauma and proximity to an explosive blast , smoking status , occupation requiring ppe , or exposures that involve routine contact with chemicals , among other factors . in summary , these are the first analyses to our knowledge to define and quantify the substantial risk of new - onset hearing loss related to military combat - related exposures . we found that combat experience was associated with a 63% increased risk for hearing loss . in addition , we also identified that individuals who reported exposure to an explosive blast or had combat - related head trauma were much more likely to report hearing loss . this study also demonstrated the validity of self - reported hearing loss , when queried in the context of the millennium cohort study , in defining this important health outcome . from a clinical perspective , the 6-fold increase in risk of hearing loss after combat - related head trauma deserves further attention . a multidisciplinary approach to treatment of patients with combat - related head trauma should take into account possible overlapping symptoms with blast - related comorbidities including ptsd , dizziness and imbalance , and speech and language problems , in order to identify and properly manage auditory system outcomes . preventive strategies should include early detection and monitoring of hearing loss , based on pre - deployment and post - deployment audiograms , to inform clinical practice guidelines , as well as development of improved and more acceptable hearing protection , protective head gear , and possible identification of effective otoprotectants .

breast cancer is the most common malignancy in women , constituting more than 25% of cancers in this group . the incidence of breast cancer around the world greatly varies , with highest rates in the north america , western , and northern europe and australia / new zealand ( 82.599.4 per 100,000 women ) . breast cancer metastasizes most often to axillary lymph nodes , but may involve any organ . metastasis to serosal surfaces involves primarily the pleural cavity [ 2 , 3 ] , and infrequently , the pericardial and peritoneal cavities . pleural effusions may occur at any point of time in the clinical course and may be the sole manifestation of metastatic disease . this condition is associated with a poor median survival of less than 1 year [ 5 , 6 ] . in recent years , we have reported on the differential expression of metastasis - related molecules , including proteases , angiogenic molecules , signaling molecules , inhibitors of apoptosis , and transcription factors , in breast carcinoma effusions compared to patient - matched primary carcinomas [ 710 ] . the global view of cellular transcriptional activity using gene array technology is required to identify clusters of genetic markers that explain the complex biological processes involved in carcinoma progression to effusions . at present , only one study in which breast carcinoma effusions were compared with primary carcinomas is available . analyzed the gene expression signature of 19 effusions and compared them to 4 primary carcinomas , 8 cells lines and 4 specimens consisting of benign breast tissue . effusions could be differentiated into two categories , one resembling cells lines and expressing cd24 , cd44 and cytokeratins 8 , 18 and 19 , the other expressing metastasis - associated genes , such as s100a4 , upa receptor , vimentin and cxcr4 . the present study compared the gene expression signatures of 10 effusions and 10 primary breast carcinomas . selected differentially expressed genes of pathways related to adhesion , interaction with the extracellular matrix ( ecm ) and regulation of the actin cytoskeleton were validated on mrna and protein level , and their clinical relevance was analyzed in a larger series of breast carcinoma effusions . our data demonstrate that in agreement with our previous observations , breast carcinoma cells in effusions are markedly different from their counterparts in primary carcinomas . this bears relevance to validation of novel therapeutic targets and stratification of patients with respect to treatment response and survival . ten effusions ( 7 pleural , 2 peritoneal , 1 pericardial ) from patients with primary breast carcinoma ( 9 of infiltrating ductal type , 1 lobular ) were submitted for routine diagnostic purposes to the department of pathology at the norwegian radium hospital during the period 19992005 . submitted specimens were processed immediately upon arrival , and pellets were used for preparation of paraffin - embedded cell blocks and for freezing in equal volumes of rpmi supplemented with 20% fetal calf serum and 20% dmso . all specimens underwent morphological evaluation and were further characterized using immunohistochemistry , as previously detailed . all effusions had > 50% carcinoma cells of the total cellular content , ranging between 80100% of cells in 8/10 specimens . ten primary breast carcinomas of infiltrating duct type were retrieved from snap - frozen archival material stored at 70c at the department of pathology , norwegian radium hospital . morphological evaluation of frozen sections from the studied specimens was performed in all cases in order to ensure the presence of a predominant carcinoma cell population and absence of necrosis . eleven samples were prepared from each group ( one effusion and one primary carcinoma with two samples each ) . ( 1 ) eleven pair - wise competitive hybridizations of cdna target sample from randomly chosen patients from the effusion group and sample from randomly chosen patients from primary solid tumor group . ( 2 ) six pair - wise hybridizations of cdna samples from randomly chosen patients from the effusion group and pooled reference sample from four primary solid tumors . biopsies from primary tumors and effusions were lysed using sv- total isolation kit ( promega , madison , wis , usa ) and total rna was extracted according to the manufacturer 's guidelines . the quantity and quality of the total rna preparations were assessed using a nanodrop nd-1000 ( nanodrop , wilmington , de , usa ) combined with agarose gel electrophoresis and only samples with ribosomal 28s/18s ratio near 2 were selected for array analysis . twenty micrograms total rna was mixed with 2 g oligo dt ( amersham biosciences , piscataway , nj , usa ) and rnase - free water to a total volume of 16.9 l , incubated 10 minutes at 75c and cooled on ice . six microliters of first strand buffer , 3 l of 0.1 m dtt , 2 l of superscript ii rt ( invitrogen , carlsbad , calif , usa ) , 1.2 l 25x aminoallyl ( aa - autp)/d - ntp mix ( sigma , haverhill , uk ) and 1 l of rnase inhibitor ( amersham biosciences ) were added to each reaction tube . additional 1 l of superscript ii rt was added following incubation for 60 minutes at 42c . free rna was disassembled with mixture of 10 l 1 m naoh and 10 l 0.5 m edta and underwent neutralization with 25 l 1 m hepes . unincorporated aa - autp and free amines were removed using microcon ym-30 spin column ( millipore , bedford , mass , usa ) and cdna yield were measured by nanodrop spectrophotometer . the cdna target samples were labeled by cy3 or cy5 fluorescent dyes ( amersham biosciences ) according to the sample group and resuspended in 0.1 m carbonate buffer ( ph 8.6 ) . the samples were incubated in the dark for 1 hour in rt and diluted in 35 l naoac 100 mm ( ph 5.2 ) . the free dyes were removed using qiaquik pcr clean up kit ( qiagen , valencia , ca , usa ) , washed with 5 mm phosphate buffer ( ph 8.0 , 80% ethanol ) and eluted with 4 mm phosphate elution buffer ( ph 8.5 ) . the hybridization efficiency and the yield of the dye incorporation were calculated using labeled cdna calculator ( http://www.pangloss.com/seidel/protocols/percent_inc.html ) . equal amount of cy3/cy5 labeled cdna were mixed and dried in the speed - vac . the slides , with printed array - ready oligo set for the human genome version 3.0 ( qiagen ) containing 34,580 longmer probes , representing 24,650 genes and 37,123 gene transcripts , were blocked with 1%bsa in 0.1% sds 5xssc buffer , washed in distilled water and dried at 1000 rpm for 2 minutes . the mixed pair of samples was resuspended in hybridization buffer ( 25% formamide , 0.1% sds in 5xssc ) supplied with 0.02 g t - rna and denatured at 95c . the arrays were hybridized in a water bath , in sealed , watertight hybridization chambers ( dietech , ford city , pa , usa ) for 1618 hours at 42c . after hybridization , the slides were rinsed in a coupling jar containing 2 ssc 0.1% sds , followed by washing for 5 minutes in 1 ssc , then for 5 minutes in 0.2 ssc , and finally for 10 minutes in 0.05 ssc . griding and analysis of images was performed using gene pix 6.0 software package ( molecular devices , sunnyvale , calif , usa ) . the fluorescent intensities of cy5 and cy3 for each target spot were adjusted so that the mean cy3/cy5 ratio of housekeeping genes was equal to one , a design allowing more precise analysis of differentially expressed genes . statistical analysis of microarrays was preformed by the genomic data analysis unit of hadassah medical school , hebrew university of jerusalem . the quality assurance , calibration , data normalization ( lowess ) and volcano plot for gpr files were performed by custom built package written in matlab r2007a . an additional statistical analysis and clustering were carried out using the spotfire ( somerville , ma ) and partek ( st . louis , mo ) software packages . gene annotations and specific pathways were fingered out using online free access programs such as : go annotation ( http://www.geneontology.org/ ) , onto - express , pathway - express ( intelligent systems and bioinformatics laboratory , computer science department , wayne state university ) . total rna from specimens analyzed for bcar1 , vil2 and dcn expression was extracted using tri - reagent ( sigma ) according to the manufacturer 's guidelines . 0.5 g total rna was reverse - transcribed using the m - mlv reverse transcriptase ( promega ) with incubation of 2 hours at 37c , followed by 5 minutes at 95c , and diluted to 1 : 5 with rnase - free water . rt - pcr was performed on complementary dna samples using a dna thermal cycler ( eppendorf mastercycler gradient , eppendorf , hamburg , germany ) with reddymix pcr master mix ( abgene , surrey , uk ) . sense 5-ggg - cca - cag - gac - atc - tat - gat-3 , antisense 5-gag - gaa - cgt - cgt - aga - ctg - cg-3 ( amplicon size , 318 base pairs [ bp ] ) . sense 5-gtt - ttc - ccc - agt - tgt - aat - agt - gcc-3 , antisense 5-tgc - ctt - tgc - aaa - gct - ttt - att - tca-3 ( amplicon size , 995 bp ) . conditions were as follows : bcar1 : 95c for 3 minutes , denaturation at 95 for 15 seconds , annealing at 59 for 30 seconds , extension at 72 for 20 seconds , 34 cycles ; vil2 : 95 for 3 minutes , denaturation at 95 for 15 seconds , annealing at 64 for 30 seconds , extension at 72 for 20 seconds , 33 cycles . products were separated on 1.5% agarose gels , isolated using the invisorb spin dna extraction kit ( invitek gmbh , berlin , germany ) and sequenced . gels were photographed by the kodak edas 290 system ( kodak , rochester , ny , usa ) . densitometer analysis of films was performed using a computerized image analysis ( nih image 1.63 ) program . bcar1 and vil2 mrna levels were established by calculating the target molecule/28s ratio ( all cases scored for band intensity compared to control ) . qrt - pcr was preformed using the mx3000p qpcr system ( stratagene , calif , usa ) . oligonucleotide primers were designed in the primer express program ( applied biosystems , foster city , calif , usa ) . primer sequences for dcn were 5-tcc - gct - gaa - gag - ctc - agg - aat-3 for the forward primer , and 5-cct - tga - gga - atg - ctg - gtg - ata - ttg-3 for the reverse primer . the primers for rplpo normalizer gene were : 5-cca - act - act - tcc - tta - aga - tca - tcc - aac - ta-3 for the forward primer and 5-aca - tgc - gga - tct - gct - gca-3 for the reverse primer . one of the primers in each primer pair was designed in exon - exon boundaries region in order to minimize the dna contamination noise . the specificity of primer binding was analyzed by blast ( http://blast.ncbi.nlm.nih.gov/ ) with human genomic + transcript ( human g + t ) database for highly similar sequences ( megablast ) . the primer optimal concentration and the sensitivity , efficiency , and accuracy of qpcr were calibrated by amplifying serial geometric dilutions of pooled sample consisted from five primary tumor and five effusion cdna samples . 0.1 g of cdna product from the reverse transcriptase reaction were amplified using dynamo sybr green qpcr kit with rox passive reference dye ( finnzymes oy , espoo , finland ) according to the manufacturer 's instructions . absence of primer - dimers and non - specific products was verified by single product peak in the qpcr dissociation curve . in addition , the pcr product was separated by gel electrophoresis and sequenced ( hebrew university facilities ) . formalin - fixed paraffin - embedded sections were available from 52 breast carcinoma effusions ( 47 pleural , 4 peritoneal , 1 pericardial ) from 51 female patients ( one patient with 2 effusions ) aged 3386 ( mean = 59 ) years with histologically verified breast cancer . in 27 cases , the primary carcinoma was additionally available for analysis . slides from the primary breast carcinoma specimens were available in our archives for 45 cases . these were diagnosed as infiltrating duct carcinoma ( 38 ) , lobular carcinoma ( 5 ) or mixed duct and lobular carcinoma ( 2 ) . in the remaining 6 cases , these 79 above - described specimens were manually immunostained for p130cas , phospho - ezrin ( p - ezrin ) , and claudin-4 . the monoclonal mouse p130cas antibody ( clone cas-14 ) was purchased from neomarkers ( labvision corporation , fremont , calif , usa ) . a monoclonal mouse p - ezrin antibody was purchased from bd pharmingen ( san jose , calif , usa ) . the rabbit polyclonal claudin-4 antibody was purchased from zymed ( san francisco , calif , usa ) . all slides underwent pretreatment in a microwave oven for 20 minutes ( p - ezrin and claudin-4 slides in tris / edta buffer , ph = 9 - 9.1 , p130 slides in citrate buffer , ph = 6 ) . visualization was achieved using the envision + peroxidase system ( dako a / s , glostrup , denmark ) . negative controls consisted of sections that underwent similar staining procedures with isotype - matched mouse antibody , normal goat igg or non - relevant rabbit immunoglobulins according to the antibody host species . positive controls consisted of a breast carcinoma biopsy that demonstrated immunoreactivity for the studied antigens in a pilot study . staining was considered positive only when localized to the cell membrane in a linear pattern for p - ezrin and claudin-4 , and when present in the cytoplasm for the p130cas reaction . staining extent was scored on a scale of 04 , as follows : 0 = no staining , 1 = staining of 15% , 2 = staining of 625% , 3 = staining of 2675% , 4 = staining of 76100% of cells . slides were scored by a surgical pathologist experienced in effusion cytology and breast pathology ( bd ) . frozen specimens were thawed and subsequently lysed in 1% np-40 , 20 mm tris hcl ( ph 7.5 ) , 137 mm nacl , 0.5 mm edta , 10% glycerol , 1 mm phenyl - methylsulfonyl fluoride , 1 g / ml aprotinin , 2 g / ml leupeptin , 1 mm sodium orthovanadate , and 0.1% sds . 25 g of a total protein from each sample were separated by electrophoresis through sds-10% polyacrylamide gels under reducing conditions . after electrophoresis , proteins were transferred to immobilon transfer membranes ( millipore , bedford , mass , usa ) . membranes were blocked in 5% non fat dry milk ( nfdm ) in 0.1% tween tbs ( tbst ) and incubated overnight at 4c in 5% bsa tbst containing anti - p - ezrin ( thr ) rabbit mab ( cell signaling technology inc . , danvers , mass , usa ) . after incubation , membranes were washed and incubated for 1 hour with peroxidase - conjugated affinipure goat anti - rabbit igg ( jackson immunoresearch , west grove , pa , usa ) in tbst containing 5% bsa . membranes were developed using the enhanced chemiluminescence kit ( pierce , rockford , ill , usa ) , according to manufacturer 's specifications . membranes were then washed , stripped in 0.2 m glycine , 0.1% sds , and 1% tween 20 ( ph 2.2 ) , blocked in tbst containing 5% nfdm , and incubated overnight at 4c in 5% bsa in tbst containing a rabbit polyclonal anti - ezrin ab ( abcam , cambridge , uk ) . total ezrin activity was normalized to -actin activity measured using rabbit anti--actin polyclonal antibody ( cell signaling technology inc . ) . levels of phosphrylation of the torc1 substrate p70s6k were analyzed using goat anti - p - p70s6k and rabbit anti - p70 s6k antibodies ( santa cruz biotechnology , inc . secondary peroxidase - conjugated donkey anti - goat igg ( santa cruz biotechnology , inc . ) was used for anti - p - p70s6k detection . densitometer analysis of films was performed using a computerized image analysis program ( nih image 1.63 ) . ihc and ib results were analyzed using the spss - pc package , version 15.0 ( chicago , ill , usa ) . comparative analyses of tumor cell expression results in all effusions versus primary tumors were performed using the mann - whitney u test . the same test was applied for analysis of the relationship between protein expression in effusions and clinicopathologic parameters . the wilcoxon signed ranks test was applied for patient - matched analysis in the 27 cases with effusion and primary tumor . univariate analysis for disease - free survival ( dfs ) and overall survival ( os ) for 44 patients with clinical data were executed using the kaplan - meier method and log - rank test . for this analysis , expression categories were grouped as focal ( 25% of cells ) or diffuse ( > 25% of cells ) . we have previously shown that effusions constitute a unique form of breast carcinoma metastasis with mrna and protein expression patterns that differ from primary tumors and solid metastases [ 710 ] . in the present study , we compared the global expression profile of breast carcinoma cells in effusions with that of primary carcinomas . figure 1(a ) shows volcano plot of global gene expression in effusions and primary tumors . differences of 15 fold in gene expression with cut - off p - value < .05 were defined as significant . we identified 255 significantly down - regulated and 96 significantly up - regulated genes in effusion samples ( total = 351 ) . louis , mon , usa ) is a technique used to reduce multidimensional data sets to lower dimensions and to highlight their similarities and differences . pca analysis of six effusion and primary tumor samples was performed using the set of 351 genes that were differentially expressed in effusions and primary carcinomas ( figure 1(b ) , supplementary table 1 , available at doi:10.1155/2010/969084 . ) . the analysis showed that this gene set effectively separates tumors at these two anatomic sites . we additionally performed random pca analysis of the gene expression pattern in all 11 effusions and 11 primary tumor pairs ( figure 1(c ) ) . the analysis was performed using a set of 342 genes that showed trend of up- or downregulation in those patients . the difference between this gene number and the above - detailed 351 genes results from the fact that two different analyses were performed , the first being a pool versus individual specimen analysis , the second of individual case versus individual case . three patterns were identified : ( 1 ) unique for primary tumors ; ( 2 ) unique for effusions and ( 3 ) samples with overlapping gene expression . in order to understand the biological function of the genes that were up- or down - regulated in effusions , we used the go annotation ( http://www.geneontology.org/ ) and pathway - express ( 14 , 15 ) programs . we found multiple pathways involved in cell maintenance that are altered in effusions in comparison to primary tumors ( table 2 ) . it can be seen that the pathways involved in focal adhesion , ecm - receptor interaction and regulation of the actin cytoskeleton are highly involved in phenotypic transformation of carcinoma cells in primary tumors to those in effusions . some of the differentially - expressed genes were found to participate in the specific pathways listed above . other differentially - expressed genes could not be classified as components of a specific pathway , but are of great clinical impact in breast carcinoma , for example , er with a 3.23-fold downregulation in effusions and mta3 , an estrogen - sensitive gene involved in e - cadherin regulation , with a 2.42- fold downregulation in effusions . the significantly altered genes in effusions ( t - test/ anova , p - value < .05 ) were selected for performing upgma hierarchical clustering ( figure 2 ) . of these 10 clusters , cluster g included genes that were strongly up - regulated in effusions , while cluster j genes were strongly down - regulated in comparison to primary tumors . we found some clinically - relevant genes , such as krt8 , cldn4 and vil2 in cluster g , while cluster j included cldn19 , the ecm genes col1a1 , col22a1 , col5a2 and the itga7 and itga5 integrin genes . since cell motility and cell - ecm interactions may have a major effect on the metastatic potential of carcinoma cells in effusions , we focused on genes participating in regulation of the actin cytoskeleton , focal adhesion , and ecm - receptor interactions in validation of the array results . the genes focused on were the following : dcn , which encodes for the small cellular or pericellular matrix proteoglycan decorin and was found to be significantly down - regulated in effusions ; vil2 , encoding for ezrin , which controls the actin cytoskeleton dynamics ; bcar , encoding for the integrin signaling adaptor protein p130cas . in addition , tuberous sclerosis 1 ( tsc1 ) , also known as the tumor suppressor hamartin , the main inhibitor of the mtor signaling pathway [ 15 , 16 ] , was one of the genes that were found to be down - regulated in effusions in comparison to primary tumors . thus , we decided to analyze mtor activity in effusions compared to solid primary tumors . validation was by semiquantitative and quantitative rt - pcr , western blotting and immunohistochemistry , using an enlarged set of effusions and primary carcinomas . dcn expression levels were analyzed in 29 effusions and 35 primary carcinomas using qrt - pcr . dcn levels were significantly higher in primary tumors ( p < .0001 , figure 3(a ) ) , in agreement with the gene array results . semiquantitative rt - pcr analysis of 35 primary tumors and of 29 effusions showed significantly higher up - regulation of bcar1 in effusions ( p < .0001 , figure 3(b ) ) . immunostaining of 52 effusions and 26 of the 27 primary carcinomas ( one unsatisfactory reaction ) for p130cas showed its presence in tumor cells in 50/52 effusions and 24/26 primary carcinomas ( figures 4(a ) and 4(b ) ) . comparative analysis showed no significant difference in staining extent at these two anatomic sites ( p > .05 ) . os for the 44 patients with survival data ranged from 2393 months ( mean = 90 months ) , while dfs ranged from 0336 months ( mean = 55 months ) . in survival analysis , higher p130cas expression in effusions was associated with a trend for poor os ( p = .062 ) and dfs ( p = .098 ; figure 5 ) . semiquantitative rt - pcr analysis of 43 primary tumors and 25 effusions showed significantly higher vil2 expression in effusions ( p = .0021 , figure 3(c ) ) . protein levels and phosphorylation extent of ezrin were analyzed by western blotting using phospho- and pan - specific antibodies . pan - ezrin protein level was significantly higher in effusions ( p = .004 , figure 3(d ) ) , whereas p - ezrin levels did not significantly differ . although the fraction of phosphorylated protein did not significantly differ , the total amount of the protein was up - regulated in effusions ( p = .004 ) . thus , the absolute phosphorylated ezrin levels were higher in effusions compared to primary tumors . immunostaining of 51 of the 52 effusions ( one unsatisfactory reaction ) and 27 primary carcinomas showed significantly higher p - ezrin expression in effusions compared to primary carcinomas ( p < .001 in analysis of all cases , as well as patient - matched specimens ) , as evidenced by score = 4 staining in 49/51 effusions and only 2/27 primary carcinomas ( figures 4(c)4(e ) ) . ezrin was not analyzed for survival in view of the practically uniform score = 4 staining in effusions . immunostaining of 52 effusions and 23 of the 27 primary carcinomas ( 4 unsatisfactory reactions ) for claudin-4 showed its presence in tumor cells in 51/52 effusions and 20/23 primary carcinomas ( figures 4(f)4(i ) ) . however , staining extent was higher in effusions , a difference that was significant in analysis of all cases ( p = .002 ) , and showed a trend in matched specimen analysis ( p = .062 ) . claudin-4 protein expression was unrelated to os or dfs ( p > .05 ) . tsc1 showed a 2.3-fold downregulation in effusions compared to primary tumors in the array analysis . analysis of the phosphorylation level of the rapamycin sensitive mtor complex 1 ( mtorc1 ) substrate p70s6k showed that in spite of the tsc1 downregulation , the levels of p70s6k phosphorylation were lower in effusions compared to primary tumors ( p = .003 , figure 6 ) . breast carcinoma metastasis to the serosal cavities represents an advanced stage in tumor progression and is associated with extensive alterations at the molecular level , involving clinically established targets such as her-2 and hormone receptors , as well as other cancer - associated molecules [ 710 ] . despite the fact that breast carcinoma is one of the most extensively studied cancer forms , little effort has been directed towards understanding the biology of tumor cells in malignant effusions . the major aim of the present study was to characterize a general expression fingerprint that distinguishes effusions from primary tumors . our data show that 351 genes among 24,650 gene transcripts are significantly altered in effusions in comparison to primary carcinomas . many of these genes are involved in ecm - receptor interaction , focal adhesion and regulation of the actin cytoskeleton pathways , which define the metastatic potential of carcinoma cells by enhancing their motility and leading to anoikis escape in the absence of ecm molecules . the gene expression profile correlated with phenotypic change during the transition of breast carcinoma cells from the solid tumor to suspended cell clusters in pleural effusions . carcinoma cells in effusions showed down - regulated ecm encoding molecules such as decorin , fibronectin , collagens i , xxii and v , concomitantly with the downregulation of the ecm - binding receptors , integrins 5 and 7 . thus , it appears as if the cells in effusions lose the requirement for interaction with matrix components , possibly by a compensatory signaling mechanism within the cells . the second goal of the study was to highlight molecules with multiple functional influences on the metastatic potential of cells in effusions . this protein provides a functional link between the plasma membrane and the actin cytoskeleton by interacting with the cytoplasmic domains of adhesion membrane proteins and regulation of cytoskeleton polymerization through the rho pathway activation . moreover , ezrin promotes growth and survival via akt / mtor pathway activation in ewing 's sarcoma cell lines . a recent study provided additional information regarding the role of ezrin in elevation of the metastatic potential of carcinoma cells , by showing that its downregulation of the cell - cell adhesion molecule e - cadherin , and suggesting that ezrin is associated indirectly with the e - cadherin/-catenin complex by regulating src activation . screening of a broad spectrum of human cancers , including breast , lung and prostate tumors showed high expression of ezrin in tumors of mesenchymal origin and in primary breast carcinomas . moreover , ezrin expression was shown to be strongly associated with poor prognosis in breast carcinoma . in agreement with the latter report , higher vil2 mrna expression in effusions was associated with poor disease - free survival in our cohort ( data not shown ) . this finding requires further investigation , as it was obtained in analysis of only 17 effusions . the up - regulation of ezrin in effusions was validated using rt - pcr , western blotting and ihc . we found that the up - regulation of ezrin in effusions is associated with expression of the functionally active t567 phosphorylated ezrin [ 17 , 18 ] at the plasma membrane . this gene is not only up - regulated at the mrna and protein levels , but is also more active in effusions compared to solid tumors . since the increase in ezrin activation may influence multiple metastasis - associated cell functions [ 17 , 18 ] , the therapeutic targeting of this protein may prove beneficial in effusion therapy . statistical analysis of the array results showed that the tsc1 gene , encoding a protein involved in mtorc1 inhibition , is down - regulated in effusions in comparison to primary tumors . moreover , there is evidence that mtor activation can lead to anchorage - independent growth of carcinoma cells , making it a potentially important factor for cell survival in effusions . rapamycin analogues , such as temsirolimus ( cci-779 ) or everolimus ( rad-001 ) that target mtor are now in different stages of clinical trials for anti - cancer therapy as a single agent or as additive treatment having synergetic effect with er- and her2/neu- targeted therapy [ 24 , 25 ] . downregulation of the mtor inhibitor tsc1 in effusion may lead to subsequent activation of mtorc1 at this site of metastasis , suggesting that this signaling pathway may be altered along tumor progression in breast carcinoma . recent studies demonstrate that tsc1 directly interacts with ezrin and through this interaction regulates focal adhesion complex formation and causes cytoskeletal remodeling [ 26 , 27 ] . the loss of tcs1 results in loss of focal adhesions , cell rounding and progressive detachment of cells from the substrate . since effusions are characterized as clusters of detached carcinoma cells , the parallel dysregulation of tsc1 and ezrin expression may play a critical role in effusion formation . the relative phosphorylation extent of the mtorc1 substrate p70s6k [ 28 , 29 ] was measured in order to analyze the effect of tsc1 downregulation on the mtorc1 activity . in spite of the downregulation of the mtorc1 inhibitor tsc1 , the extent of p70s6k phosphorylation remains low in the effusions in comparison to primary tumors . one possible explanation is that other substrates of mtor may be relevant and p70s6k may be a minor effector of this pathway in effusions . hormone receptor status and the relevance of adjuvant hormonal therapy at different stages of the disease are central in breast carcinoma research . we have previously shown that er is down - regulated in effusions in comparison to primary tumors . in the present study we observed bcar1 gene up - regulation in effusions . high bcar1 expression was reported to be associated with a poor response to first - line tamoxifen therapy in patients with recurrent disease and with an increased rate of relapse . moreover the up - regulation of p130cas in effusions can be a result of population enrichment by resistant cells due to tamoxifen treatment . thus , bcar1 expression status in effusions must be taken under consideration while choosing therapeutic regimen in patients with breast carcinoma effusions . the up - regulation of p130cas can lead to subsequent activation of rac pathway and actin cytoskeleton rearrangements [ 34 , 35 ] . this can elevate the metastatic potential of the cells in effusions by enhancing cell migration and leading to anoikis escape , as has been shown in in vitro systems [ 36 , 37 ] . in contrast to the established importance of er- as a breast cancer marker , the prognostic and predictive relevance of er- remains unclear . several previous reports have shown correlation between low er- expression and advanced disease stage and shorter survival in various tumors , including gynecological carcinomas [ 3840 ] . in the present study we found downregulation of er- in effusions . since breast carcinoma effusions constitute stage iv disease , this observation is concordant with the above - detailed publications . low er- levels in effusions may contribute to tamoxifen resistance , as had been shown in er - positive primary breast carcinomas . thus , the expression levels of er- may influence decisions regarding therapeutic regimens for patients with this form of metastatic disease . claudins are a family of tight junction ( tj)-specific integral membrane proteins , including more than 20 members to date . tjs , located between epithelial or endothelial cells , at the apical region of the adjacent lateral membranes , control the paracellular transport of solutes and maintain cell polarity by blocking the free diffusion of proteins and lipids between the apical and basolateral domains of the plasma membrane [ 4244 ] . tj filaments also contain occludin , the first tj - specific integral membrane protein identified , yet it has been shown that claudins are essential and sufficient to form tj strands . the structure of claudins consists of intracellular amino and carboxy termini , four transmembrane domains , and two extracellular loops mediating interactions between claudins on adjacent cells [ 4244 ] . the second extracellular loop serves as a binding site for clostridium perfringens enterotoxin in claudin-3 and -4 . the carboxy terminus of most claudins contains potential serine and/or thereonine phosphorylation sites and a pdz - binding motif , to which the tj cytoplasmic scaffolding proteins zo-1 , -2 and -3 bind . we have recently shown that several claudin family members are upregulated in ovarian carcinoma effusions compared to corresponding primary carcinomas . in the present study , we found upregulation of claudin-4 in breast carcinoma effusions compared to primary carcinomas , suggesting that members of this family are upregulated at this anatomic site in multiple epithelial malignancies . our observations are in agreement with a recent study in which claudin-4 expression was shown to be associated with high grade and poor prognosis in breast carcinoma . the previously discovered role of claudin-3 and claudin-4 in cell motility and increased mmp-2 activity suggests that this may be yet another metastasis - promoting molecule in breast carcinoma effusions . in conclusion , gene array analysis of breast carcinoma effusions and primary carcinomas showed differences in expression of multiple genes regulating cell motility , invasion and metastasis . the study of effusions and the way they differ from solid tumors will expand our knowledge regarding tumor progression in general , as well as regarding malignancies affecting this anatomic site in particular , and may have an impact on treatment modalities and prognostic models .

the detection of breast carcinoma cells in effusions is associated with rapidly fatal outcome , but these cells are poorly characterized at the molecular level . this study compared the gene array signatures of breast carcinoma cells in primary carcinomas and effusions . the genetic signature of 10 primary tumors and 10 effusions was analyzed using the array - ready oligo set for the human genome platform . results for selected genes were validated using pcr , western blotting , and immunohistochemistry . array analysis identified 255 significantly downregulated and 96 upregulated genes in the effusion samples . the majority of differentially expressed genes were part of pathways involved in focal adhesion , extracellular matrix - cell interaction , and the regulation of the actin cytoskeleton . genes that were upregulated in effusions included krt8 , bcar1 , cldn4 , vil2 , while dcn , cldn19 , itga7 , and itga5 were downregulated at this anatomic site . pcr , western blotting , and immunohistochemistry confirmed the array findings for bcar1 , cldn4 , vil2 , and dcn . our data show that breast carcinoma cells in primary carcinomas and effusions have different gene expression signatures , and differentially express a large number of molecules related to adhesion , motility , and metastasis . these differences may have a critical role in designing therapy and in prognostication for patients with metastatic disease localized to the serosal cavities .

1. Introduction 2. Materials and Methods 3. Statistical Analysis 4. Results 5. Discussion

the incidence of breast cancer around the world greatly varies , with highest rates in the north america , western , and northern europe and australia / new zealand ( 82.599.4 per 100,000 women ) . pleural effusions may occur at any point of time in the clinical course and may be the sole manifestation of metastatic disease . this condition is associated with a poor median survival of less than 1 year [ 5 , 6 ] . in recent years , we have reported on the differential expression of metastasis - related molecules , including proteases , angiogenic molecules , signaling molecules , inhibitors of apoptosis , and transcription factors , in breast carcinoma effusions compared to patient - matched primary carcinomas [ 710 ] . the global view of cellular transcriptional activity using gene array technology is required to identify clusters of genetic markers that explain the complex biological processes involved in carcinoma progression to effusions . at present , only one study in which breast carcinoma effusions were compared with primary carcinomas is available . analyzed the gene expression signature of 19 effusions and compared them to 4 primary carcinomas , 8 cells lines and 4 specimens consisting of benign breast tissue . the present study compared the gene expression signatures of 10 effusions and 10 primary breast carcinomas . selected differentially expressed genes of pathways related to adhesion , interaction with the extracellular matrix ( ecm ) and regulation of the actin cytoskeleton were validated on mrna and protein level , and their clinical relevance was analyzed in a larger series of breast carcinoma effusions . our data demonstrate that in agreement with our previous observations , breast carcinoma cells in effusions are markedly different from their counterparts in primary carcinomas . ten effusions ( 7 pleural , 2 peritoneal , 1 pericardial ) from patients with primary breast carcinoma ( 9 of infiltrating ductal type , 1 lobular ) were submitted for routine diagnostic purposes to the department of pathology at the norwegian radium hospital during the period 19992005 . all effusions had > 50% carcinoma cells of the total cellular content , ranging between 80100% of cells in 8/10 specimens . biopsies from primary tumors and effusions were lysed using sv- total isolation kit ( promega , madison , wis , usa ) and total rna was extracted according to the manufacturer 's guidelines . the quantity and quality of the total rna preparations were assessed using a nanodrop nd-1000 ( nanodrop , wilmington , de , usa ) combined with agarose gel electrophoresis and only samples with ribosomal 28s/18s ratio near 2 were selected for array analysis . free rna was disassembled with mixture of 10 l 1 m naoh and 10 l 0.5 m edta and underwent neutralization with 25 l 1 m hepes . the hybridization efficiency and the yield of the dye incorporation were calculated using labeled cdna calculator ( http://www.pangloss.com/seidel/protocols/percent_inc.html ) . equal amount of cy3/cy5 labeled cdna were mixed and dried in the speed - vac . the slides , with printed array - ready oligo set for the human genome version 3.0 ( qiagen ) containing 34,580 longmer probes , representing 24,650 genes and 37,123 gene transcripts , were blocked with 1%bsa in 0.1% sds 5xssc buffer , washed in distilled water and dried at 1000 rpm for 2 minutes . the fluorescent intensities of cy5 and cy3 for each target spot were adjusted so that the mean cy3/cy5 ratio of housekeeping genes was equal to one , a design allowing more precise analysis of differentially expressed genes . total rna from specimens analyzed for bcar1 , vil2 and dcn expression was extracted using tri - reagent ( sigma ) according to the manufacturer 's guidelines . 0.5 g total rna was reverse - transcribed using the m - mlv reverse transcriptase ( promega ) with incubation of 2 hours at 37c , followed by 5 minutes at 95c , and diluted to 1 : 5 with rnase - free water . primer sequences for dcn were 5-tcc - gct - gaa - gag - ctc - agg - aat-3 for the forward primer , and 5-cct - tga - gga - atg - ctg - gtg - ata - ttg-3 for the reverse primer . the primer optimal concentration and the sensitivity , efficiency , and accuracy of qpcr were calibrated by amplifying serial geometric dilutions of pooled sample consisted from five primary tumor and five effusion cdna samples . in the remaining 6 cases , these 79 above - described specimens were manually immunostained for p130cas , phospho - ezrin ( p - ezrin ) , and claudin-4 . positive controls consisted of a breast carcinoma biopsy that demonstrated immunoreactivity for the studied antigens in a pilot study . staining was considered positive only when localized to the cell membrane in a linear pattern for p - ezrin and claudin-4 , and when present in the cytoplasm for the p130cas reaction . levels of phosphrylation of the torc1 substrate p70s6k were analyzed using goat anti - p - p70s6k and rabbit anti - p70 s6k antibodies ( santa cruz biotechnology , inc . ihc and ib results were analyzed using the spss - pc package , version 15.0 ( chicago , ill , usa ) . comparative analyses of tumor cell expression results in all effusions versus primary tumors were performed using the mann - whitney u test . the same test was applied for analysis of the relationship between protein expression in effusions and clinicopathologic parameters . univariate analysis for disease - free survival ( dfs ) and overall survival ( os ) for 44 patients with clinical data were executed using the kaplan - meier method and log - rank test . we have previously shown that effusions constitute a unique form of breast carcinoma metastasis with mrna and protein expression patterns that differ from primary tumors and solid metastases [ 710 ] . in the present study , we compared the global expression profile of breast carcinoma cells in effusions with that of primary carcinomas . figure 1(a ) shows volcano plot of global gene expression in effusions and primary tumors . we identified 255 significantly down - regulated and 96 significantly up - regulated genes in effusion samples ( total = 351 ) . pca analysis of six effusion and primary tumor samples was performed using the set of 351 genes that were differentially expressed in effusions and primary carcinomas ( figure 1(b ) , supplementary table 1 , available at doi:10.1155/2010/969084 . ) we additionally performed random pca analysis of the gene expression pattern in all 11 effusions and 11 primary tumor pairs ( figure 1(c ) ) . three patterns were identified : ( 1 ) unique for primary tumors ; ( 2 ) unique for effusions and ( 3 ) samples with overlapping gene expression . in order to understand the biological function of the genes that were up- or down - regulated in effusions , we used the go annotation ( http://www.geneontology.org/ ) and pathway - express ( 14 , 15 ) programs . we found multiple pathways involved in cell maintenance that are altered in effusions in comparison to primary tumors ( table 2 ) . it can be seen that the pathways involved in focal adhesion , ecm - receptor interaction and regulation of the actin cytoskeleton are highly involved in phenotypic transformation of carcinoma cells in primary tumors to those in effusions . some of the differentially - expressed genes were found to participate in the specific pathways listed above . other differentially - expressed genes could not be classified as components of a specific pathway , but are of great clinical impact in breast carcinoma , for example , er with a 3.23-fold downregulation in effusions and mta3 , an estrogen - sensitive gene involved in e - cadherin regulation , with a 2.42- fold downregulation in effusions . the significantly altered genes in effusions ( t - test/ anova , p - value < .05 ) were selected for performing upgma hierarchical clustering ( figure 2 ) . of these 10 clusters , cluster g included genes that were strongly up - regulated in effusions , while cluster j genes were strongly down - regulated in comparison to primary tumors . we found some clinically - relevant genes , such as krt8 , cldn4 and vil2 in cluster g , while cluster j included cldn19 , the ecm genes col1a1 , col22a1 , col5a2 and the itga7 and itga5 integrin genes . since cell motility and cell - ecm interactions may have a major effect on the metastatic potential of carcinoma cells in effusions , we focused on genes participating in regulation of the actin cytoskeleton , focal adhesion , and ecm - receptor interactions in validation of the array results . the genes focused on were the following : dcn , which encodes for the small cellular or pericellular matrix proteoglycan decorin and was found to be significantly down - regulated in effusions ; vil2 , encoding for ezrin , which controls the actin cytoskeleton dynamics ; bcar , encoding for the integrin signaling adaptor protein p130cas . in addition , tuberous sclerosis 1 ( tsc1 ) , also known as the tumor suppressor hamartin , the main inhibitor of the mtor signaling pathway [ 15 , 16 ] , was one of the genes that were found to be down - regulated in effusions in comparison to primary tumors . thus , we decided to analyze mtor activity in effusions compared to solid primary tumors . validation was by semiquantitative and quantitative rt - pcr , western blotting and immunohistochemistry , using an enlarged set of effusions and primary carcinomas . dcn levels were significantly higher in primary tumors ( p < .0001 , figure 3(a ) ) , in agreement with the gene array results . semiquantitative rt - pcr analysis of 35 primary tumors and of 29 effusions showed significantly higher up - regulation of bcar1 in effusions ( p < .0001 , figure 3(b ) ) . immunostaining of 52 effusions and 26 of the 27 primary carcinomas ( one unsatisfactory reaction ) for p130cas showed its presence in tumor cells in 50/52 effusions and 24/26 primary carcinomas ( figures 4(a ) and 4(b ) ) . os for the 44 patients with survival data ranged from 2393 months ( mean = 90 months ) , while dfs ranged from 0336 months ( mean = 55 months ) . in survival analysis , higher p130cas expression in effusions was associated with a trend for poor os ( p = .062 ) and dfs ( p = .098 ; figure 5 ) . semiquantitative rt - pcr analysis of 43 primary tumors and 25 effusions showed significantly higher vil2 expression in effusions ( p = .0021 , figure 3(c ) ) . although the fraction of phosphorylated protein did not significantly differ , the total amount of the protein was up - regulated in effusions ( p = .004 ) . thus , the absolute phosphorylated ezrin levels were higher in effusions compared to primary tumors . immunostaining of 51 of the 52 effusions ( one unsatisfactory reaction ) and 27 primary carcinomas showed significantly higher p - ezrin expression in effusions compared to primary carcinomas ( p < .001 in analysis of all cases , as well as patient - matched specimens ) , as evidenced by score = 4 staining in 49/51 effusions and only 2/27 primary carcinomas ( figures 4(c)4(e ) ) . ezrin was not analyzed for survival in view of the practically uniform score = 4 staining in effusions . immunostaining of 52 effusions and 23 of the 27 primary carcinomas ( 4 unsatisfactory reactions ) for claudin-4 showed its presence in tumor cells in 51/52 effusions and 20/23 primary carcinomas ( figures 4(f)4(i ) ) . however , staining extent was higher in effusions , a difference that was significant in analysis of all cases ( p = .002 ) , and showed a trend in matched specimen analysis ( p = .062 ) . tsc1 showed a 2.3-fold downregulation in effusions compared to primary tumors in the array analysis . analysis of the phosphorylation level of the rapamycin sensitive mtor complex 1 ( mtorc1 ) substrate p70s6k showed that in spite of the tsc1 downregulation , the levels of p70s6k phosphorylation were lower in effusions compared to primary tumors ( p = .003 , figure 6 ) . breast carcinoma metastasis to the serosal cavities represents an advanced stage in tumor progression and is associated with extensive alterations at the molecular level , involving clinically established targets such as her-2 and hormone receptors , as well as other cancer - associated molecules [ 710 ] . despite the fact that breast carcinoma is one of the most extensively studied cancer forms , little effort has been directed towards understanding the biology of tumor cells in malignant effusions . the major aim of the present study was to characterize a general expression fingerprint that distinguishes effusions from primary tumors . our data show that 351 genes among 24,650 gene transcripts are significantly altered in effusions in comparison to primary carcinomas . many of these genes are involved in ecm - receptor interaction , focal adhesion and regulation of the actin cytoskeleton pathways , which define the metastatic potential of carcinoma cells by enhancing their motility and leading to anoikis escape in the absence of ecm molecules . the gene expression profile correlated with phenotypic change during the transition of breast carcinoma cells from the solid tumor to suspended cell clusters in pleural effusions . carcinoma cells in effusions showed down - regulated ecm encoding molecules such as decorin , fibronectin , collagens i , xxii and v , concomitantly with the downregulation of the ecm - binding receptors , integrins 5 and 7 . thus , it appears as if the cells in effusions lose the requirement for interaction with matrix components , possibly by a compensatory signaling mechanism within the cells . the second goal of the study was to highlight molecules with multiple functional influences on the metastatic potential of cells in effusions . this protein provides a functional link between the plasma membrane and the actin cytoskeleton by interacting with the cytoplasmic domains of adhesion membrane proteins and regulation of cytoskeleton polymerization through the rho pathway activation . a recent study provided additional information regarding the role of ezrin in elevation of the metastatic potential of carcinoma cells , by showing that its downregulation of the cell - cell adhesion molecule e - cadherin , and suggesting that ezrin is associated indirectly with the e - cadherin/-catenin complex by regulating src activation . screening of a broad spectrum of human cancers , including breast , lung and prostate tumors showed high expression of ezrin in tumors of mesenchymal origin and in primary breast carcinomas . moreover , ezrin expression was shown to be strongly associated with poor prognosis in breast carcinoma . in agreement with the latter report , higher vil2 mrna expression in effusions was associated with poor disease - free survival in our cohort ( data not shown ) . the up - regulation of ezrin in effusions was validated using rt - pcr , western blotting and ihc . we found that the up - regulation of ezrin in effusions is associated with expression of the functionally active t567 phosphorylated ezrin [ 17 , 18 ] at the plasma membrane . this gene is not only up - regulated at the mrna and protein levels , but is also more active in effusions compared to solid tumors . statistical analysis of the array results showed that the tsc1 gene , encoding a protein involved in mtorc1 inhibition , is down - regulated in effusions in comparison to primary tumors . moreover , there is evidence that mtor activation can lead to anchorage - independent growth of carcinoma cells , making it a potentially important factor for cell survival in effusions . downregulation of the mtor inhibitor tsc1 in effusion may lead to subsequent activation of mtorc1 at this site of metastasis , suggesting that this signaling pathway may be altered along tumor progression in breast carcinoma . since effusions are characterized as clusters of detached carcinoma cells , the parallel dysregulation of tsc1 and ezrin expression may play a critical role in effusion formation . in spite of the downregulation of the mtorc1 inhibitor tsc1 , the extent of p70s6k phosphorylation remains low in the effusions in comparison to primary tumors . hormone receptor status and the relevance of adjuvant hormonal therapy at different stages of the disease are central in breast carcinoma research . we have previously shown that er is down - regulated in effusions in comparison to primary tumors . in the present study we observed bcar1 gene up - regulation in effusions . high bcar1 expression was reported to be associated with a poor response to first - line tamoxifen therapy in patients with recurrent disease and with an increased rate of relapse . moreover the up - regulation of p130cas in effusions can be a result of population enrichment by resistant cells due to tamoxifen treatment . thus , bcar1 expression status in effusions must be taken under consideration while choosing therapeutic regimen in patients with breast carcinoma effusions . the up - regulation of p130cas can lead to subsequent activation of rac pathway and actin cytoskeleton rearrangements [ 34 , 35 ] . this can elevate the metastatic potential of the cells in effusions by enhancing cell migration and leading to anoikis escape , as has been shown in in vitro systems [ 36 , 37 ] . in the present study we found downregulation of er- in effusions . thus , the expression levels of er- may influence decisions regarding therapeutic regimens for patients with this form of metastatic disease . tjs , located between epithelial or endothelial cells , at the apical region of the adjacent lateral membranes , control the paracellular transport of solutes and maintain cell polarity by blocking the free diffusion of proteins and lipids between the apical and basolateral domains of the plasma membrane [ 4244 ] . we have recently shown that several claudin family members are upregulated in ovarian carcinoma effusions compared to corresponding primary carcinomas . in the present study , we found upregulation of claudin-4 in breast carcinoma effusions compared to primary carcinomas , suggesting that members of this family are upregulated at this anatomic site in multiple epithelial malignancies . our observations are in agreement with a recent study in which claudin-4 expression was shown to be associated with high grade and poor prognosis in breast carcinoma . in conclusion , gene array analysis of breast carcinoma effusions and primary carcinomas showed differences in expression of multiple genes regulating cell motility , invasion and metastasis . the study of effusions and the way they differ from solid tumors will expand our knowledge regarding tumor progression in general , as well as regarding malignancies affecting this anatomic site in particular , and may have an impact on treatment modalities and prognostic models .

aging progresses and opportunities of medical care for elderly patients older than 80 years are increasing with the extension of life expectancy . in particular , the increase in cancer patients is a global challenge ; international agency for research of cancer estimated that the number of cancer patients expect to increase from 12.8 million patients in 2010 to 26 million patients in 2030 1 . japan has the most aged population in the world : the proportion of patients more than 80 years old continues to increase , from 0.7% in 1963 , to 4.9% in 1990 , and 7.8% in 2002 2 . the life expectancy of japanese at 80yearold was 8.5 years for a male and 11.4 years for a female in 2012 3 . about one million new cases of stomach cancer were estimated to have occurred globally ( 989,000 cases , 7.8% of total cancer cases ) , making it currently the fourth most common malignancy in the world , behind cancers of the lung , breast , and colorectum . more than 70% of stomach cancer cases ( 714,000 cases ) occur in developing countries ( 467,000 in men , 247,000 in women ) , and half of stomach cancers occur in eastern asia 4 . the number of new patients diagnosed with gastric cancer in 2002 was estimated to be 106,760 5 . although gastrectomy for elderly patients is increasing , the surgical procedure in the elderly must be decided carefully by assessing the patient 's tolerance of surgical stress because elderly patients have declining organ capacity and the quality of life postoperatively may suffer 6 . surgeons are concerned about the possibility of postoperative complications or hospital death when performing surgery for elderly patients with comorbidities , and often hesitate to treat these patients . it is important to consider the fact that elderly patients often have comorbidities and agerelated physiological problems , such as organ dysfunction . there have been several reports of shortterm outcomes of surgery in elderly patients 7 , 8 , 9 . however , a few reports of the evaluation of longterm outcomes including the cause of death were available . the aim of this study was to clarify the perioperative mortality and longterm survival of gastrectomy for elderly patients with gastric cancer , and to determine an appropriate postoperative treatment by assessing the longterm outcomes . surgical and pathological data of 95 patients at more than 80 yearsold and 366 patients at 60s yearsold who had undergone gastrectomy for histologically confirmed gastric adenocarcinoma from january 2003 to december 2010 at osaka city university hospital were retrospectively analyzed in this study . clinicopathological features , complication rates and the 5year survival of the two patient groups were compared . coronary disease was assigned to patients who were diagnosed with angina or myocardial infarction and who underwent stent placement , bypass surgery , or medical therapy . cerebrovascular disease was assigned to patients who were diagnosed with cerebral infarction or cerebral hemorrhage . diabetes mellitus was assigned to patients using oral hypoglycemic drugs or insulin , or to those with hba1c 4.3 , which are the criteria in our institution . pulmonary disease was assigned to patients with a history of chronic obstructive pulmonary disease ( copd ) , pulmonary tuberculosis or pulmonary resection . renal disease was assigned to patients with egfr less than 60 ml / min/1.73 m 10 . age , sex , comorbidity , hemoglobin , and american society of anesthesiology physical status classification ( asaps ) were obtained from preoperative anethesiology records . tumor depth , lymph node metastasis , pathological stage , and resectability of the tumor were evaluated . the pathological diagnosis and classification status were determined according to the 14th edition of japanese gastric cancer association 11 . the decision on the type of operation , e.g. , proximal gastric resection , total gastrectomy , or distal gastrectomy depended on tumor location , infiltration depth , and histological type . generally , all gastrectomies were combined with lymphadenectomy according to japanese gastric cancer treatment guidelines 12 . d1 or d1 + lymph node dissection was performed for early gastric cancer , and d2 lymph node dissection was performed for advanced gastric cancer . but patients with potentially fatal comorbidities underwent limited lymph node dissections to reduce postoperative morbidity or mortality by reducing operation time and blood loss . a postoperative complication was defined as grade ii or more , according to the clavien dindo classification method 13 . hospital discharge was decided based on consideration of the following criteria : ( i ) no requirement for intravenous medication or nutrition , ( ii ) no requirement for bed side care , ( iii ) no clinical sign of a complication , ( iv ) no elevated inflammatory reaction on laboratory data , ( v ) tolerable pain with no or only oral analgesics , ( vi ) the ability to fully ambulate without assistance , ( vii ) oral intake of more than half of given meal without vomiting or diarrhea , and ( viii ) a willingness of the patient and the family to discharge home . we recommended being admitted to a care hospital if even one of these criteria does not meet though condition was stable . longterm outcome was defined as the 5year overall survival ( os ) and disease specific survival ( dss ) at each stage . the categorical variables were presented as numbers and percentages and data for the groups were compared using the test . continuous variables with normal distributions were expressed as means and standard deviations and the means were compared using the mann the dss was defined as the time from the operation until death , only for tumor relapse . all reported p values were twosided ; p < 0.05 was considered to be significant . the statistical analyzes were performed using the jmp software program , version 10 ( sas institute , cary , nc , usa ) . surgical and pathological data of 95 patients at more than 80 yearsold and 366 patients at 60s yearsold who had undergone gastrectomy for histologically confirmed gastric adenocarcinoma from january 2003 to december 2010 at osaka city university hospital were retrospectively analyzed in this study . clinicopathological features , complication rates and the 5year survival of the two patient groups were compared . coronary disease was assigned to patients who were diagnosed with angina or myocardial infarction and who underwent stent placement , bypass surgery , or medical therapy . cerebrovascular disease was assigned to patients who were diagnosed with cerebral infarction or cerebral hemorrhage . diabetes mellitus was assigned to patients using oral hypoglycemic drugs or insulin , or to those with hba1c 4.3 , which are the criteria in our institution . pulmonary disease was assigned to patients with a history of chronic obstructive pulmonary disease ( copd ) , pulmonary tuberculosis or pulmonary resection . renal disease was assigned to patients with egfr less than 60 ml / min/1.73 m 10 . age , sex , comorbidity , hemoglobin , and american society of anesthesiology physical status classification ( asaps ) were obtained from preoperative anethesiology records . tumor depth , lymph node metastasis , pathological stage , and resectability of the tumor were evaluated . the pathological diagnosis and classification status were determined according to the 14th edition of japanese gastric cancer association 11 . the decision on the type of operation , e.g. , proximal gastric resection , total gastrectomy , or distal gastrectomy depended on tumor location , infiltration depth , and histological type . generally , all gastrectomies were combined with lymphadenectomy according to japanese gastric cancer treatment guidelines 12 . d1 or d1 + lymph node dissection was performed for early gastric cancer , and d2 lymph node dissection was performed for advanced gastric cancer . but patients with potentially fatal comorbidities underwent limited lymph node dissections to reduce postoperative morbidity or mortality by reducing operation time and blood loss . a postoperative complication was defined as grade ii or more , according to the clavien dindo classification method 13 . hospital discharge was decided based on consideration of the following criteria : ( i ) no requirement for intravenous medication or nutrition , ( ii ) no requirement for bed side care , ( iii ) no clinical sign of a complication , ( iv ) no elevated inflammatory reaction on laboratory data , ( v ) tolerable pain with no or only oral analgesics , ( vi ) the ability to fully ambulate without assistance , ( vii ) oral intake of more than half of given meal without vomiting or diarrhea , and ( viii ) a willingness of the patient and the family to discharge home . we recommended being admitted to a care hospital if even one of these criteria does not meet though condition was stable . longterm outcome was defined as the 5year overall survival ( os ) and disease specific survival ( dss ) at each stage . the categorical variables were presented as numbers and percentages and data for the groups were compared using the test . continuous variables with normal distributions were expressed as means and standard deviations and the means were compared using the mann the dss was defined as the time from the operation until death , only for tumor relapse . differences between the curves were analyzed by the logrank test . all reported p values were twosided ; the statistical analyzes were performed using the jmp software program , version 10 ( sas institute , cary , nc , usa ) . the clinical characteristics of both groups of patients are presented in table i. the elderly group comprised 55 males and 40 females with a mean age of 82.8 2.3 . of this group , the most common comorbidity was renal disease ( 42.1% ) , followed by hypertension ( 41.1% ) . more elderly patients than control had multiple comorbidities ( p < 0.001 ) . the asa physical status of the elderly patients was 1.1% in class i , 89.5% in class ii , and 9.5% in class iii , which was significantly different from that of the control group ( p < 0.001 ) . comparison of clinicopathological factors between the two groups tnm stage and perioperative outcomes are shown in table ii . of the elderly group , regarding the tumor depth , 38.9% were t1 , 11.6% were t2 , 9.5% were t3 , and 40% were t4 , which was not significantly different from the control group ( p = 0.0864 ) . in the elderly group , regarding lymph node metastasis status , 57.9% were n0 , 10.5% were n1 , 16.8% were n2 , and 14.7% were n3 , which was not significantly different from the control group ( p = 0.2750 ) . pathological staging results for the elderly group revealed that 45.3% were stage i , 17.9% were stage ii , 21.1% were stage iii , and 15.8% were stage iv , which was not significantly different from the control group ( p = 0.2577 ) . in the elderly group , regarding extent of resection , 82.1% were r0 , 10.5% were r1 , and 7.4% were r2 , which was not significantly different from the control group ( p = 0.0771 ) . surgical procedures performed in the elderly group included distal gastrectomy ( 69.5% ) , total gastrectomy ( 29.5% ) , and partial gastrectomy ( 1.1% ) , which was not significantly different from the control group ( p = 0.4903 ) . the proportion of d2 dissection was 37.9% in the elderly group and 50.3% in the control group . there was a significant difference between the groups ( p = 0.0314 ) . table iii shows that postoperative complications were not significantly different between the groups : 23.2% in the elderly group and 23.2% in the control group ( p = 0.9004 ) . of the elderly group , by the clavien dindo classification system 14.7% were grade ii , 7.4% were grade iiia / iiib , and 1.1% were grade v , which was not significantly different from the control group ( p = 0.5981 ) . postoperative pneumonia occurred in 4.2% of the elderly group and in 1.6% of the control group ; the incidence trended higher among the elderly , but the difference was not significant . in the elderly group , complication rate ( clavien dindo 3 ) was 13.9% in d2 dissection and 5.1% in less d2 dissection . complication rate was higher in d2 dissection than in less d2 dissection but there was no significant difference between the groups ( p = 0.1508 ) . the average postoperative stay for the elderly group was 19.3 days , which was not significantly different from 20.0 days for the control group ( p = 0.6599 ) . adjuvant chemotherapy was 9.5% in the elderly , which was significantly less than 29.0% of the control group ( p = 0.0012 ) . comparison of clinicopathological factors between the two groups dg , distal gastrectomy ; tg , total gastrectomy ; pg , proxymal gastrectomy comparison of shortterm outcomes of the two groups mortality was one case in the elderly group and two cases in the control group . except mortality cases , 89 patients ( 94.7% ) in the elderly group and 356 patients ( 97.8% ) in the control group could discharge home on foot . five patients ( 5.3% ) in the elderly group and eight patients ( 2.2% ) in the control group were admitted to care hospitals . there was no significant difference of the rate ( p = 0.1043 ) to discharge home between two groups ( table iii ) . survival rates were as follows : 5year os at stages i , ii , iii , and iv were 76% , 17% , 28% , and 15% in the elderly group and 95% , 83% , 54% , and 12% in the control group . there was no significant difference in 5year os of stage i and iv patients between the elderly and control groups , but the elderly stage ii and iii patients had significantly poorer prognosis ( fig . the stage ii and iii elderly patients fared worse than the corresponding controls for 5year dss ( fig . , it can be seen that the 5year os of stage i patients was worse than the 5year dss ( 5year os / dss ; 76.2%/100% , p = 0.0165 ) , whereas , there was no difference between the 5year os and dss for stage ii , iii , and iv patients ( 5year os / dss , stage ii ; 17.0%/31.3% , p = 0.5309 , stage iii ; 27.7%/32.2% , p = 0.9815 , and stage iv ; 15.7%/17.1% , p = 0.9960 ) . survival of elderly patients in stage ii and iii disease who did or did not undergo adjuvant chemotherapy is shown in figure 3 . there was no significant difference in 5year os between both groups ( p = 0.5244 ) . survival curves of os in the elderly and control group which were calculated by the kaplan meier method . survival curves of dss in the elderly and control group which were calculated by the kaplan meier method . survival curves of os in stage ii and iii patients who did or did not undergo adjuvant chemotherapy in the elderly group by the kaplan meier method . the number of deaths was 42 in the elderly group and 87 in the control group . as shown in figure 4 , the numbers of deaths in stages i / ii / iii / iv were 8/9/12/13 , respectively , in the elderly group and 19/11/23/34 , respectively , in the control group . the rates of relapse death were 0% ( for stage i ) , 56% ( ii ) , 92% ( iii ) , and 92% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 64% , 78% , and 100% . the rates of other disease death were 75% ( for stage i ) , 22% ( ii ) , 8% ( iii ) , and 8% ( iv ) in the elderly group ; in the control group , the corresponding rates were 33% , 0% , 17% , and 0% . the rates of other cancer death were 25% ( for stage i ) , 22% ( ii ) , 0% ( iii ) , and 0% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 36% , 4% , and 0% . there were no significant differences in the proportion of patients who die due to recurrence , other malignant disease and other disease between the elderly group and the control group ( p = 0.1950 ) ( fig . the clinical characteristics of both groups of patients are presented in table i. the elderly group comprised 55 males and 40 females with a mean age of 82.8 2.3 . of this group , the most common comorbidity was renal disease ( 42.1% ) , followed by hypertension ( 41.1% ) . more elderly patients than control had multiple comorbidities ( p < 0.001 ) . the asa physical status of the elderly patients was 1.1% in class i , 89.5% in class ii , and 9.5% in class iii , which was significantly different from that of the control group ( p < 0.001 ) . comparison of clinicopathological factors between the two groups tnm stage and perioperative outcomes are shown in table ii . of the elderly group , regarding the tumor depth , 38.9% were t1 , 11.6% were t2 , 9.5% were t3 , and 40% were t4 , which was not significantly different from the control group ( p = 0.0864 ) . in the elderly group , regarding lymph node metastasis status , 57.9% were n0 , 10.5% were n1 , 16.8% were n2 , and 14.7% were n3 , which was not significantly different from the control group ( p = 0.2750 ) . pathological staging results for the elderly group revealed that 45.3% were stage i , 17.9% were stage ii , 21.1% were stage iii , and 15.8% were stage iv , which was not significantly different from the control group ( p = 0.2577 ) . in the elderly group , regarding extent of resection , 82.1% were r0 , 10.5% were r1 , and 7.4% were r2 , which was not significantly different from the control group ( p = 0.0771 ) . surgical procedures performed in the elderly group included distal gastrectomy ( 69.5% ) , total gastrectomy ( 29.5% ) , and partial gastrectomy ( 1.1% ) , which was not significantly different from the control group ( p = 0.4903 ) . the proportion of d2 dissection was 37.9% in the elderly group and 50.3% in the control group . there was a significant difference between the groups ( p = 0.0314 ) . table iii shows that postoperative complications were not significantly different between the groups : 23.2% in the elderly group and 23.2% in the control group ( p = 0.9004 ) . of the elderly group , by the clavien dindo classification system 14.7% were grade ii , 7.4% were grade iiia / iiib , and 1.1% were grade v , which was not significantly different from the control group ( p = 0.5981 ) . postoperative pneumonia occurred in 4.2% of the elderly group and in 1.6% of the control group ; the incidence trended higher among the elderly , but the difference was not significant . in the elderly group , complication rate ( clavien dindo 3 ) was 13.9% in d2 dissection and 5.1% in less d2 dissection . complication rate was higher in d2 dissection than in less d2 dissection but there was no significant difference between the groups ( p = 0.1508 ) . the average postoperative stay for the elderly group was 19.3 days , which was not significantly different from 20.0 days for the control group ( p = 0.6599 ) . adjuvant chemotherapy was 9.5% in the elderly , which was significantly less than 29.0% of the control group ( p = 0.0012 ) . comparison of clinicopathological factors between the two groups dg , distal gastrectomy ; tg , total gastrectomy ; pg , proxymal gastrectomy comparison of shortterm outcomes of the two groups mortality was one case in the elderly group and two cases in the control group . except mortality cases , 89 patients ( 94.7% ) in the elderly group and 356 patients ( 97.8% ) in the control group could discharge home on foot . five patients ( 5.3% ) in the elderly group and eight patients ( 2.2% ) in the control group were admitted to care hospitals . there was no significant difference of the rate ( p = 0.1043 ) to discharge home between two groups ( table iii ) . survival rates were as follows : 5year os at stages i , ii , iii , and iv were 76% , 17% , 28% , and 15% in the elderly group and 95% , 83% , 54% , and 12% in the control group . there was no significant difference in 5year os of stage i and iv patients between the elderly and control groups , but the elderly stage ii and iii patients had significantly poorer prognosis ( fig . the stage ii and iii elderly patients fared worse than the corresponding controls for 5year dss ( fig . , it can be seen that the 5year os of stage i patients was worse than the 5year dss ( 5year os / dss ; 76.2%/100% , p = 0.0165 ) , whereas , there was no difference between the 5year os and dss for stage ii , iii , and iv patients ( 5year os / dss , stage ii ; 17.0%/31.3% , p = 0.5309 , stage iii ; 27.7%/32.2% , p = 0.9815 , and stage iv ; 15.7%/17.1% , p = 0.9960 ) . survival of elderly patients in stage ii and iii disease who did or did not undergo adjuvant chemotherapy is shown in figure 3 . there was no significant difference in 5year os between both groups ( p = 0.5244 ) . survival curves of os in the elderly and control group which were calculated by the kaplan meier method . survival curves of dss in the elderly and control group which were calculated by the kaplan meier method . survival curves of os in stage ii and iii patients who did or did not undergo adjuvant chemotherapy in the elderly group by the kaplan meier method . the number of deaths was 42 in the elderly group and 87 in the control group . as shown in figure 4 , the numbers of deaths in stages i / ii / iii / iv were 8/9/12/13 , respectively , in the elderly group and 19/11/23/34 , respectively , in the control group . the rates of relapse death were 0% ( for stage i ) , 56% ( ii ) , 92% ( iii ) , and 92% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 64% , 78% , and 100% . the rates of other disease death were 75% ( for stage i ) , 22% ( ii ) , 8% ( iii ) , and 8% ( iv ) in the elderly group ; in the control group , the corresponding rates were 33% , 0% , 17% , and 0% . the rates of other cancer death were 25% ( for stage i ) , 22% ( ii ) , 0% ( iii ) , and 0% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 36% , 4% , and 0% . there were no significant differences in the proportion of patients who die due to recurrence , other malignant disease and other disease between the elderly group and the control group ( p = 0.1950 ) ( fig . current japanese gastric cancer guidelines describe the appropriate therapy for patients with gastric cancer , but there is no clear description regarding surgical treatment of the elderly 11 . in the present study , we evaluated the difference of operative mortality and longterm survival of surgical treatment for elderly patients with gastric carcinoma to determine an appropriate postoperative treatment for elderly patients . the egfr was lower in the elderly than the control group , and more elderly patients had two or more comorbidities , especially systemic diseases such as pulmonary and cardiovascular disease . however , there was no significant difference in the incidence of postoperative complications between the groups . these results suggest that gastrectomy can be carried out safely in patients aged 80 and older through careful monitoring of the postoperative status . the percentage of comorbidity of 7079 years old patients that performed gastrectomy in the same period as the current study was higher than that of control group and lower than that of the elderly . the postoperative complication rate of 7079 years old patients was not significantly different from that of both groups ( data not shown ) . many studies have shown that concomitant illness , advanced stage , prolonged operative time , excessive blood loss , and age are risk factors for the occurrence of complications after gastrectomy 14 , 15 , 16 , 17 . in contrast , others reported that the incidence was similar 19 , 20 , 21 , so this topic remains controversial . wu cw et al . 6 reported a complication rate of 2535% in the elderly group , significantly more than in the younger group . 18 reported that the complications after gastrectomy for elderly patients were likely to be fatal or severe compared to those in younger patients . on the other hand , katai et al . 20 also reported that gastrectomy can be carried out safely in elderly patients and that the short and longterm outcomes in elderly patients were comparable to those in younger patients . this safety might be due to advancements in perioperative management such as anesthesiology , intensive care , surgical techniques , and devices of surgical tools . in the current study , there was no significant difference in survival between d2 and less d2 dissection in stage ii and stage iii of the elderly group ( data not shown ) . previous report showed the incidence of postoperative pneumonia in elderly patients with gastrectomy to be 216% [ 7 , 8 , 9 , 19 , 20 , 22 , 23 , 24 , 25 ] . some reports concluded that there were no differences in postoperative pulmonary complication between elderly and younger groups 20 , 24 , 25 , 26 , 27 . however , postoperative pneumonia in these reports trended higher in elderly groups , consistent with the current results . it was reported that patients aged 85 and older were at high risk for postoperative pneumonia 7 , 8 . 7 reported that postoperative pneumonia occurred significantly more often in patients aged 85 and older than in patients aged 7584 . recently , performance of laparoscopic gastrectomy for early gastric cancer has been common in asia ; after shortterm followup , this procedure was reported to be safe and feasible in the elderly 9 , 23 , 24 . 24 reported that postoperative respiratory complications were quite low in the elderly group despite the fact that many had preoperative respiratory disease . laparoscopy with a small incision and earlier start of postoperative walk might be useful for preventing postoperative pneumonia for elderly patients . in this study , analysis of os and dss in stage ii and iii patients revealed a poorer prognosis for the elderly group than the control group . the reason for this may be that less adjuvant chemotherapy was used . only nine of stage ii and iii patients underwent adjuvant chemotherapy of 3 months or more . in japan , the standard treatment regimen for stage ii and iii in patients aged 2080 years is surgery and adjuvant chemotherapy 28 . in this study , whether patients aged 80 and older underwent adjuvant chemotherapy depended on a discussion between the patient and attending physician . attending physicians tended to be likely to accept the desire of patients to reject chemotherapy until their physical strength recovered sufficiently . another reason for poor os in stage ii patients is thought to be that there are many deaths due to other disease and other cancers , and it is thought that there is a risk for developing a new cancer after surgery in the elderly . thus , followup encompassing problems of the whole body , as well as cancer recurrence , is important . to our knowledge , there is no evidence regarding postoperative chemotherapy for elderly patients aged 80 and older in japan . a phase iii clinical trial of adjuvant chemotherapy and chemotherapy for unresectable gastric cancer was performed , but patients aged 80 and over were excluded 28 , 29 . 30 reported that the incidences of grade iii hematological and nonhematological toxicities of s1 adjuvant chemotherapy for the elderly were < 5% , and that this regimen was safe and feasible and tsushima et al . 31 reported that s1 or s1 plus cisplatin for elderly patients presented a high risk of hematological toxicities , but was feasible . however , there were only a few patients aged 80 and older in these studies . in the present study , of the elderly stage ii and iii patients , there was no significant difference in prognosis between the patients who underwent adjuvant chemotherapy and those who did not do . this result might be related to the fact that various kinds of chemotheraputic regimens were performed and the period of administration were different in this study . another reason for this result is that patient 's illness was strongly involved in decision making of undergoing or discontinuing adjuvant chemotherapy . it seems that the elderly patients , in consultation with their physicians , accepted a slightly different goal of therapy than the younger patients who are certainly understandable . a large clinical trial assessing not only the safety and feasibility of adjuvant chemotherapy but also the effect for longterm outcomes in patients aged 80 and older is required . because this study was retrospective , we could not draw a definitive conclusion about the usefulness of d2 dissection and adjuvant chemotherapy for the elderly patients . however our results suggested that there was no benefit of d2 dissection and adjuvant chemotherapy in survival for the elderly stage ii and iii patients . i would advocate for a novel neoadjuvant systemic therapy in the elderly if they were known to harbor stage ii or stage iii gastric cancers . in the current study , there was a significant difference between os and dss in elderly stage i patients . in the two cases of death from other malignant disease , the second primary malignancies were diagnosed after gastrectomy . thus , postoperative examinations should be performed with consideration for the possible incidence of other malignant diseases . in contrast , there was no significant difference between os and dss in the stage ii , iii , and iv elderly patients . our data showed that 56% of deaths in stage ii were from recurrence and 44% were due to other disease and malignant death . these results suggest that we should make an effort to not only to prevent recurrence but also to manage accompanying illness and to screen for other malignant disease . thus , we should be aware of recurrence during followup . however , one problem is that patients aged 80 and older often have renal dysfunction . 30 reported that treatment events of s1 , such as delay and dose reduction , occurred more frequently in elderly than in nonelderly patients . thus , for the elderly , development of anticancer drug which has less toxicity , for example molecular target therapy , is desired . because this study was retrospective , we could not draw a definitive conclusion about the usefulness of d2 dissection and adjuvant chemotherapy for the elderly patients . however our results suggested that there was no benefit of d2 dissection and adjuvant chemotherapy in survival for the elderly patients with stage ii or stage iii disease . i would advocate for a novel neoadjuvant systemic therapy in the elderly if they were known to harbor stage ii or stage iii gastric cancers . in the present study , we demonstrated that there was no significant difference of the severity of complication and the rate to discharge home on foot between two groups , suggesting that elderly patients could be underwent gastrectomy without falling their activity . however , the percentage ( 5.3% ) of patients admitted to care hospitals was higher in the elderly group than those ( 2.2% ) in the control group . in future study , therefore , it might be necessary to clarify whether the elderly patients could recover their activity to the preoperative baseline . followup with attention to accompanying illness and other malignant disease of stage i elderly patients is needed . in stage ii and iii disease patients , a novel drug which is acceptable for the elderly is needed as a postoperative therapy .

backgroundthe aim of this study was to clarify the operative mortality and longterm survival of gastrectomy for elderly patients with gastric cancer.methodsa total of 461 patients who underwent gastrectomy for gastric cancer in our hospital were classified as elderly group ( 80 yearsold , 95 patients ) and control group ( 6069 yearsold , 366 patients).resultsthe frequency of comorbidities was significantly ( p < 0.05 ) higher in elderly group ( 74.7% ) than that in the control group ( 49.5% ) . no significant difference of the postoperative complication rate was found between the elderly group ( 23.2% ) and the control group ( 23.2% ) . adjuvant chemotherapy was 9.5% in the elderly group , which was significantly less than 29.0% of the control group ( p < 0.05 ) . stage ii and iii elderly patients had worse disease specific survival ( dss ) than controls did . in the elderly , overall survival ( os ) was significantly worse than dss in stage i patients ( p < 0.05).conclusionsthe operative complication rate of elderly patients was comparable to the control group . comorbidity and occurrence of secondary malignant disease should be followed for elderly patients at stage i. for stage ii and iii disease patients , a novel drug which is acceptable for the elderly is needed as a postoperative therapy . j. surg . oncol . 2015 111:848854 . 2015 the authors . journal of surgical oncology published by wiley periodicals , inc .

INTRODUCTION MATERIALS AND METHODS Patients Comorbidity Data ShortTerm Outcome LongTerm Outcome Statistical Analysis RESULTS Clinicopathological Characteristics of Patients With Gastric Cancer Patient Survival DISCUSSION CONCLUSIONS

although gastrectomy for elderly patients is increasing , the surgical procedure in the elderly must be decided carefully by assessing the patient 's tolerance of surgical stress because elderly patients have declining organ capacity and the quality of life postoperatively may suffer 6 . the aim of this study was to clarify the perioperative mortality and longterm survival of gastrectomy for elderly patients with gastric cancer , and to determine an appropriate postoperative treatment by assessing the longterm outcomes . surgical and pathological data of 95 patients at more than 80 yearsold and 366 patients at 60s yearsold who had undergone gastrectomy for histologically confirmed gastric adenocarcinoma from january 2003 to december 2010 at osaka city university hospital were retrospectively analyzed in this study . longterm outcome was defined as the 5year overall survival ( os ) and disease specific survival ( dss ) at each stage . surgical and pathological data of 95 patients at more than 80 yearsold and 366 patients at 60s yearsold who had undergone gastrectomy for histologically confirmed gastric adenocarcinoma from january 2003 to december 2010 at osaka city university hospital were retrospectively analyzed in this study . longterm outcome was defined as the 5year overall survival ( os ) and disease specific survival ( dss ) at each stage . more elderly patients than control had multiple comorbidities ( p < 0.001 ) . the asa physical status of the elderly patients was 1.1% in class i , 89.5% in class ii , and 9.5% in class iii , which was significantly different from that of the control group ( p < 0.001 ) . of the elderly group , regarding the tumor depth , 38.9% were t1 , 11.6% were t2 , 9.5% were t3 , and 40% were t4 , which was not significantly different from the control group ( p = 0.0864 ) . in the elderly group , regarding lymph node metastasis status , 57.9% were n0 , 10.5% were n1 , 16.8% were n2 , and 14.7% were n3 , which was not significantly different from the control group ( p = 0.2750 ) . pathological staging results for the elderly group revealed that 45.3% were stage i , 17.9% were stage ii , 21.1% were stage iii , and 15.8% were stage iv , which was not significantly different from the control group ( p = 0.2577 ) . in the elderly group , regarding extent of resection , 82.1% were r0 , 10.5% were r1 , and 7.4% were r2 , which was not significantly different from the control group ( p = 0.0771 ) . surgical procedures performed in the elderly group included distal gastrectomy ( 69.5% ) , total gastrectomy ( 29.5% ) , and partial gastrectomy ( 1.1% ) , which was not significantly different from the control group ( p = 0.4903 ) . the proportion of d2 dissection was 37.9% in the elderly group and 50.3% in the control group . table iii shows that postoperative complications were not significantly different between the groups : 23.2% in the elderly group and 23.2% in the control group ( p = 0.9004 ) . of the elderly group , by the clavien dindo classification system 14.7% were grade ii , 7.4% were grade iiia / iiib , and 1.1% were grade v , which was not significantly different from the control group ( p = 0.5981 ) . postoperative pneumonia occurred in 4.2% of the elderly group and in 1.6% of the control group ; the incidence trended higher among the elderly , but the difference was not significant . in the elderly group , complication rate ( clavien dindo 3 ) was 13.9% in d2 dissection and 5.1% in less d2 dissection . complication rate was higher in d2 dissection than in less d2 dissection but there was no significant difference between the groups ( p = 0.1508 ) . the average postoperative stay for the elderly group was 19.3 days , which was not significantly different from 20.0 days for the control group ( p = 0.6599 ) . adjuvant chemotherapy was 9.5% in the elderly , which was significantly less than 29.0% of the control group ( p = 0.0012 ) . comparison of clinicopathological factors between the two groups dg , distal gastrectomy ; tg , total gastrectomy ; pg , proxymal gastrectomy comparison of shortterm outcomes of the two groups mortality was one case in the elderly group and two cases in the control group . except mortality cases , 89 patients ( 94.7% ) in the elderly group and 356 patients ( 97.8% ) in the control group could discharge home on foot . five patients ( 5.3% ) in the elderly group and eight patients ( 2.2% ) in the control group were admitted to care hospitals . there was no significant difference of the rate ( p = 0.1043 ) to discharge home between two groups ( table iii ) . survival rates were as follows : 5year os at stages i , ii , iii , and iv were 76% , 17% , 28% , and 15% in the elderly group and 95% , 83% , 54% , and 12% in the control group . there was no significant difference in 5year os of stage i and iv patients between the elderly and control groups , but the elderly stage ii and iii patients had significantly poorer prognosis ( fig . the stage ii and iii elderly patients fared worse than the corresponding controls for 5year dss ( fig . , it can be seen that the 5year os of stage i patients was worse than the 5year dss ( 5year os / dss ; 76.2%/100% , p = 0.0165 ) , whereas , there was no difference between the 5year os and dss for stage ii , iii , and iv patients ( 5year os / dss , stage ii ; 17.0%/31.3% , p = 0.5309 , stage iii ; 27.7%/32.2% , p = 0.9815 , and stage iv ; 15.7%/17.1% , p = 0.9960 ) . survival of elderly patients in stage ii and iii disease who did or did not undergo adjuvant chemotherapy is shown in figure 3 . survival curves of dss in the elderly and control group which were calculated by the kaplan meier method . survival curves of os in stage ii and iii patients who did or did not undergo adjuvant chemotherapy in the elderly group by the kaplan meier method . the number of deaths was 42 in the elderly group and 87 in the control group . as shown in figure 4 , the numbers of deaths in stages i / ii / iii / iv were 8/9/12/13 , respectively , in the elderly group and 19/11/23/34 , respectively , in the control group . the rates of relapse death were 0% ( for stage i ) , 56% ( ii ) , 92% ( iii ) , and 92% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 64% , 78% , and 100% . the rates of other disease death were 75% ( for stage i ) , 22% ( ii ) , 8% ( iii ) , and 8% ( iv ) in the elderly group ; in the control group , the corresponding rates were 33% , 0% , 17% , and 0% . the rates of other cancer death were 25% ( for stage i ) , 22% ( ii ) , 0% ( iii ) , and 0% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 36% , 4% , and 0% . there were no significant differences in the proportion of patients who die due to recurrence , other malignant disease and other disease between the elderly group and the control group ( p = 0.1950 ) ( fig . the asa physical status of the elderly patients was 1.1% in class i , 89.5% in class ii , and 9.5% in class iii , which was significantly different from that of the control group ( p < 0.001 ) . of the elderly group , regarding the tumor depth , 38.9% were t1 , 11.6% were t2 , 9.5% were t3 , and 40% were t4 , which was not significantly different from the control group ( p = 0.0864 ) . in the elderly group , regarding lymph node metastasis status , 57.9% were n0 , 10.5% were n1 , 16.8% were n2 , and 14.7% were n3 , which was not significantly different from the control group ( p = 0.2750 ) . pathological staging results for the elderly group revealed that 45.3% were stage i , 17.9% were stage ii , 21.1% were stage iii , and 15.8% were stage iv , which was not significantly different from the control group ( p = 0.2577 ) . in the elderly group , regarding extent of resection , 82.1% were r0 , 10.5% were r1 , and 7.4% were r2 , which was not significantly different from the control group ( p = 0.0771 ) . surgical procedures performed in the elderly group included distal gastrectomy ( 69.5% ) , total gastrectomy ( 29.5% ) , and partial gastrectomy ( 1.1% ) , which was not significantly different from the control group ( p = 0.4903 ) . the proportion of d2 dissection was 37.9% in the elderly group and 50.3% in the control group . table iii shows that postoperative complications were not significantly different between the groups : 23.2% in the elderly group and 23.2% in the control group ( p = 0.9004 ) . of the elderly group , by the clavien dindo classification system 14.7% were grade ii , 7.4% were grade iiia / iiib , and 1.1% were grade v , which was not significantly different from the control group ( p = 0.5981 ) . postoperative pneumonia occurred in 4.2% of the elderly group and in 1.6% of the control group ; the incidence trended higher among the elderly , but the difference was not significant . in the elderly group , complication rate ( clavien dindo 3 ) was 13.9% in d2 dissection and 5.1% in less d2 dissection . complication rate was higher in d2 dissection than in less d2 dissection but there was no significant difference between the groups ( p = 0.1508 ) . the average postoperative stay for the elderly group was 19.3 days , which was not significantly different from 20.0 days for the control group ( p = 0.6599 ) . adjuvant chemotherapy was 9.5% in the elderly , which was significantly less than 29.0% of the control group ( p = 0.0012 ) . comparison of clinicopathological factors between the two groups dg , distal gastrectomy ; tg , total gastrectomy ; pg , proxymal gastrectomy comparison of shortterm outcomes of the two groups mortality was one case in the elderly group and two cases in the control group . except mortality cases , 89 patients ( 94.7% ) in the elderly group and 356 patients ( 97.8% ) in the control group could discharge home on foot . five patients ( 5.3% ) in the elderly group and eight patients ( 2.2% ) in the control group were admitted to care hospitals . there was no significant difference of the rate ( p = 0.1043 ) to discharge home between two groups ( table iii ) . survival rates were as follows : 5year os at stages i , ii , iii , and iv were 76% , 17% , 28% , and 15% in the elderly group and 95% , 83% , 54% , and 12% in the control group . there was no significant difference in 5year os of stage i and iv patients between the elderly and control groups , but the elderly stage ii and iii patients had significantly poorer prognosis ( fig . the stage ii and iii elderly patients fared worse than the corresponding controls for 5year dss ( fig . , it can be seen that the 5year os of stage i patients was worse than the 5year dss ( 5year os / dss ; 76.2%/100% , p = 0.0165 ) , whereas , there was no difference between the 5year os and dss for stage ii , iii , and iv patients ( 5year os / dss , stage ii ; 17.0%/31.3% , p = 0.5309 , stage iii ; 27.7%/32.2% , p = 0.9815 , and stage iv ; 15.7%/17.1% , p = 0.9960 ) . survival of elderly patients in stage ii and iii disease who did or did not undergo adjuvant chemotherapy is shown in figure 3 . survival curves of dss in the elderly and control group which were calculated by the kaplan meier method . survival curves of os in stage ii and iii patients who did or did not undergo adjuvant chemotherapy in the elderly group by the kaplan meier method . the number of deaths was 42 in the elderly group and 87 in the control group . as shown in figure 4 , the numbers of deaths in stages i / ii / iii / iv were 8/9/12/13 , respectively , in the elderly group and 19/11/23/34 , respectively , in the control group . the rates of relapse death were 0% ( for stage i ) , 56% ( ii ) , 92% ( iii ) , and 92% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 64% , 78% , and 100% . the rates of other disease death were 75% ( for stage i ) , 22% ( ii ) , 8% ( iii ) , and 8% ( iv ) in the elderly group ; in the control group , the corresponding rates were 33% , 0% , 17% , and 0% . the rates of other cancer death were 25% ( for stage i ) , 22% ( ii ) , 0% ( iii ) , and 0% ( iv ) in the elderly group ; in the control group , the corresponding rates were 32% , 36% , 4% , and 0% . there were no significant differences in the proportion of patients who die due to recurrence , other malignant disease and other disease between the elderly group and the control group ( p = 0.1950 ) ( fig . current japanese gastric cancer guidelines describe the appropriate therapy for patients with gastric cancer , but there is no clear description regarding surgical treatment of the elderly 11 . in the present study , we evaluated the difference of operative mortality and longterm survival of surgical treatment for elderly patients with gastric carcinoma to determine an appropriate postoperative treatment for elderly patients . the egfr was lower in the elderly than the control group , and more elderly patients had two or more comorbidities , especially systemic diseases such as pulmonary and cardiovascular disease . however , there was no significant difference in the incidence of postoperative complications between the groups . the percentage of comorbidity of 7079 years old patients that performed gastrectomy in the same period as the current study was higher than that of control group and lower than that of the elderly . the postoperative complication rate of 7079 years old patients was not significantly different from that of both groups ( data not shown ) . 6 reported a complication rate of 2535% in the elderly group , significantly more than in the younger group . 20 also reported that gastrectomy can be carried out safely in elderly patients and that the short and longterm outcomes in elderly patients were comparable to those in younger patients . in the current study , there was no significant difference in survival between d2 and less d2 dissection in stage ii and stage iii of the elderly group ( data not shown ) . recently , performance of laparoscopic gastrectomy for early gastric cancer has been common in asia ; after shortterm followup , this procedure was reported to be safe and feasible in the elderly 9 , 23 , 24 . in this study , analysis of os and dss in stage ii and iii patients revealed a poorer prognosis for the elderly group than the control group . in japan , the standard treatment regimen for stage ii and iii in patients aged 2080 years is surgery and adjuvant chemotherapy 28 . another reason for poor os in stage ii patients is thought to be that there are many deaths due to other disease and other cancers , and it is thought that there is a risk for developing a new cancer after surgery in the elderly . in the present study , of the elderly stage ii and iii patients , there was no significant difference in prognosis between the patients who underwent adjuvant chemotherapy and those who did not do . because this study was retrospective , we could not draw a definitive conclusion about the usefulness of d2 dissection and adjuvant chemotherapy for the elderly patients . however our results suggested that there was no benefit of d2 dissection and adjuvant chemotherapy in survival for the elderly stage ii and iii patients . in the current study , there was a significant difference between os and dss in elderly stage i patients . in contrast , there was no significant difference between os and dss in the stage ii , iii , and iv elderly patients . thus , for the elderly , development of anticancer drug which has less toxicity , for example molecular target therapy , is desired . because this study was retrospective , we could not draw a definitive conclusion about the usefulness of d2 dissection and adjuvant chemotherapy for the elderly patients . however our results suggested that there was no benefit of d2 dissection and adjuvant chemotherapy in survival for the elderly patients with stage ii or stage iii disease . i would advocate for a novel neoadjuvant systemic therapy in the elderly if they were known to harbor stage ii or stage iii gastric cancers . in the present study , we demonstrated that there was no significant difference of the severity of complication and the rate to discharge home on foot between two groups , suggesting that elderly patients could be underwent gastrectomy without falling their activity . however , the percentage ( 5.3% ) of patients admitted to care hospitals was higher in the elderly group than those ( 2.2% ) in the control group . in future study , therefore , it might be necessary to clarify whether the elderly patients could recover their activity to the preoperative baseline . followup with attention to accompanying illness and other malignant disease of stage i elderly patients is needed . in stage ii and iii disease patients , a novel drug which is acceptable for the elderly is needed as a postoperative therapy .

partial - thickness rotator cuff tears ( ptrcts ) have the potential to cause significant pain and disability in affected patients . after failed conservative management , operative intervention is typically indicated for patients with persistent symptoms . surgical treatment is generally limited to tear debridement with or without acromioplasty or tear repair with or without acromioplasty . most authors recommend repair of tears involving 50% or more of the tendon thickness . with the advent of magnetic resonance imaging ( mri ) and shoulder arthroscopy , more unfortunately , high - quality data on the management of ptrcts are relatively lacking in the literature when compared with those available on full - thickness tears . partial - thickness rotator cuff tears are classified into three subtypes : bursal side , articular side , and intratendinous tears . among them , intratendinous tears are an important clinical entity . intratendinous tears are characterized by the absence of fiber disruption on both the bursal and articular surface of the rotator cuff . a cadaveric study reported that this type of tear was the most frequent ( 55% ) among all of the partial - thickness tears . because the intratendinous tear has no communication to the subacromial space and the glenohumeral joint , it is probably the most difficult condition to be diagnosed among the three types of the partial tear . . however , confirmation of such lesions during operation can be difficult . only a few literature described how to find the intratendinous tears during surgery . both bursal side and articular side rotator cuff tears have been extensively studied , but little has been written about intratendinous tears . although arthroscopy has led to an increase in treatment of ptrcts , there were only few case reports concerning about arthroscopic repair of intratendinous tears . in addition , no one reported the structural outcomes after arthroscopic repair of intratendinous tears . the purpose of this study was to evaluate the functional results and structural outcomes after arthroscopic repair of intratendinous ptrcts . our hypothesis was that arthroscopic repair of intratendinous ptrcts could achieve good clinical and structural results . this study received approval from the investigational review board . from february 2008 to april 2012 , 36 consecutive patients ( 36 shoulders ) with intratendinous tears underwent arthroscopic treatment . the inclusion criteria in this study were ( 1 ) symptoms lasting more than 3 months with proper conservative treatment and ( 2 ) no major associated pathology that would need to be addressed at the time of arthroscopic surgery , such as a frozen shoulder or bankart lesion . three patients with frozen shoulder were excluded from this study . therefore , 33 patients met the inclusion criteria and were retrospectively studied . all of the 33 patients ( 33 shoulders ) were available for evaluation of clinical follow - up . the mean age at the time of surgery was 42.9 9.9 years ( range , 2261 years ) . a total of 18 ( 54.5% ) patients had repair of the dominant shoulder , with 15 left and 18 right shoulders involved . the active range of motion ( rom ) was 155 ( range , 60180 ) in flexion , 156 ( range , 45180 ) in abduction , 43 ( range , 1555 ) in external rotation , and active internal rotation was l3 ( range , t7gluteus ) . preoperatively , all of the patients received bilateral radiographs of anteroposterior and supraspinatus outlet views and a noncontrast mri examination . oblique sagittal , oblique coronal and transverse , t2-weighted fat - depressed , fast spin echo images were acquired for all shoulders . the diagnostic signs of intratendinous tears included a defect within the rotator cuff and fluid - intensity signals within the tendons , which did not connect to the surface of the tendon . depending on the serial oblique coronal images , we could estimate the distance between the center of the tear and the long head of the biceps tendon ( bt ) ( slice thickness was 4 mm ) . this enabled us to identify the tear in the operation more easily . before operation , conservative therapy comprised rest , modification of activities , local application of heat or cold , nonsteroidal anti - inflammatory medication , subacromial steroid injection , gentle exercises for maintaining and increasing rom , and muscle - strengthening exercises . all of the arthroscopic procedures were performed with the patient under general anesthesia in the beach - chair position . diagnostic arthroscopy was performed , and intra - articular pathology was treated in the appropriate manner . the tear was then localized preliminarily under arthroscopic visualization . according to the distance between the long head of the bt and the tear , which was estimated on preoperative mri , we percutaneously penetrated the cuff with a spinal needle near tendon insertion to the humeral head . to determine this distance as accurately as possible ( errors can be made as a result of the magnifying effect of the arthroscope ) , the width of the long head of the bt ( approximately 6 mm ) was taken as a guide . a no . 1 polydioxynone ( pds ) suture ( ethicon , somerville , nj , usa ) was introduced through the spinal needle , and the needle was then removed [ figure 1 ] . arthroscopic view from a posterior glenohumeral portal of a right shoulder shows introduction of a polydioxynone marking suture through supraspinatus tendon . hh : humeral head ; bt : biceps tendon ( note : all arthroscopic views are of right shoulders oriented in the beach - chair position ) . when looking through the posterolateral portal , marking suture could be found in the subacromial space . careful evaluation of the cuff insertion was then performed around the pds suture [ figure 2 ] . the bursal surface was intact in all of the patients , but some areas of the tendon insertion appeared soft and lax as the tendon was palpated using a hooked probe [ figure 3 ] . after inserting the probe into the center of the suspected lesion within the midsubstance of the tendon , a cavity within the tendon could be felt and the probe could easily touch the bone trough on the greater tuberosity [ figure 4 ] . arthroscopic view from a posterolateral subacromial portal shows inspection of the intact bursal side tendon with hooked probe around the polydioxynone suture . arthroscopic view from a posterolateral subacromial portal shows that the bursal side tendon appeared soft and lax . after inserting the probe into the tendon , a cavity within the tendon could be felt , and the bone trough of the greater tuberosity could be palpated easily . arthroscopic view from a posterolateral subacromial portal . after confirming the intratendinous tear , the bursal side tendon was incised . all the degenerative tissues of the tendon were removed until normal articular side tendon fibers were identified as being inserted into the greater tuberosity [ figure 5 ] . a cancellous bed was prepared at the site of the repair by removal of a thin layer of cortical bone with a power burr to promote healing of the reattached cuff . standard single - row repair was performed with one or two suture anchors according to tear length [ figure 6 ] . arthroscopic view from a posterolateral subacromial portal shows the intratendinous tear with the intact articular side tendon . * the arm was maintained in a sling at 15 of abduction and neutral rotation for 6 weeks . six weeks later , active rom exercises that progressively applied loads to the repair construct were allowed . strengthening exercises that focused on restoring power and endurance to the healed rotator cuff muscles began after 3 months and were continued until 46 months postoperatively . the university of california at los angeles ( ucla ) shoulder score and the constant score were used before the operation and at the final evaluation . tendon healing was classified into five types according to sugaya 's criteria as follows : type i , sufficient thickness compared with a normal cuff with homogenously low intensity on each image ; type ii , sufficient thickness compared with a partial high - intensity area ; type iii , insufficient thickness with less than half the thickness without discontinuity , suggesting a partial - thickness , delaminated tear ; type iv , presence of a minor discontinuity in only one or two slices on both oblique coronal and sagittal images , suggesting a small , full - thickness tear ; and type v , presence of a major discontinuity observed in more than two slices on both oblique coronal and sagittal images , suggesting a medium or large , full - thickness tear . preoperative and postoperative clinical scores were compared using the paired student 's t - test . postoperative clinical scores between patients with intact cuff and patients with retears were compared using an independent t - test . from february 2008 to april 2012 , 36 consecutive patients ( 36 shoulders ) with intratendinous tears underwent arthroscopic treatment . the inclusion criteria in this study were ( 1 ) symptoms lasting more than 3 months with proper conservative treatment and ( 2 ) no major associated pathology that would need to be addressed at the time of arthroscopic surgery , such as a frozen shoulder or bankart lesion . all of the 33 patients ( 33 shoulders ) were available for evaluation of clinical follow - up . the mean age at the time of surgery was 42.9 9.9 years ( range , 2261 years ) . a total of 18 ( 54.5% ) patients had repair of the dominant shoulder , with 15 left and 18 right shoulders involved . the active range of motion ( rom ) was 155 ( range , 60180 ) in flexion , 156 ( range , 45180 ) in abduction , 43 ( range , 1555 ) in external rotation , and active internal rotation was l3 ( range , t7gluteus ) . preoperatively , all of the patients received bilateral radiographs of anteroposterior and supraspinatus outlet views and a noncontrast mri examination . oblique sagittal , oblique coronal and transverse , t2-weighted fat - depressed , fast spin echo images were acquired for all shoulders . the diagnostic signs of intratendinous tears included a defect within the rotator cuff and fluid - intensity signals within the tendons , which did not connect to the surface of the tendon . depending on the serial oblique coronal images , we could estimate the distance between the center of the tear and the long head of the biceps tendon ( bt ) ( slice thickness was 4 mm ) . before operation , all of the patients received conservative treatment for at least 3 months . conservative therapy comprised rest , modification of activities , local application of heat or cold , nonsteroidal anti - inflammatory medication , subacromial steroid injection , gentle exercises for maintaining and increasing rom , and muscle - strengthening exercises . all of the arthroscopic procedures were performed with the patient under general anesthesia in the beach - chair position . diagnostic arthroscopy was performed , and intra - articular pathology was treated in the appropriate manner . the tear was then localized preliminarily under arthroscopic visualization . according to the distance between the long head of the bt and the tear , which was estimated on preoperative mri , we percutaneously penetrated the cuff with a spinal needle near tendon insertion to the humeral head . to determine this distance as accurately as possible ( errors can be made as a result of the magnifying effect of the arthroscope ) , the width of the long head of the bt ( approximately 6 mm ) was taken as a guide . a no . 1 polydioxynone ( pds ) suture ( ethicon , somerville , nj , usa ) was introduced through the spinal needle , and the needle was then removed [ figure 1 ] . arthroscopic view from a posterior glenohumeral portal of a right shoulder shows introduction of a polydioxynone marking suture through supraspinatus tendon . hh : humeral head ; bt : biceps tendon ( note : all arthroscopic views are of right shoulders oriented in the beach - chair position ) . when looking through the posterolateral portal , marking suture could be found in the subacromial space . careful evaluation of the cuff insertion was then performed around the pds suture [ figure 2 ] . the bursal surface was intact in all of the patients , but some areas of the tendon insertion appeared soft and lax as the tendon was palpated using a hooked probe [ figure 3 ] . after inserting the probe into the center of the suspected lesion within the midsubstance of the tendon , a cavity within the tendon could be felt and the probe could easily touch the bone trough on the greater tuberosity [ figure 4 ] . arthroscopic view from a posterolateral subacromial portal shows inspection of the intact bursal side tendon with hooked probe around the polydioxynone suture . arthroscopic view from a posterolateral subacromial portal shows that the bursal side tendon appeared soft and lax . after inserting the probe into the tendon , a cavity within the tendon could be felt , and the bone trough of the greater tuberosity could be palpated easily . arthroscopic view from a posterolateral subacromial portal . after confirming the intratendinous tear , the bursal side tendon was incised . all the degenerative tissues of the tendon were removed until normal articular side tendon fibers were identified as being inserted into the greater tuberosity [ figure 5 ] . a cancellous bed was prepared at the site of the repair by removal of a thin layer of cortical bone with a power burr to promote healing of the reattached cuff . standard single - row repair was performed with one or two suture anchors according to tear length [ figure 6 ] . arthroscopic view from a posterolateral subacromial portal shows the intratendinous tear with the intact articular side tendon . * the arm was maintained in a sling at 15 of abduction and neutral rotation for 6 weeks . six weeks later , active rom exercises that progressively applied loads to the repair construct were allowed . strengthening exercises that focused on restoring power and endurance to the healed rotator cuff muscles began after 3 months and were continued until 46 months postoperatively . the university of california at los angeles ( ucla ) shoulder score and the constant score were used before the operation and at the final evaluation . tendon healing was classified into five types according to sugaya 's criteria as follows : type i , sufficient thickness compared with a normal cuff with homogenously low intensity on each image ; type ii , sufficient thickness compared with a partial high - intensity area ; type iii , insufficient thickness with less than half the thickness without discontinuity , suggesting a partial - thickness , delaminated tear ; type iv , presence of a minor discontinuity in only one or two slices on both oblique coronal and sagittal images , suggesting a small , full - thickness tear ; and type v , presence of a major discontinuity observed in more than two slices on both oblique coronal and sagittal images , suggesting a medium or large , full - thickness tear . preoperative and postoperative clinical scores were compared using the paired student 's t - test . postoperative clinical scores between patients with intact cuff and patients with retears were compared using an independent t - test . preoperative plain radiographs showed type ii acromion in 26 patients and type iii acromion in seven patients . on oblique , coronal t2-weighted fat - depressed images from 33 preoperative mris , 26 patients showed an area of high - signal intensity within the insertion of the supraspinatus tendon , supporting the diagnosis of intratendinous tears [ figure 7 ] . two patients showed a tendon defect , three showed high signal on the bursal surface of the tendon , and two showed tendon degeneration . these were interpreted as two full - thickness tears , three bursal side partial tears , and two normal tendons preoperatively . the intratendinous tears , which were confirmed under the arthroscopy , were found within the supraspinatus tendon in all of the patients . the coracoacromial ligament surface was fibrillated or rough in all of the patients , indicating the presence of subacromial impingement . some intra - articular lesions were defined and treated , including two repairs and one debridement of superior labrum anterior and posterior lesion , one debridement of partial rupture of the long head of the bt , one debridement for partial tear of the subscapularis tendon , and three debridements of labrum lesions . seventeen patients had no pain , 13 patients felt light pain or discomfort occasionally while three patients felt pain during strenuous exercise . the active rom was 178 ( range , 160180 ) in flexion , 176 ( range , 150180 ) in abduction , 46 ( range , 4050 ) in external rotation , and active internal rotation was t12 ( range t7l3 ) . both scoring systems reflected significant improvement in the status of the shoulder when the preoperative scores were compared with those at the time of the final follow - up [ table 1 ] . the mean postoperative ucla score and constant score were significantly higher compared with that preoperatively ( p < 0.001 ) . no significant difference in either postoperative score was found between patients with an intact cuff and those with a cuff retear ( ucla : p = 0.696 , constant : p = 0.834 ) [ table 2 ] . preoperative and postoperative clinical scores ( n = 33 ) ucla : university of california at los angeles . comparison of functional outcomes between patients with an intact rotator cuff and those with a retear ucla : university of california at los angeles . a total of 27 ( 81.8% ) patients received postoperative mri , which was performed at a mean of 15.2 months after surgery ( 645 months ) . there were four type i [ 14.8% , figure 8a ] , 18 type ii [ 66.7% , figure 8b ] , and five type iii retears [ 18.5% , figure 8c ] . in this study overall , there were 22 intact repaired cuff tendons ( sugaya 's type i or ii ) and five partial tears ( sugaya 's type iii ) . ( a ) type i , sufficient thickness with homogenously low intensity ( white arrow ) ; ( b ) type ii , sufficient thickness with partial high intensity ( white arrow ) ; ( c ) type iii , insufficient thickness without discontinuity ( white arrow ) . preoperative plain radiographs showed type ii acromion in 26 patients and type iii acromion in seven patients . on oblique , coronal t2-weighted fat - depressed images from 33 preoperative mris , 26 patients showed an area of high - signal intensity within the insertion of the supraspinatus tendon , supporting the diagnosis of intratendinous tears [ figure 7 ] . two patients showed a tendon defect , three showed high signal on the bursal surface of the tendon , and two showed tendon degeneration . these were interpreted as two full - thickness tears , three bursal side partial tears , and two normal tendons preoperatively . the intratendinous tears , which were confirmed under the arthroscopy , were found within the supraspinatus tendon in all of the patients . the coracoacromial ligament surface was fibrillated or rough in all of the patients , indicating the presence of subacromial impingement . some intra - articular lesions were defined and treated , including two repairs and one debridement of superior labrum anterior and posterior lesion , one debridement of partial rupture of the long head of the bt , one debridement for partial tear of the subscapularis tendon , and three debridements of labrum lesions . seventeen patients had no pain , 13 patients felt light pain or discomfort occasionally while three patients felt pain during strenuous exercise . the active rom was 178 ( range , 160180 ) in flexion , 176 ( range , 150180 ) in abduction , 46 ( range , 4050 ) in external rotation , and active internal rotation was t12 ( range t7l3 ) . both scoring systems reflected significant improvement in the status of the shoulder when the preoperative scores were compared with those at the time of the final follow - up [ table 1 ] . the mean postoperative ucla score and constant score were significantly higher compared with that preoperatively ( p < 0.001 ) . no significant difference in either postoperative score was found between patients with an intact cuff and those with a cuff retear ( ucla : p = 0.696 , constant : p = 0.834 ) [ table 2 ] . preoperative and postoperative clinical scores ( n = 33 ) ucla : university of california at los angeles . comparison of functional outcomes between patients with an intact rotator cuff and those with a retear ucla : university of california at los angeles . a total of 27 ( 81.8% ) patients received postoperative mri , which was performed at a mean of 15.2 months after surgery ( 645 months ) . there were four type i [ 14.8% , figure 8a ] , 18 type ii [ 66.7% , figure 8b ] , and five type iii retears [ 18.5% , figure 8c ] . in this study overall , there were 22 intact repaired cuff tendons ( sugaya 's type i or ii ) and five partial tears ( sugaya 's type iii ) . ( a ) type i , sufficient thickness with homogenously low intensity ( white arrow ) ; ( b ) type ii , sufficient thickness with partial high intensity ( white arrow ) ; ( c ) type iii , insufficient thickness without discontinuity ( white arrow ) . to the best of our knowledge , this is the largest study to examine the clinical and anatomical results of intratendinous ptrcts after arthroscopic repair . the combination of decompression and repair with preservation of as much of the intact articular tendon fiber as possible yields a satisfactory clinical outcome . postoperative mri also showed a high rate of healing of the repaired tendon among our patients . partial - thickness rotator cuff tear is more difficult to diagnose than a full - thickness tear . in particular , preoperative diagnosis of intratendinous tears may be the most challenging among the three subtypes of ptrcts . however , in recent years , advancement in mri , specifically sequence alteration and differential arm positioning , have greatly improved its accuracy in identifying intratendinous tears . one is a linear high signal within the tendon , which is parallel to the direction of tendon fibers . the other is a focal defect at the tendon insertion , which has no communication with either surface of the tendon . in the current study , 26 of 33 ( 78.8% ) patients had been diagnosed with intratendinous tears by preoperative mri , showing that mri is an effective way of diagnosing intratendinous tears . although preoperative diagnosis has become more accurate , intraoperative localization of the tears remains problematic . this difficulty is due to the absence of overt tendon disruption on both the bursal and articular surfaces of the cuff . in uchiyama 's study , a definitive diagnosis was established by a longitudinal split of the supraspinatus tendon in the area of softening , fraying , edema , erosion , and redness . itoi and tabata reported three cases where intratendinous tear was suspected when the cuff was soft , fluffy , and bulged when the arm was elevated . bubble sign as facilitating the diagnosis of intratendinous tears . in our study , with the help of the marking suture , the inspected area could be minimized , and the tears could be found quickly . the intratendinous tear was excised in 15 patients , including the whole lesion and a small portion of the greater tuberosity . the tendon defect was then closed by side - to - side sutures and transosseous sutures . itoi and tabata reported three cases in which a full - thickness cuff involving the tear was resected and repaired . few studies have investigated the effectiveness of acromioplasty alone for the treatment of intratendinous tears . fukuda et al . showed in their histological study that partial - thickness tears have essentially no ability to heal themselves over time . intratendinous tears biopsied at the time of operative intervention show granulation tissue with rounded , avascular tissue margins without evidence of healing . biomechanical studies have shown that in the presence of a partial - thickness tear , the strain patterns within the remaining intact rotator cuff change , potentially predisposing the tissue to tear propagation . according to the results of these studies , tendon repair with acromioplasty may be more suitable . in the current study , intratendinous tears were converted to bursal side tears after the normal bursal side tendon was incised . some authors have proposed to complete a full thickness tear and repair it , whereas others believe that the normal articular sided tissue should be reserved because it can protect the repaired bursal side tendon and offer a good opportunity for healing of the repaired tendon . in addition , with preservation of an intact articular side tendon , some authors tend to perform a full - layer repair whereas others prefer to repair the outer layer only . in the present study , we preserved the healthy articular side tendon as much as possible and repaired the bursal flap back to the bone . good clinical and structural results showed that our technique was effective for intratendinous tears . to the best of our knowledge , few studies have examined structural outcomes after arthroscopic repair of intratendinous ptrct . in uchiyama low signal in tendon was found in 7 patients . with regard to our repair technique , koh et al . reported good structural integrity ( 88% healing rate ) on mri after arthroscopic full - layer repair . kim et al . described satisfactory structural integrity ( 89% ) after either conversion to a full - thickness tear repair or only repairing the detached layer . in this study , 22 ( 81.5% ) repaired tendons were intact . the healing rate is consistent with these studies . whether the tendon integrity affect the clinical result is under debate . some authors believed that retearing had no effect on the clinical results , especially in small tears . our results are consistent with these reports . however , further studies are needed to clarify the reason of this interesting finding . first , this was a retrospective study with a relatively small number of patients and a relatively short time of follow - up . therefore , this study does not provide a clear understanding of the requirement for treatment of intratendinous rotator cuff tears . second , because we did not perform subacromial decompression alone to the intratendinous tears , we could not compare both results . third , postoperative mri scans were taken at 6 months to 4 years ( i.e. , the postoperative period varied ) . therefore , the structural integrity that we achieved might not match the clinical outcomes at the final follow - up .

background : partial - thickness rotator cuff tears ( ptrcts ) are being diagnosed more often because of high - resolution magnetic resonance imaging ( mri ) . compared with articular and bursal side tears , there have been few studies about evaluating the clinical and structural outcomes after intratendinous tear repair.methods:from 2008 to 2012 , 33 consecutive patients with intratendinous ptrcts underwent arthroscopic repair . all of them were retrospectively evaluated . the university of california at los angeles ( ucla ) and constant scores were evaluated before operation and at the final follow - up . postoperative cuff integrity was determined using mri according to sugaya 's classification.results:at the 2-year follow - up , the average ucla score increased from 16.7 1.9 to 32.5 3.5 , and the constant score increased from 66.2 10.5 to 92.4 6.9 ( p < 0.001 ) . twenty seven patients received follow - up mri examinations at an average of 15.2 months after surgery . of these 27 patients , 22 ( 81.5% ) had a healed tendon , and five patients had partial tears . there was no association between functional and anatomic results.conclusions:for intratendinous ptrct , clinical outcomes and tendon healing showed good results at a minimum 2-year after arthroscopic repair .

I M Patient selection Preoperative clinical features Preoperative images Conservative treatment Surgical technique Rehabilitation Clinical and magnetic resonance imaging evaluations Statistical analysis R Preoperative imaging findings Intraoperative findings Clinical outcomes Magnetic resonance imaging outcomes D

partial - thickness rotator cuff tears ( ptrcts ) have the potential to cause significant pain and disability in affected patients . surgical treatment is generally limited to tear debridement with or without acromioplasty or tear repair with or without acromioplasty . with the advent of magnetic resonance imaging ( mri ) and shoulder arthroscopy , more unfortunately , high - quality data on the management of ptrcts are relatively lacking in the literature when compared with those available on full - thickness tears . partial - thickness rotator cuff tears are classified into three subtypes : bursal side , articular side , and intratendinous tears . a cadaveric study reported that this type of tear was the most frequent ( 55% ) among all of the partial - thickness tears . because the intratendinous tear has no communication to the subacromial space and the glenohumeral joint , it is probably the most difficult condition to be diagnosed among the three types of the partial tear . both bursal side and articular side rotator cuff tears have been extensively studied , but little has been written about intratendinous tears . although arthroscopy has led to an increase in treatment of ptrcts , there were only few case reports concerning about arthroscopic repair of intratendinous tears . in addition , no one reported the structural outcomes after arthroscopic repair of intratendinous tears . the purpose of this study was to evaluate the functional results and structural outcomes after arthroscopic repair of intratendinous ptrcts . our hypothesis was that arthroscopic repair of intratendinous ptrcts could achieve good clinical and structural results . from february 2008 to april 2012 , 36 consecutive patients ( 36 shoulders ) with intratendinous tears underwent arthroscopic treatment . therefore , 33 patients met the inclusion criteria and were retrospectively studied . all of the 33 patients ( 33 shoulders ) were available for evaluation of clinical follow - up . the mean age at the time of surgery was 42.9 9.9 years ( range , 2261 years ) . a total of 18 ( 54.5% ) patients had repair of the dominant shoulder , with 15 left and 18 right shoulders involved . the active range of motion ( rom ) was 155 ( range , 60180 ) in flexion , 156 ( range , 45180 ) in abduction , 43 ( range , 1555 ) in external rotation , and active internal rotation was l3 ( range , t7gluteus ) . preoperatively , all of the patients received bilateral radiographs of anteroposterior and supraspinatus outlet views and a noncontrast mri examination . depending on the serial oblique coronal images , we could estimate the distance between the center of the tear and the long head of the biceps tendon ( bt ) ( slice thickness was 4 mm ) . before operation , conservative therapy comprised rest , modification of activities , local application of heat or cold , nonsteroidal anti - inflammatory medication , subacromial steroid injection , gentle exercises for maintaining and increasing rom , and muscle - strengthening exercises . all of the arthroscopic procedures were performed with the patient under general anesthesia in the beach - chair position . according to the distance between the long head of the bt and the tear , which was estimated on preoperative mri , we percutaneously penetrated the cuff with a spinal needle near tendon insertion to the humeral head . to determine this distance as accurately as possible ( errors can be made as a result of the magnifying effect of the arthroscope ) , the width of the long head of the bt ( approximately 6 mm ) was taken as a guide . 1 polydioxynone ( pds ) suture ( ethicon , somerville , nj , usa ) was introduced through the spinal needle , and the needle was then removed [ figure 1 ] . the bursal surface was intact in all of the patients , but some areas of the tendon insertion appeared soft and lax as the tendon was palpated using a hooked probe [ figure 3 ] . after inserting the probe into the center of the suspected lesion within the midsubstance of the tendon , a cavity within the tendon could be felt and the probe could easily touch the bone trough on the greater tuberosity [ figure 4 ] . arthroscopic view from a posterolateral subacromial portal shows inspection of the intact bursal side tendon with hooked probe around the polydioxynone suture . arthroscopic view from a posterolateral subacromial portal shows that the bursal side tendon appeared soft and lax . after inserting the probe into the tendon , a cavity within the tendon could be felt , and the bone trough of the greater tuberosity could be palpated easily . after confirming the intratendinous tear , the bursal side tendon was incised . a cancellous bed was prepared at the site of the repair by removal of a thin layer of cortical bone with a power burr to promote healing of the reattached cuff . standard single - row repair was performed with one or two suture anchors according to tear length [ figure 6 ] . arthroscopic view from a posterolateral subacromial portal shows the intratendinous tear with the intact articular side tendon . strengthening exercises that focused on restoring power and endurance to the healed rotator cuff muscles began after 3 months and were continued until 46 months postoperatively . the university of california at los angeles ( ucla ) shoulder score and the constant score were used before the operation and at the final evaluation . tendon healing was classified into five types according to sugaya 's criteria as follows : type i , sufficient thickness compared with a normal cuff with homogenously low intensity on each image ; type ii , sufficient thickness compared with a partial high - intensity area ; type iii , insufficient thickness with less than half the thickness without discontinuity , suggesting a partial - thickness , delaminated tear ; type iv , presence of a minor discontinuity in only one or two slices on both oblique coronal and sagittal images , suggesting a small , full - thickness tear ; and type v , presence of a major discontinuity observed in more than two slices on both oblique coronal and sagittal images , suggesting a medium or large , full - thickness tear . preoperative and postoperative clinical scores were compared using the paired student 's t - test . postoperative clinical scores between patients with intact cuff and patients with retears were compared using an independent t - test . from february 2008 to april 2012 , 36 consecutive patients ( 36 shoulders ) with intratendinous tears underwent arthroscopic treatment . the inclusion criteria in this study were ( 1 ) symptoms lasting more than 3 months with proper conservative treatment and ( 2 ) no major associated pathology that would need to be addressed at the time of arthroscopic surgery , such as a frozen shoulder or bankart lesion . all of the 33 patients ( 33 shoulders ) were available for evaluation of clinical follow - up . the mean age at the time of surgery was 42.9 9.9 years ( range , 2261 years ) . a total of 18 ( 54.5% ) patients had repair of the dominant shoulder , with 15 left and 18 right shoulders involved . preoperatively , all of the patients received bilateral radiographs of anteroposterior and supraspinatus outlet views and a noncontrast mri examination . depending on the serial oblique coronal images , we could estimate the distance between the center of the tear and the long head of the biceps tendon ( bt ) ( slice thickness was 4 mm ) . before operation , all of the patients received conservative treatment for at least 3 months . conservative therapy comprised rest , modification of activities , local application of heat or cold , nonsteroidal anti - inflammatory medication , subacromial steroid injection , gentle exercises for maintaining and increasing rom , and muscle - strengthening exercises . all of the arthroscopic procedures were performed with the patient under general anesthesia in the beach - chair position . diagnostic arthroscopy was performed , and intra - articular pathology was treated in the appropriate manner . according to the distance between the long head of the bt and the tear , which was estimated on preoperative mri , we percutaneously penetrated the cuff with a spinal needle near tendon insertion to the humeral head . 1 polydioxynone ( pds ) suture ( ethicon , somerville , nj , usa ) was introduced through the spinal needle , and the needle was then removed [ figure 1 ] . the bursal surface was intact in all of the patients , but some areas of the tendon insertion appeared soft and lax as the tendon was palpated using a hooked probe [ figure 3 ] . after inserting the probe into the center of the suspected lesion within the midsubstance of the tendon , a cavity within the tendon could be felt and the probe could easily touch the bone trough on the greater tuberosity [ figure 4 ] . arthroscopic view from a posterolateral subacromial portal shows inspection of the intact bursal side tendon with hooked probe around the polydioxynone suture . after inserting the probe into the tendon , a cavity within the tendon could be felt , and the bone trough of the greater tuberosity could be palpated easily . after confirming the intratendinous tear , the bursal side tendon was incised . arthroscopic view from a posterolateral subacromial portal shows the intratendinous tear with the intact articular side tendon . the university of california at los angeles ( ucla ) shoulder score and the constant score were used before the operation and at the final evaluation . tendon healing was classified into five types according to sugaya 's criteria as follows : type i , sufficient thickness compared with a normal cuff with homogenously low intensity on each image ; type ii , sufficient thickness compared with a partial high - intensity area ; type iii , insufficient thickness with less than half the thickness without discontinuity , suggesting a partial - thickness , delaminated tear ; type iv , presence of a minor discontinuity in only one or two slices on both oblique coronal and sagittal images , suggesting a small , full - thickness tear ; and type v , presence of a major discontinuity observed in more than two slices on both oblique coronal and sagittal images , suggesting a medium or large , full - thickness tear . preoperative and postoperative clinical scores were compared using the paired student 's t - test . postoperative clinical scores between patients with intact cuff and patients with retears were compared using an independent t - test . preoperative plain radiographs showed type ii acromion in 26 patients and type iii acromion in seven patients . on oblique , coronal t2-weighted fat - depressed images from 33 preoperative mris , 26 patients showed an area of high - signal intensity within the insertion of the supraspinatus tendon , supporting the diagnosis of intratendinous tears [ figure 7 ] . two patients showed a tendon defect , three showed high signal on the bursal surface of the tendon , and two showed tendon degeneration . these were interpreted as two full - thickness tears , three bursal side partial tears , and two normal tendons preoperatively . the intratendinous tears , which were confirmed under the arthroscopy , were found within the supraspinatus tendon in all of the patients . the coracoacromial ligament surface was fibrillated or rough in all of the patients , indicating the presence of subacromial impingement . some intra - articular lesions were defined and treated , including two repairs and one debridement of superior labrum anterior and posterior lesion , one debridement of partial rupture of the long head of the bt , one debridement for partial tear of the subscapularis tendon , and three debridements of labrum lesions . the active rom was 178 ( range , 160180 ) in flexion , 176 ( range , 150180 ) in abduction , 46 ( range , 4050 ) in external rotation , and active internal rotation was t12 ( range t7l3 ) . both scoring systems reflected significant improvement in the status of the shoulder when the preoperative scores were compared with those at the time of the final follow - up [ table 1 ] . the mean postoperative ucla score and constant score were significantly higher compared with that preoperatively ( p < 0.001 ) . no significant difference in either postoperative score was found between patients with an intact cuff and those with a cuff retear ( ucla : p = 0.696 , constant : p = 0.834 ) [ table 2 ] . preoperative and postoperative clinical scores ( n = 33 ) ucla : university of california at los angeles . comparison of functional outcomes between patients with an intact rotator cuff and those with a retear ucla : university of california at los angeles . a total of 27 ( 81.8% ) patients received postoperative mri , which was performed at a mean of 15.2 months after surgery ( 645 months ) . there were four type i [ 14.8% , figure 8a ] , 18 type ii [ 66.7% , figure 8b ] , and five type iii retears [ 18.5% , figure 8c ] . in this study overall , there were 22 intact repaired cuff tendons ( sugaya 's type i or ii ) and five partial tears ( sugaya 's type iii ) . on oblique , coronal t2-weighted fat - depressed images from 33 preoperative mris , 26 patients showed an area of high - signal intensity within the insertion of the supraspinatus tendon , supporting the diagnosis of intratendinous tears [ figure 7 ] . two patients showed a tendon defect , three showed high signal on the bursal surface of the tendon , and two showed tendon degeneration . these were interpreted as two full - thickness tears , three bursal side partial tears , and two normal tendons preoperatively . the intratendinous tears , which were confirmed under the arthroscopy , were found within the supraspinatus tendon in all of the patients . the coracoacromial ligament surface was fibrillated or rough in all of the patients , indicating the presence of subacromial impingement . some intra - articular lesions were defined and treated , including two repairs and one debridement of superior labrum anterior and posterior lesion , one debridement of partial rupture of the long head of the bt , one debridement for partial tear of the subscapularis tendon , and three debridements of labrum lesions . the active rom was 178 ( range , 160180 ) in flexion , 176 ( range , 150180 ) in abduction , 46 ( range , 4050 ) in external rotation , and active internal rotation was t12 ( range t7l3 ) . both scoring systems reflected significant improvement in the status of the shoulder when the preoperative scores were compared with those at the time of the final follow - up [ table 1 ] . the mean postoperative ucla score and constant score were significantly higher compared with that preoperatively ( p < 0.001 ) . no significant difference in either postoperative score was found between patients with an intact cuff and those with a cuff retear ( ucla : p = 0.696 , constant : p = 0.834 ) [ table 2 ] . preoperative and postoperative clinical scores ( n = 33 ) ucla : university of california at los angeles . comparison of functional outcomes between patients with an intact rotator cuff and those with a retear ucla : university of california at los angeles . a total of 27 ( 81.8% ) patients received postoperative mri , which was performed at a mean of 15.2 months after surgery ( 645 months ) . there were four type i [ 14.8% , figure 8a ] , 18 type ii [ 66.7% , figure 8b ] , and five type iii retears [ 18.5% , figure 8c ] . in this study overall , there were 22 intact repaired cuff tendons ( sugaya 's type i or ii ) and five partial tears ( sugaya 's type iii ) . to the best of our knowledge , this is the largest study to examine the clinical and anatomical results of intratendinous ptrcts after arthroscopic repair . partial - thickness rotator cuff tear is more difficult to diagnose than a full - thickness tear . one is a linear high signal within the tendon , which is parallel to the direction of tendon fibers . the other is a focal defect at the tendon insertion , which has no communication with either surface of the tendon . in the current study , 26 of 33 ( 78.8% ) patients had been diagnosed with intratendinous tears by preoperative mri , showing that mri is an effective way of diagnosing intratendinous tears . itoi and tabata reported three cases where intratendinous tear was suspected when the cuff was soft , fluffy , and bulged when the arm was elevated . in our study , with the help of the marking suture , the inspected area could be minimized , and the tears could be found quickly . the intratendinous tear was excised in 15 patients , including the whole lesion and a small portion of the greater tuberosity . showed in their histological study that partial - thickness tears have essentially no ability to heal themselves over time . intratendinous tears biopsied at the time of operative intervention show granulation tissue with rounded , avascular tissue margins without evidence of healing . biomechanical studies have shown that in the presence of a partial - thickness tear , the strain patterns within the remaining intact rotator cuff change , potentially predisposing the tissue to tear propagation . according to the results of these studies , tendon repair with acromioplasty may be more suitable . in the current study , intratendinous tears were converted to bursal side tears after the normal bursal side tendon was incised . some authors have proposed to complete a full thickness tear and repair it , whereas others believe that the normal articular sided tissue should be reserved because it can protect the repaired bursal side tendon and offer a good opportunity for healing of the repaired tendon . good clinical and structural results showed that our technique was effective for intratendinous tears . to the best of our knowledge , few studies have examined structural outcomes after arthroscopic repair of intratendinous ptrct . reported good structural integrity ( 88% healing rate ) on mri after arthroscopic full - layer repair . described satisfactory structural integrity ( 89% ) after either conversion to a full - thickness tear repair or only repairing the detached layer . in this study , 22 ( 81.5% ) repaired tendons were intact . some authors believed that retearing had no effect on the clinical results , especially in small tears . first , this was a retrospective study with a relatively small number of patients and a relatively short time of follow - up . therefore , this study does not provide a clear understanding of the requirement for treatment of intratendinous rotator cuff tears . second , because we did not perform subacromial decompression alone to the intratendinous tears , we could not compare both results . therefore , the structural integrity that we achieved might not match the clinical outcomes at the final follow - up .

heart failure ( hf ) represents an increasing clinical and public health burden in the united states as its prevalence and incidence continue to rise , especially among elderly americans . hf has emerged as the single leading diagnosis for hospitalization in the elderly and hospitalization rates are higher among blacks than among whites , with 83% of hf patients hospitalized at least once and 43% hospitalized at least 4 times over a mean duration of 4.7 years . the burden of hf is likely to increase because of the increasing age of the american population , earlier age of onset of hf and improved treatment and survival from cardiovascular ( cv ) diseases , including myocardial infarction ( mi ) and hypertension . carotid imt is a well established marker of subclinical atherosclerosis , which indicates early manifestation of atherosclerosis in the carotid arteries , and is associated with future cv events , asymptomatic myocardial ischemia , and changes in risk factors induced by therapeutic interventions . there is evidence of a direct relationship between increased carotid imt and reduced left ventricular ( lv ) systolic and diastolic function assessed by myocardial strain in asymptomatic individuals without previous clinical cv disease . elderly patients with hf differ from younger patients with hf in terms of several biologic characteristics , including the relatively large proportion of elderly patients with hf who have preserved systolic function . using data from the cardiovascular health study , which included subjects 65 years or older , atherosclerosis as measured by carotid imt was shown to predict overt systolic and diastolic hf . the association of carotid imt with incident hf in middleaged adults , who are at a lower risk of hf than older adults , is not well known . previous crosssectional analysis using data from the aric cohort showed that participants with hf had a higher mean carotid imt than participants without hf . subclinical vascular disease as determined by an increase in carotid imt may be a determinant of risk for future hf , but this has not been studied . in the present study , we examine the hypothesis that subclinical atherosclerosis , assessed by mean carotid imt , is associated with incident hf beyond what is explained by major cvd risk factors in middleaged whites and blacks . we tested this hypothesis using data from the atherosclerosis risk in communities ( aric ) study cohort . the aric study is communitybased prospective study which enrolled 15 792 participants aged 45 to 64 years at the time of their baseline assessment ( 19871989 ) from 4 communities : forsyth county , north carolina ; suburban minneapolis , minnesota ; washington county , maryland ; and jackson , mississippi . in the cohort , response rates at baseline were 46% in jackson and 65% to 67% for the other communities . successful contact with living cohort members through an annual phone interview was above 93% . during the followup period , annual phone interviews were conducted with participants to inquire about events ( including hf ) , and hospital records were surveyed for identification and classification of hf events . the institutional review boards from each site approved the aric study . our analysis involved the use of data from the baseline examination . excluded from the sample were : ( 1 ) participants who were missing all far wall values of carotid imt at baseline ( n=607 ) ; ( 2 ) racial groups other than black or white because of their limited numbers ( n=48 ) ; ( 3 ) participants with missing criteria needed to define baseline glucose status ( n=148 ) ; ( 4 ) participants with missing data needed to define hf at baseline ( n=287 ) ; ( 5 ) prevalent cases of hf at baseline , either by selfreported current intake of hf medication , or those with stage 3 or manifest hf by gothenburg criteria ( n=752 ) . the gothenburg criterion ( table 1 ) is composed of 3 scores , ( 1 ) cardiac , ( 2 ) pulmonary , and ( 3 ) therapy . to have stage 3 or manifest hf all current medications ( taken within the last 2 weeks ) were brought into the clinic and documented . atrial fibrillation was diagnosed by visual inspection of a 2minute rhythm strip from leads v1 , ii , and v5 using standardized methodology . aric study participants provided information on demographic and behavioral variables and medical history to a trained interviewer at each visit . race , gender , educational level , current alcohol use , and smoking status were determined by selfreport at baseline . atherosclerosis of the common carotid arteries was measured by noninvasive , highresolution bmode ultrasound . in this study , technicians scanned 3 specified segments of the extra cranial carotid arteries bilaterally : the common carotid , the bifurcation , and the internal carotid . the common carotid intima and media was assessed in a 1cm segment proximal to the dilatation of the carotid bulb . the intima and media of the bifurcation and internal carotid were assessed over the 1cm segments proximal and distal to the flow divider , respectively . the mean intimalmedial thickness of the far wall of the 6 segments was used as a general indicator of atherosclerosis in the carotid artery . the ultrasound examinations were performed according to a standardized protocol by trained , certified sonographers subject to semiannual evaluation . ultrasound images were recorded on a videotape and forwarded to a central reading center for interpretation . for each of the 6 images , the imt was measured over a 1cm segment at 1mm increments ( total of 11 measurements ) . a mean wall thickness for each segment this protocol produces a single index of atherosclerosis with improved precision provided by the averaging of multiple imt measurements . in a randomly selected subset of 855 participants , the betweenreader reliability coefficients ranged from 0.78 to 0.93 and coefficients of variation ranged from 13.1% to 18.3% ( 80% of duplicate scans differed by < 0.267 mm ) . incident hf was defined as the first occurrence of either ( 1 ) an hf hospitalization that included an international classification of diseases ( icd ) , ninth revision , discharge code of 428.x ( 428.0 to 428.9 ) in any position , or ( 2 ) a death certificate with a 428 ( hf ) or icd , 10th revision , code i50 ( hf ) in any position . all hospitalizations occurring in cohort participants were identified either through generated computer programs , or review of hospital discharge indexes or information elicited during the annual followup interview . hospitalizations eligible based on set criteria ( discharge codes , discharge dates , race sex , and age ) but were found upon review to have been hospitalized for less than 24 hours were not abstracted . abstractors made copies of sections of the medical record ( discharge summary , history and physical report , admission note , and imaging reports ) for use by the aric heart failure mortality and morbidity classification committee ( hf mmcc ) . the interabstractor agreement rate for determining whether or not to conduct detailed abstraction in a quality control sample was 99% . the aric community surveillance database was also searched for possible hf events occurring among cohort participants that were not reported at the annual followup visit or may be otherwise missed . beginning in 2005 , the aric study conducted continuous , retrospective surveillance of hospital discharges for hf for all residents aged 55 and older in the 4 aric communities via annual hospital discharge indices . each hospitalization eligible for full abstraction was independently reviewed by centrally trained and certified physicians on the aric hf mmcc and classified into 1 of 5 categories : definite acute decompensated hf , possible acute decompensated hf , chronic stable hf , hf unlikely , or unclassifiable . hf cases occurring prior to 2005 were not adjudicated . the % agreement between any hf event adjudicated by the hf mmcc and icd9 code 428 was 75% , similar to that with the framingham followup time for those with incident hf events was defined by the time from their baseline exam ( visit 1 ) until the incident event . the end of followup time for those without hf was ( 1 ) december 31 , 2009 , ( 2 ) date of last contact for those lost to followup , or ( 3 ) date of death , whichever occurred first . prevalent chd at baseline was defined as a selfreported history of mi , mi from adjudicated baseline ecg data , a history of physiciandiagnosed mi , a prior coronary reperfusion procedure . incident chd was defined as any case of hospitalized mi , fatal chd , or ecgdiagnosed mi by the end of the study period . three systolic and diastolic blood pressures were taken with participants in the sitting position after 5 minutes of rest using a randomzero sphygmomanometer . certified technicians measured height , weight , and waist circumference . body mass index was calculated as weight ( in kg ) divided by the square of the height ( in meters ) . fasting serum glucose was measured by the optimized direct analysis in real time ( dart ) glucose reagent method . mmol / l ( 126 mg / dl ) , selfreported previous physician diagnosis , or use of diabetes medication ( oral hypoglycemic agents and/or insulin ) , or a nonfasting glucose of 11 mmol / l ( 200 mg / dl ) . mmol / l ( 100 to 125 mg / dl ) in accordance with the 2004 american diabetes association definition . normal fasting glucose was defined as any other participant who does not meet the criteria for dm and ifg . unadjusted differences in baseline characteristics between participants with and without hf were assessed using the student 's t test ( for continuous variables ) and chisquare test ( for categorical variables ) . the association of carotid imt with incident heart failure was assessed using cox proportional hazard models with sequential adjustment for covariates . hazard ratios and 95% confidence intervals for cox regression analysis are presented per unit standard deviation increase and by quartiles of imt . in multivariable analysis , the base model ( unadjusted model ) was sequentially adjusted for covariates of interest . a second model was adjusted for age , gender , race , and level of education ( demographic model ) . a third model was further adjusted for systolic and diastolic blood pressures , bmi , waist circumference , hdl cholesterol , ldl cholesterol , triglycerides , smoking status and amount in packyears , alcohol consumption , serum creatinine , and medications for hypertension and dyslipidemia ( clinical and biologic model ) . finally , a fourth model was adjusted for prevalent and incident chd ( chd model ) . for the fourth model , covariates were chosen based on their associations in the present cohort and in prior published studies . effect modification between race , gender , and carotid imt was examined using 2way interaction . this was achieved by including the product the imtrace and imtgender terms in the model . in the presence of a significant interaction ( p for interaction < 0.05 ) separate cox regression models included carotid imt as a continuous variable ( zscores ) and as a categorical variable ( quartiles ) . the proportional hazard assumption was assessed by visually examining the log ( log survival ) plots and timecovariate interaction terms . kaplanmeier plots were used to illustrate the overall survival probability and cumulative incidence of hf by subgroups , and group comparison was done using the logrank test . all analyses were performed using sas 9.2 ( sas institute inc , cary , nc ) . the aric study is communitybased prospective study which enrolled 15 792 participants aged 45 to 64 years at the time of their baseline assessment ( 19871989 ) from 4 communities : forsyth county , north carolina ; suburban minneapolis , minnesota ; washington county , maryland ; and jackson , mississippi . in the cohort , response rates at baseline were 46% in jackson and 65% to 67% for the other communities . successful contact with living cohort members through an annual phone interview was above 93% . during the followup period , annual phone interviews were conducted with participants to inquire about events ( including hf ) , and hospital records were surveyed for identification and classification of hf events . the institutional review boards from each site approved the aric study . our analysis involved the use of data from the baseline examination . excluded from the sample were : ( 1 ) participants who were missing all far wall values of carotid imt at baseline ( n=607 ) ; ( 2 ) racial groups other than black or white because of their limited numbers ( n=48 ) ; ( 3 ) participants with missing criteria needed to define baseline glucose status ( n=148 ) ; ( 4 ) participants with missing data needed to define hf at baseline ( n=287 ) ; ( 5 ) prevalent cases of hf at baseline , either by selfreported current intake of hf medication , or those with stage 3 or manifest hf by gothenburg criteria ( n=752 ) . the gothenburg criterion ( table 1 ) is composed of 3 scores , ( 1 ) cardiac , ( 2 ) pulmonary , and ( 3 ) therapy . to have stage 3 or manifest hf all current medications ( taken within the last 2 weeks ) were brought into the clinic and documented . atrial fibrillation was diagnosed by visual inspection of a 2minute rhythm strip from leads v1 , ii , and v5 using standardized methodology . aric study participants provided information on demographic and behavioral variables and medical history to a trained interviewer at each visit . race , gender , educational level , current alcohol use , and smoking status were determined by selfreport at baseline . atherosclerosis of the common carotid arteries was measured by noninvasive , highresolution bmode ultrasound . in this study , technicians scanned 3 specified segments of the extra cranial carotid arteries bilaterally : the common carotid , the bifurcation , and the internal carotid . the common carotid intima and media was assessed in a 1cm segment proximal to the dilatation of the carotid bulb . the intima and media of the bifurcation and internal carotid were assessed over the 1cm segments proximal and distal to the flow divider , respectively . the mean intimalmedial thickness of the far wall of the 6 segments was used as a general indicator of atherosclerosis in the carotid artery . the ultrasound examinations were performed according to a standardized protocol by trained , certified sonographers subject to semiannual evaluation . ultrasound images were recorded on a videotape and forwarded to a central reading center for interpretation . for each of the 6 images , the imt was measured over a 1cm segment at 1mm increments ( total of 11 measurements ) . a mean wall thickness for each segment this protocol produces a single index of atherosclerosis with improved precision provided by the averaging of multiple imt measurements . in a randomly selected subset of 855 participants , the betweenreader reliability coefficients ranged from 0.78 to 0.93 and coefficients of variation ranged from 13.1% to 18.3% ( 80% of duplicate scans differed by < 0.267 mm ) . incident hf was defined as the first occurrence of either ( 1 ) an hf hospitalization that included an international classification of diseases ( icd ) , ninth revision , discharge code of 428.x ( 428.0 to 428.9 ) in any position , or ( 2 ) a death certificate with a 428 ( hf ) or icd , 10th revision , code i50 ( hf ) in any position . all hospitalizations occurring in cohort participants were identified either through generated computer programs , or review of hospital discharge indexes or information elicited during the annual followup interview . hospitalizations eligible based on set criteria ( discharge codes , discharge dates , race sex , and age ) but were found upon review to have been hospitalized for less than 24 hours were not abstracted . abstractors made copies of sections of the medical record ( discharge summary , history and physical report , admission note , and imaging reports ) for use by the aric heart failure mortality and morbidity classification committee ( hf mmcc ) . the interabstractor agreement rate for determining whether or not to conduct detailed abstraction in a quality control sample was 99% . the aric community surveillance database was also searched for possible hf events occurring among cohort participants that were not reported at the annual followup visit or may be otherwise missed . beginning in 2005 , the aric study conducted continuous , retrospective surveillance of hospital discharges for hf for all residents aged 55 and older in the 4 aric communities via annual hospital discharge indices . each hospitalization eligible for full abstraction was independently reviewed by centrally trained and certified physicians on the aric hf mmcc and classified into 1 of 5 categories : definite acute decompensated hf , possible acute decompensated hf , chronic stable hf , hf unlikely , or unclassifiable . hf cases occurring prior to 2005 were not adjudicated . the % agreement between any hf event adjudicated by the hf mmcc and icd9 code 428 was 75% , similar to that with the framingham followup time for those with incident hf events was defined by the time from their baseline exam ( visit 1 ) until the incident event . the end of followup time for those without hf was ( 1 ) december 31 , 2009 , ( 2 ) date of last contact for those lost to followup , or ( 3 ) date of death , whichever occurred first . prevalent chd at baseline was defined as a selfreported history of mi , mi from adjudicated baseline ecg data , a history of physiciandiagnosed mi , a prior coronary reperfusion procedure . incident chd was defined as any case of hospitalized mi , fatal chd , or ecgdiagnosed mi by the end of the study period . three systolic and diastolic blood pressures were taken with participants in the sitting position after 5 minutes of rest using a randomzero sphygmomanometer . certified technicians measured height , weight , and waist circumference . body mass index was calculated as weight ( in kg ) divided by the square of the height ( in meters ) . fasting serum glucose was measured by the optimized direct analysis in real time ( dart ) glucose reagent method . mmol / l ( 126 mg / dl ) , selfreported previous physician diagnosis , or use of diabetes medication ( oral hypoglycemic agents and/or insulin ) , or a nonfasting glucose of 11 mmol / l ( 200 mg / dl ) . mmol / l ( 100 to 125 mg / dl ) in accordance with the 2004 american diabetes association definition . normal fasting glucose was defined as any other participant who does not meet the criteria for dm and ifg . unadjusted differences in baseline characteristics between participants with and without hf were assessed using the student 's t test ( for continuous variables ) and chisquare test ( for categorical variables ) . the association of carotid imt with incident heart failure was assessed using cox proportional hazard models with sequential adjustment for covariates . hazard ratios and 95% confidence intervals for cox regression analysis are presented per unit standard deviation increase and by quartiles of imt . in multivariable analysis , the base model ( unadjusted model ) was sequentially adjusted for covariates of interest . a second model was adjusted for age , gender , race , and level of education ( demographic model ) . a third model was further adjusted for systolic and diastolic blood pressures , bmi , waist circumference , hdl cholesterol , ldl cholesterol , triglycerides , smoking status and amount in packyears , alcohol consumption , serum creatinine , and medications for hypertension and dyslipidemia ( clinical and biologic model ) . finally , a fourth model was adjusted for prevalent and incident chd ( chd model ) . for the fourth model , covariates were chosen based on their associations in the present cohort and in prior published studies . effect modification between race , gender , and carotid imt was examined using 2way interaction . this was achieved by including the product the imtrace and imtgender terms in the model . in the presence of a significant interaction ( p for interaction < 0.05 ) separate cox regression models included carotid imt as a continuous variable ( zscores ) and as a categorical variable ( quartiles ) . the proportional hazard assumption was assessed by visually examining the log ( log survival ) plots and timecovariate interaction terms . kaplanmeier plots were used to illustrate the overall survival probability and cumulative incidence of hf by subgroups , and group comparison was done using the logrank test . all analyses were performed using sas 9.2 ( sas institute inc , cary , nc ) . among the 13 590 participants in this study , there were 2008 ( 14.8% of total cohort ) incident cases of hf over a median followup period of 20.6 years . table 2 compares the baseline demographic and clinical characteristics of participants with and without incident hf . compared with participants without hf , participants with hf were older ( 56.6 versus 53.7 years ) , more likely to be male ( 52.5% versus 44.1% ) , and more likely to be hypertensive ( 50.3% versus 28.6% ) . participants who developed hf over the course of followup were also more likely , at baseline , to have an abnormal lipid profile , were current smokers but less likely to be current drinkers and to have completed a college education . the baseline prevalence of chd and incidence of chd were higher among subjects with hf ( p<0.0001 ) . baseline demographic and clinical characteristics by incident heart failure status of aric participants data are meanstandard deviation ( sd ) , or number ( percentages ) . bmi indicates body mass index ; chd , coronary heart disease ; hdl , high density lipoprotein ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein ; lvh , left ventricular hypertrophy . mean carotid imt was significantly higher for subjects with hf ( 0.810.23 versus 0.710.17 ; p<0.0001 ) , table 2 . compared with subjects in the first ( lowest ) quartile of carotid imt ( imt0.61 mm ) , the unadjusted incidence rates of hf increased across the second , third , and fourth quartiles ( table 3 ) . the rate in the highest quartile of carotid imt was 15.42 cases per 1000 personyears ( about 4 times the rate in the first quartile , and twice the rate in the third quartile ) . figure 1 shows a kaplanmeier plot of the overall survival probability of hf over time as a function of carotid imt quartiles . there was a significant difference in eventfree survival between quartiles of imt ( p<0.0001 ) ( figure 1 ) . of note , participants in the fourth quartile had a significantly lower survival ; 74% of those in the fourth quartile were hffree at 20 years , compared with 92% in the first quartile . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure across quartiles of carotid imt ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness ; pyrs , personyears ; q , quartile ; ref . , reference . * p<0.0001 , p=0.001 ; p<0.0001 for trend in all 4 models . for each model , the hr for q2 , q3 , and q4 are in comparison to the reference model , q1 . model 1 : unadjusted ; model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . kaplanmeier curves illustrating the hffree survival probability over time as a function of quartiles of carotid imt . cimtquartiles indicates quartiles of carotid imt ; q1 , first quartile ; q2 , second quartile ; q3 , third quartile ; q4 , fourth quartile . hf indicates heart failure ; imt , intimamedia thickness . in the multivariable analysis , we estimated the hr of hf across quartiles of carotid imt ( table 3 ) . after adjustment for demographic factors , the second , third , and fourth quartiles of carotid imt were significantly associated with hf compared with the first quartile of imt ( hr for fourth quartile 2.59 , 95% ci : 2.23 to 2.99 ) . the strength of this association was reduced , but significantly persisted for the fourth quartile of carotid imt after further adjustments for cvd risk factors ( hr 1.65 , 95% ci : 1.42 , 1.93 , < 0.0001 ) and chd ( hr 1.60 , 95% ci : 1.37 to 1.87 , p<0.0001 ) . there was a significant trend in the relationship between carotid imt and hf across quartiles of imt ( p<0.0001 ) in all 4 models . even after accounting for death from other causes as a competing risk event , the cumulative incidence functions of hf across quartiles of carotid imt remained significant ( p<0.0001 ) ( figure 2 ) . a , analysis does not take into account death from other causes as a competing risk . quartilescimt indicates quartiles of carotid imt ; q1 , first quartile ; q2 , second quartile ; q3 , third quartile ; q4 , fourth quartile . the hazard ratios ( hrs ) and 95% confidence intervals of hf per unit sd ( 0.18 mm ) increase in imt are given in table 4 . no interactions were observed by gender or race ( the p values for the imtrace and imtgender interaction terms in the unadjusted model were 0.97 and 0.11 , respectively ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt bmi indicates body mass index ; chd , coronary heart disease ; ci confidence interval ; hdl , high density lipoprotein ; hr , hazard ratio ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein . model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . mean brachial pp was higher among those with hf than those without the condition ( 53.115.5 mm hg versus 46.312.7 mm hg ; p<0.0001 , respectively ) . in univariate analysis , carotid imt correlated directly with brachial pp ( r=0.25 , p<0.0001 ) . when brachial pp was included in multivariable models , no significant modification of hf risk was observed . overall mean carotid imt was not significantly different between blacks and whites ( 0.73 mm0.16 versus 0.72 mm0.19 , respectively , p=0.16 ) . the unadjusted incidence rate of hf was higher for blacks than whites ( 10.8 versus 7.22 cases per 1000 personyears ) ( table 5 ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt by race ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness . model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . in multivariable analysis , carotid imt although the strength of the association decreased progressively in both blacks and whites with adjustment of more covariates , the association was only slightly attenuated ; for example , in blacks the hr was 1.54 per unit sd increase in imt ( 95% ci , 1.45 to 1.65 ) for the unadjusted model and 1.23 ( 95% ci , 1.12 to 1.32 ) after adjustment for demographic , clinical , and biological variables , and 1.22 ( 95% ci , 1.12 to 1.32 ) after adjustment for chd . the results of the association of increasing carotid imt and incident hf by gender paralleled that by race . among the 13 590 participants in this study , there were 2008 ( 14.8% of total cohort ) incident cases of hf over a median followup period of 20.6 years . table 2 compares the baseline demographic and clinical characteristics of participants with and without incident hf . compared with participants without hf , participants with hf were older ( 56.6 versus 53.7 years ) , more likely to be male ( 52.5% versus 44.1% ) , and more likely to be hypertensive ( 50.3% versus 28.6% ) . participants who developed hf over the course of followup were also more likely , at baseline , to have an abnormal lipid profile , were current smokers but less likely to be current drinkers and to have completed a college education . the baseline prevalence of chd and incidence of chd were higher among subjects with hf ( p<0.0001 ) . baseline demographic and clinical characteristics by incident heart failure status of aric participants data are meanstandard deviation ( sd ) , or number ( percentages ) . bmi indicates body mass index ; chd , coronary heart disease ; hdl , high density lipoprotein ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein ; lvh , left ventricular hypertrophy . mean carotid imt was significantly higher for subjects with hf ( 0.810.23 versus 0.710.17 ; p<0.0001 ) , table 2 . compared with subjects in the first ( lowest ) quartile of carotid imt ( imt0.61 mm ) , the unadjusted incidence rates of hf increased across the second , third , and fourth quartiles ( table 3 ) . the rate in the highest quartile of carotid imt was 15.42 cases per 1000 personyears ( about 4 times the rate in the first quartile , and twice the rate in the third quartile ) . figure 1 shows a kaplanmeier plot of the overall survival probability of hf over time as a function of carotid imt quartiles . there was a significant difference in eventfree survival between quartiles of imt ( p<0.0001 ) ( figure 1 ) . of note , participants in the fourth quartile had a significantly lower survival ; 74% of those in the fourth quartile were hffree at 20 years , compared with 92% in the first quartile . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure across quartiles of carotid imt ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness ; pyrs , personyears ; q , quartile ; ref . , reference . * p<0.0001 , p=0.001 ; p<0.0001 for trend in all 4 models . for each model , the hr for q2 , q3 , and q4 are in comparison to the reference model , q1 . model 1 : unadjusted ; model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . kaplanmeier curves illustrating the hffree survival probability over time as a function of quartiles of carotid imt . cimtquartiles indicates quartiles of carotid imt ; q1 , first quartile ; q2 , second quartile ; q3 , third quartile ; q4 , fourth quartile . , we estimated the hr of hf across quartiles of carotid imt ( table 3 ) . after adjustment for demographic factors , the second , third , and fourth quartiles of carotid imt were significantly associated with hf compared with the first quartile of imt ( hr for fourth quartile 2.59 , 95% ci : 2.23 to 2.99 ) . the strength of this association was reduced , but significantly persisted for the fourth quartile of carotid imt after further adjustments for cvd risk factors ( hr 1.65 , 95% ci : 1.42 , 1.93 , < 0.0001 ) and chd ( hr 1.60 , 95% ci : 1.37 to 1.87 , p<0.0001 ) . there was a significant trend in the relationship between carotid imt and hf across quartiles of imt ( p<0.0001 ) in all 4 models . even after accounting for death from other causes as a competing risk event , the cumulative incidence functions of hf across quartiles of carotid imt remained significant ( p<0.0001 ) ( figure 2 ) . quartilescimt indicates quartiles of carotid imt ; q1 , first quartile ; q2 , second quartile ; q3 , third quartile ; q4 , fourth quartile . similar results were observed with carotid imt modeled as a continuous variable . the hazard ratios ( hrs ) and 95% confidence intervals of hf per unit sd ( 0.18 mm ) no interactions were observed by gender or race ( the p values for the imtrace and imtgender interaction terms in the unadjusted model were 0.97 and 0.11 , respectively ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt bmi indicates body mass index ; chd , coronary heart disease ; ci confidence interval ; hdl , high density lipoprotein ; hr , hazard ratio ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein . model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . mean brachial pp was higher among those with hf than those without the condition ( 53.115.5 mm hg versus 46.312.7 mm hg ; p<0.0001 , respectively ) . in univariate analysis , carotid imt correlated directly with brachial pp ( r=0.25 , p<0.0001 ) . when brachial pp was included in multivariable models , no significant modification of hf risk was observed . overall mean carotid imt was not significantly different between blacks and whites ( 0.73 mm0.16 versus 0.72 mm0.19 , respectively , p=0.16 ) . the unadjusted incidence rate of hf was higher for blacks than whites ( 10.8 versus 7.22 cases per 1000 personyears ) ( table 5 ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt by race ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness . model 2 : adjusted for age , gender , race , education ; model 3 : model 2 plus systolic and diastolic blood pressure , bmi , waist circumference , ldl , hdl , triglycerides , smoking , alcohol , hypertension and cholesterol medication , serum creatinine ; model 4 : model 3 plus prevalent and incident chd . in multivariable analysis , carotid imt although the strength of the association decreased progressively in both blacks and whites with adjustment of more covariates , the association was only slightly attenuated ; for example , in blacks the hr was 1.54 per unit sd increase in imt ( 95% ci , 1.45 to 1.65 ) for the unadjusted model and 1.23 ( 95% ci , 1.12 to 1.32 ) after adjustment for demographic , clinical , and biological variables , and 1.22 ( 95% ci , 1.12 to 1.32 ) after adjustment for chd . the results of the association of increasing carotid imt and incident hf by gender paralleled that by race . the objective of this study was to investigate the association of carotid imt with incident hf among middleaged adults . carotid imt was significantly associated with incident hf among whites and blacks , after adjusting for demographic and major cvd risk factors , and chd . this relationship was comparable in both blacks and whites , as well as in males and females . carotid imt is a well validated measure of preclinical atherosclerotic lesions . in this populationbased study similar mean far wall estimates have been reported in other populations of similar age groups ; in the carotid atherosclerosis progression study and malmo diet and cancer study , mean far wall imt was 0.730.16 mm and 0.770.15 mm , respectively . the present study has shown that carotid imt is associated with incident hf , with imt modeled as both a continuous and categorical variable , even after taking into account associations explained by age , gender , race , blood pressure , bmi , waist circumference , hdl cholesterol , ldl cholesterol , triglycerides , cigarette smoking , alcohol , and serum creatinine , as well as medications for dyslipidemia and hypertension . our result is consistent with that described by engstrom et al , who reported a significant association of increased imt and hf hospitalizations in a sample of 4691 subjects with 75 cases of hf . moreover , our results remained significant even after adjustment for prevalent and incident cases of chd , which has been reported to be a major cause of hf . our study 's finding that the association of carotid imt with hf remained significant after adjustment for prevalent and incident chd , as well as blood pressure , and other traditional cvd risk factors suggests that carotid imt may be associated with hf through a mechanism that is different from that causing discrete clinical episodes of myocardial ischemia or infarction . this concept is strengthened by demonstrating that results were only slightly attenuated after adjustment for chd . first , increasing carotid imt leads to structural changes of the artery wall , which result in the deposition of collagen in the intracellular matrix and a decrease in arterial distensibility , causing increased pressure afterload , pressure wave propagation , and eventually diastolic dysfunction . we found carotid imt to be directly correlated with brachial pulse pressure ( pp ) , but no significant change was observed when brachial pp was added to the multivariable models . boutouyrie et al reported a similar correlation between brachial pp and carotid pp with carotid imt . however , only carotid pp significantly influenced carotid wall thickness , indicating that carotid pp ( a measure of arterial stiffening ) may be superior to brachial pp in exploring the mechanistic relationship between imt and hf . a second potential mechanism relates to the finding from prior studies that increasing common carotid imt was associated with reduced myocardial flow reserve in adults with and without chd . some prospective and crosssectional studies have established an association between increasing carotid imt and regional lv myocardial systolic and diastolic dysfunction , a subclinical marker and strong predictor of hf . it may be necessary to further characterize hf cases ( hfpef versus hfref ) in their relation to carotid imt to fully understand the different mechanisms that could contribute to hf in subjects with increased carotid imt . baseline echocardiographic data is not available in the aric study and cardiac ultrasound data at the time of incident hf ( obtained by review of medical records ) is available only for events occurring after january 2005 . as such , hf type in our sample bourassa et al , using data from the studies of left ventricular dysfunction ( solvd ) , reported that ischemic heart disease accounts for a majority of hf cases ( 73% ) in whites than in blacks ( 36% ) . another third of cases in blacks was due to hypertensive heart disease , compared with only 4% in whites . despite these known racial differences in the etiology of hf , we found that an association of increasing carotid imt with incident hf was observed in both whites and blacks , and to comparable degrees . to the best of our knowledge , our study is the first to investigate the association of carotid imt with incident hf in a large middleaged cohort with more than 2 decades of followup and more than 2000 cases of incident hf . the use of a middleaged population that is at a lower risk of hf compared with the elderly is particularly important . first , we did not adjust our analyses for novel biomarkers that could potentially influence the relationship between carotid imt and hf , such as nterminal probrain natriuretic peptide ( ntprobnp ) and highsensitivity creactive protein . however , in a prior published study , the association of carotid imt with hf remained significant , even after adjustment for these markers . second , the use of hospital discharge codes and death registers to recruit hf cases may have underestimated hf incidence in the population . however , the fact that increased carotid imt has been shown to be associated with reduced systolic and diastolic myocardial strain in asymptomatic individuals suggests an association of carotid imt with less severe cases of hf . our analyses included both adjudicated and nonadjudicated cases of hf . in a separate sensitivity analysis ( data not shown ) , there was no significant difference in the hazard ratios of hf prior to and after the adjudication process . third , our cohort comprised of only blacks and whites , so caution should be made when generalizing these results to other race / ethnic groups . finally , in our study , carotid imt estimates used were based on mean far wall measurements only . depending on the segment of the carotid artery measured ( common , bifurcation , or internal carotid ) , the portion of the artery wall measured ( far or near wall ) , or the variable used ( mean or maximum imt ) , different studies may report conflicting results on the association between imt and cvd risk . therefore caution should be exercised when comparing our findings to other studies employing a different ultrasound protocol . in this cohort of middleaged blacks and whites , we have demonstrated that increasing thickness of the carotid intima and media is associated with the incidence of hf even beyond the risks accounted for by traditional cvd risk factors and chd . carotid imt is appealing because it has been shown in several populationbased studies to predict future risk of myocardial infarction and stroke , and our study shows that hf is also predicted by increasing carotid imt . carotid imt is a reliable , lowcost , lowrisk indicator of early atherosclerosis currently used in cardiovascular research . measurement of carotid imt can be quickly and easily accomplished in a clinical setting , and with modern ultrasound scanners this procedure becomes more efficient and reproducible . findings from our study indicate that carotid imt may mediate hf through a mechanism that is different from myocardial ischemia or infarction . carotid imt may be useful in future hf risk prediction among middleaged blacks and whites . carotid imt is appealing because it has been shown in several populationbased studies to predict future risk of myocardial infarction and stroke , and our study shows that hf is also predicted by increasing carotid imt . carotid imt is a reliable , lowcost , lowrisk indicator of early atherosclerosis currently used in cardiovascular research . measurement of carotid imt can be quickly and easily accomplished in a clinical setting , and with modern ultrasound scanners this procedure becomes more efficient and reproducible . findings from our study indicate that carotid imt may mediate hf through a mechanism that is different from myocardial ischemia or infarction . carotid imt may be useful in future hf risk prediction among middleaged blacks and whites . the authors thank the staff and participants of the aric study for their important contributions .

backgroundincreased carotid intimamedia thickness ( imt ) is associated with subclinical left ventricular myocardial dysfunction , suggesting a possible role of carotid imt in heart failure ( hf ) risk determination.methods and resultsmean far wall carotid imt , measured by bmode ultrasound , was available for 13 590 atherosclerosis risk in communities study participants aged 45 to 64 years and free of hf at baseline . hf was defined using icd9 428 and icd10 i50 codes from hospitalization records and death certificates . the association between carotid imt and incident hf was assessed using cox proportional hazards analysis with models adjusted for demographic variables , major cvd risk factors , and interim chd . there were 2008 incident hf cases over a median followup of 20.6 years ( 8.1 cases per 1000 personyears ) . mean imt was higher in those with hf than in those without ( 0.81 mm0.23 versus 0.71 mm0.17 , p<0.001 ) . unadjusted rate of hf for the fourth compared with the first quartile of imt was 15.4 versus 3.9 per 1000 personyears ; p<0.001 . in multivariable analysis , after adjustment , each standard deviation increase in imt was associated with incident hf ( hr 1.20 [ 95% ci : 1.16 to 1.25 ] ) . after adjustment , the top quartile of imt was associated with hf ( hr 1.60 [ 95% ci : 1.37 to 1.87 ] ) . results were similar across race and gender groups.conclusionsincreasing carotid imt is associated with incident hf in middleaged whites and blacks , beyond risks explained by major cvd risk factors and chd . this suggests that carotid imt may be associated with hf through mechanisms different from myocardial ischemia or infarction .

Introduction Methods Selection and Description of Participants Data Collection and Study Variables Measurement of Carotid IMT Ascertainment of Heart Failure Measurement of Other Study Variables Statistical Analysis Results Baseline Characteristics of Participants Association of Carotid IMT With Incident HF Relationship Between Carotid IMT and Brachial Pulse Pressure (PP) Association of Carotid IMT with Incident HF by Race Discussion Conclusion Implications Acknowledgments

heart failure ( hf ) represents an increasing clinical and public health burden in the united states as its prevalence and incidence continue to rise , especially among elderly americans . carotid imt is a well established marker of subclinical atherosclerosis , which indicates early manifestation of atherosclerosis in the carotid arteries , and is associated with future cv events , asymptomatic myocardial ischemia , and changes in risk factors induced by therapeutic interventions . the association of carotid imt with incident hf in middleaged adults , who are at a lower risk of hf than older adults , is not well known . subclinical vascular disease as determined by an increase in carotid imt may be a determinant of risk for future hf , but this has not been studied . in the present study , we examine the hypothesis that subclinical atherosclerosis , assessed by mean carotid imt , is associated with incident hf beyond what is explained by major cvd risk factors in middleaged whites and blacks . the aric study is communitybased prospective study which enrolled 15 792 participants aged 45 to 64 years at the time of their baseline assessment ( 19871989 ) from 4 communities : forsyth county , north carolina ; suburban minneapolis , minnesota ; washington county , maryland ; and jackson , mississippi . excluded from the sample were : ( 1 ) participants who were missing all far wall values of carotid imt at baseline ( n=607 ) ; ( 2 ) racial groups other than black or white because of their limited numbers ( n=48 ) ; ( 3 ) participants with missing criteria needed to define baseline glucose status ( n=148 ) ; ( 4 ) participants with missing data needed to define hf at baseline ( n=287 ) ; ( 5 ) prevalent cases of hf at baseline , either by selfreported current intake of hf medication , or those with stage 3 or manifest hf by gothenburg criteria ( n=752 ) . incident hf was defined as the first occurrence of either ( 1 ) an hf hospitalization that included an international classification of diseases ( icd ) , ninth revision , discharge code of 428.x ( 428.0 to 428.9 ) in any position , or ( 2 ) a death certificate with a 428 ( hf ) or icd , 10th revision , code i50 ( hf ) in any position . abstractors made copies of sections of the medical record ( discharge summary , history and physical report , admission note , and imaging reports ) for use by the aric heart failure mortality and morbidity classification committee ( hf mmcc ) . the % agreement between any hf event adjudicated by the hf mmcc and icd9 code 428 was 75% , similar to that with the framingham followup time for those with incident hf events was defined by the time from their baseline exam ( visit 1 ) until the incident event . the association of carotid imt with incident heart failure was assessed using cox proportional hazard models with sequential adjustment for covariates . in multivariable analysis , the base model ( unadjusted model ) was sequentially adjusted for covariates of interest . the aric study is communitybased prospective study which enrolled 15 792 participants aged 45 to 64 years at the time of their baseline assessment ( 19871989 ) from 4 communities : forsyth county , north carolina ; suburban minneapolis , minnesota ; washington county , maryland ; and jackson , mississippi . during the followup period , annual phone interviews were conducted with participants to inquire about events ( including hf ) , and hospital records were surveyed for identification and classification of hf events . excluded from the sample were : ( 1 ) participants who were missing all far wall values of carotid imt at baseline ( n=607 ) ; ( 2 ) racial groups other than black or white because of their limited numbers ( n=48 ) ; ( 3 ) participants with missing criteria needed to define baseline glucose status ( n=148 ) ; ( 4 ) participants with missing data needed to define hf at baseline ( n=287 ) ; ( 5 ) prevalent cases of hf at baseline , either by selfreported current intake of hf medication , or those with stage 3 or manifest hf by gothenburg criteria ( n=752 ) . incident hf was defined as the first occurrence of either ( 1 ) an hf hospitalization that included an international classification of diseases ( icd ) , ninth revision , discharge code of 428.x ( 428.0 to 428.9 ) in any position , or ( 2 ) a death certificate with a 428 ( hf ) or icd , 10th revision , code i50 ( hf ) in any position . the % agreement between any hf event adjudicated by the hf mmcc and icd9 code 428 was 75% , similar to that with the framingham followup time for those with incident hf events was defined by the time from their baseline exam ( visit 1 ) until the incident event . the association of carotid imt with incident heart failure was assessed using cox proportional hazard models with sequential adjustment for covariates . hazard ratios and 95% confidence intervals for cox regression analysis are presented per unit standard deviation increase and by quartiles of imt . in multivariable analysis , the base model ( unadjusted model ) was sequentially adjusted for covariates of interest . among the 13 590 participants in this study , there were 2008 ( 14.8% of total cohort ) incident cases of hf over a median followup period of 20.6 years . compared with participants without hf , participants with hf were older ( 56.6 versus 53.7 years ) , more likely to be male ( 52.5% versus 44.1% ) , and more likely to be hypertensive ( 50.3% versus 28.6% ) . bmi indicates body mass index ; chd , coronary heart disease ; hdl , high density lipoprotein ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein ; lvh , left ventricular hypertrophy . mean carotid imt was significantly higher for subjects with hf ( 0.810.23 versus 0.710.17 ; p<0.0001 ) , table 2 . compared with subjects in the first ( lowest ) quartile of carotid imt ( imt0.61 mm ) , the unadjusted incidence rates of hf increased across the second , third , and fourth quartiles ( table 3 ) . the rate in the highest quartile of carotid imt was 15.42 cases per 1000 personyears ( about 4 times the rate in the first quartile , and twice the rate in the third quartile ) . figure 1 shows a kaplanmeier plot of the overall survival probability of hf over time as a function of carotid imt quartiles . of note , participants in the fourth quartile had a significantly lower survival ; 74% of those in the fourth quartile were hffree at 20 years , compared with 92% in the first quartile . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure across quartiles of carotid imt ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness ; pyrs , personyears ; q , quartile ; ref . in the multivariable analysis , we estimated the hr of hf across quartiles of carotid imt ( table 3 ) . after adjustment for demographic factors , the second , third , and fourth quartiles of carotid imt were significantly associated with hf compared with the first quartile of imt ( hr for fourth quartile 2.59 , 95% ci : 2.23 to 2.99 ) . the strength of this association was reduced , but significantly persisted for the fourth quartile of carotid imt after further adjustments for cvd risk factors ( hr 1.65 , 95% ci : 1.42 , 1.93 , < 0.0001 ) and chd ( hr 1.60 , 95% ci : 1.37 to 1.87 , p<0.0001 ) . there was a significant trend in the relationship between carotid imt and hf across quartiles of imt ( p<0.0001 ) in all 4 models . even after accounting for death from other causes as a competing risk event , the cumulative incidence functions of hf across quartiles of carotid imt remained significant ( p<0.0001 ) ( figure 2 ) . the hazard ratios ( hrs ) and 95% confidence intervals of hf per unit sd ( 0.18 mm ) increase in imt are given in table 4 . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt bmi indicates body mass index ; chd , coronary heart disease ; ci confidence interval ; hdl , high density lipoprotein ; hr , hazard ratio ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein . mean brachial pp was higher among those with hf than those without the condition ( 53.115.5 mm hg versus 46.312.7 mm hg ; p<0.0001 , respectively ) . overall mean carotid imt was not significantly different between blacks and whites ( 0.73 mm0.16 versus 0.72 mm0.19 , respectively , p=0.16 ) . the unadjusted incidence rate of hf was higher for blacks than whites ( 10.8 versus 7.22 cases per 1000 personyears ) ( table 5 ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt by race ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness . in multivariable analysis , carotid imt although the strength of the association decreased progressively in both blacks and whites with adjustment of more covariates , the association was only slightly attenuated ; for example , in blacks the hr was 1.54 per unit sd increase in imt ( 95% ci , 1.45 to 1.65 ) for the unadjusted model and 1.23 ( 95% ci , 1.12 to 1.32 ) after adjustment for demographic , clinical , and biological variables , and 1.22 ( 95% ci , 1.12 to 1.32 ) after adjustment for chd . the results of the association of increasing carotid imt and incident hf by gender paralleled that by race . among the 13 590 participants in this study , there were 2008 ( 14.8% of total cohort ) incident cases of hf over a median followup period of 20.6 years . bmi indicates body mass index ; chd , coronary heart disease ; hdl , high density lipoprotein ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein ; lvh , left ventricular hypertrophy . mean carotid imt was significantly higher for subjects with hf ( 0.810.23 versus 0.710.17 ; p<0.0001 ) , table 2 . compared with subjects in the first ( lowest ) quartile of carotid imt ( imt0.61 mm ) , the unadjusted incidence rates of hf increased across the second , third , and fourth quartiles ( table 3 ) . the rate in the highest quartile of carotid imt was 15.42 cases per 1000 personyears ( about 4 times the rate in the first quartile , and twice the rate in the third quartile ) . of note , participants in the fourth quartile had a significantly lower survival ; 74% of those in the fourth quartile were hffree at 20 years , compared with 92% in the first quartile . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure across quartiles of carotid imt ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness ; pyrs , personyears ; q , quartile ; ref . after adjustment for demographic factors , the second , third , and fourth quartiles of carotid imt were significantly associated with hf compared with the first quartile of imt ( hr for fourth quartile 2.59 , 95% ci : 2.23 to 2.99 ) . the strength of this association was reduced , but significantly persisted for the fourth quartile of carotid imt after further adjustments for cvd risk factors ( hr 1.65 , 95% ci : 1.42 , 1.93 , < 0.0001 ) and chd ( hr 1.60 , 95% ci : 1.37 to 1.87 , p<0.0001 ) . there was a significant trend in the relationship between carotid imt and hf across quartiles of imt ( p<0.0001 ) in all 4 models . even after accounting for death from other causes as a competing risk event , the cumulative incidence functions of hf across quartiles of carotid imt remained significant ( p<0.0001 ) ( figure 2 ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt bmi indicates body mass index ; chd , coronary heart disease ; ci confidence interval ; hdl , high density lipoprotein ; hr , hazard ratio ; hf , heart failure ; imt , intimamedia thickness ; ldl , low density lipoprotein . mean brachial pp was higher among those with hf than those without the condition ( 53.115.5 mm hg versus 46.312.7 mm hg ; p<0.0001 , respectively ) . overall mean carotid imt was not significantly different between blacks and whites ( 0.73 mm0.16 versus 0.72 mm0.19 , respectively , p=0.16 ) . the unadjusted incidence rate of hf was higher for blacks than whites ( 10.8 versus 7.22 cases per 1000 personyears ) ( table 5 ) . unadjusted and adjusted hrs ( 95% ci ) for incident heart failure per 1 sd ( 0.18 mm ) increase in carotid imt by race ci indicates confidence interval ; hf , heart failure ; hr , hazard ratio ; imt , intimamedia thickness . in multivariable analysis , carotid imt although the strength of the association decreased progressively in both blacks and whites with adjustment of more covariates , the association was only slightly attenuated ; for example , in blacks the hr was 1.54 per unit sd increase in imt ( 95% ci , 1.45 to 1.65 ) for the unadjusted model and 1.23 ( 95% ci , 1.12 to 1.32 ) after adjustment for demographic , clinical , and biological variables , and 1.22 ( 95% ci , 1.12 to 1.32 ) after adjustment for chd . the results of the association of increasing carotid imt and incident hf by gender paralleled that by race . the objective of this study was to investigate the association of carotid imt with incident hf among middleaged adults . carotid imt was significantly associated with incident hf among whites and blacks , after adjusting for demographic and major cvd risk factors , and chd . the present study has shown that carotid imt is associated with incident hf , with imt modeled as both a continuous and categorical variable , even after taking into account associations explained by age , gender , race , blood pressure , bmi , waist circumference , hdl cholesterol , ldl cholesterol , triglycerides , cigarette smoking , alcohol , and serum creatinine , as well as medications for dyslipidemia and hypertension . moreover , our results remained significant even after adjustment for prevalent and incident cases of chd , which has been reported to be a major cause of hf . our study 's finding that the association of carotid imt with hf remained significant after adjustment for prevalent and incident chd , as well as blood pressure , and other traditional cvd risk factors suggests that carotid imt may be associated with hf through a mechanism that is different from that causing discrete clinical episodes of myocardial ischemia or infarction . a second potential mechanism relates to the finding from prior studies that increasing common carotid imt was associated with reduced myocardial flow reserve in adults with and without chd . some prospective and crosssectional studies have established an association between increasing carotid imt and regional lv myocardial systolic and diastolic dysfunction , a subclinical marker and strong predictor of hf . it may be necessary to further characterize hf cases ( hfpef versus hfref ) in their relation to carotid imt to fully understand the different mechanisms that could contribute to hf in subjects with increased carotid imt . baseline echocardiographic data is not available in the aric study and cardiac ultrasound data at the time of incident hf ( obtained by review of medical records ) is available only for events occurring after january 2005 . as such , hf type in our sample bourassa et al , using data from the studies of left ventricular dysfunction ( solvd ) , reported that ischemic heart disease accounts for a majority of hf cases ( 73% ) in whites than in blacks ( 36% ) . despite these known racial differences in the etiology of hf , we found that an association of increasing carotid imt with incident hf was observed in both whites and blacks , and to comparable degrees . to the best of our knowledge , our study is the first to investigate the association of carotid imt with incident hf in a large middleaged cohort with more than 2 decades of followup and more than 2000 cases of incident hf . however , in a prior published study , the association of carotid imt with hf remained significant , even after adjustment for these markers . however , the fact that increased carotid imt has been shown to be associated with reduced systolic and diastolic myocardial strain in asymptomatic individuals suggests an association of carotid imt with less severe cases of hf . depending on the segment of the carotid artery measured ( common , bifurcation , or internal carotid ) , the portion of the artery wall measured ( far or near wall ) , or the variable used ( mean or maximum imt ) , different studies may report conflicting results on the association between imt and cvd risk . in this cohort of middleaged blacks and whites , we have demonstrated that increasing thickness of the carotid intima and media is associated with the incidence of hf even beyond the risks accounted for by traditional cvd risk factors and chd . findings from our study indicate that carotid imt may mediate hf through a mechanism that is different from myocardial ischemia or infarction . carotid imt may be useful in future hf risk prediction among middleaged blacks and whites . findings from our study indicate that carotid imt may mediate hf through a mechanism that is different from myocardial ischemia or infarction .

cells : a72/cslam and crfk / cslam cells expressing the canine signaling lymphocyte activation molecule ( slam ) were grown in dulbecco s modified eagle s medium ( dmem : life technologies , carlsbad , ca , u.s.a . ) containing 10% fetal calf serum ( fcs ) , 100 u / ml penicillin and 100 g / ml streptomycin ( life technologies ) . virus : cdv kdk-1 strain ( genotype asia-1 ) was isolated from a diseased dog in kagoshima , japan , in 1991 using vero cells and propagated in a72/cslam cells after six passages in vero cells . preparation of antigens : elisa antigen was prepared ; kdk-1- or mock - infected vero cells were lysed with lysis buffer , tsa ( 2 mm tris - hcl ph 8.0 , 140 mm sodium chloride and 0.025% sodium azide ) containing 1% triton x-100 and 0.5% sodium deoxycholate at 4c for 1 hr and then centrifuged at 20,630 g for 30 min . elisa using anti - dog igg or igm antibody as a secondary antibody : antigens were diluted to 5 g / ml with an adsorption buffer ( 0.05 m carbonate bicarbonate buffer , ph 9.6 ) , and 100 l was added per well into 96-well microplates ( maxisorp ; nunc , roskilde , denmark ) . after incubation at 37c for 2 hr , plates were placed at 4c overnight . the wells were washed three times with phosphate - buffered saline ( pbs ) and then incubated with 100 l per well of 0.1% bovine serum albumin ( bsa , fraction v ; sigma , st . after three washes with pbs containing 0.05% tween20 ( pbs - t ) , sera diluted with dilution buffer and pbs - t containing 10% fcs were added to duplicate wells , and plates were incubated at 37c for 30 min . next , wells were washed 3 times with pbs - t and incubated with 100 l per well of peroxidase - conjugated sheep anti - dog igg or goat anti - dog igm antibody ( bethyl , montgomery , tx , u.s.a . ) diluted with dilution buffer at 37c for 30 min . following washing three times with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad , hercules , ca , u.s.a . ) was added to each well . after incubation at room temperature for 30 min , the enzymatic reaction was stopped by adding 100 l of 2% oxalic acid to each well . the absorbance was measured by a spectrophotometer ( bio - rad ) , with a 415-nm filter . all results were subtracted by the value of control wells containing extract from mock - infected cells . elisa using protein a / g as a secondary antibody : the antigen was diluted to 5 g / ml with an adsorption buffer , and 100 l was added per well into 96-well microplates . after incubation at 37c for 2 hr , plates were placed at 4c overnight . the wells were washed 3 times with pbs and then incubated with 100 l per well of 1% block ace ( dainippon pharmaceutical , osaka , japan ) in pbs at 37c for 30 min . after three washes with pbs - t , 100 l of diluted sera were added to duplicate wells , and plates were incubated at 37c for 30 min . next , the wells were washed three times with pbs - t and incubated with 100 l per well of peroxidase conjugated purified recomb protein a / g ( thermo fischer scientific , rockford , il , u.s.a . ) diluted with pbs - t containing 0.4% block ace at 37c for 30 min . following three washes with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad ) was added to each well . after incubation at room temperature for 30 min , the enzymatic reaction was stopped by adding 100 l of 2% oxalic acid to each well . the absorbance was measured by a spectrophotometer ( bio - rad ) , with a 415-nm filter . all results were subtracted by the value of control wells containing extract from mock - infected cells . sera from dogs experimentally infected with cdv : three dogs ( beagle , female , 3 months old ; narc , japan ) were orally , intranasally and ocularly inoculated with 10 ml of viral solution containing 10 plaque - forming units ( pfu ) of cdv kochio1a . cdv kochio1a was isolated from a dead masked palm civet from kochi prefecture , japan , in 2008 . blood samples were collected from the cephalic vein under anesthesia with ketamine on days 0 , 3 , 6 , 9 , 12 , 15 , 21 and 27 post challenge . vn test : vn test to kdk-1 was performed by 75% plaque - reduction neutralization test ( prnt75 ) using our established cell line , crfk / cslam . to determine vn titers , sera were diluted to 1:5 and then serially diluted 2-fold with dmem containing 2% fcs . diluted sera were mixed with equal volumes of virus solution containing 100 pfu of kdk-1 , followed by incubation at 37c for 1 then , 50 l of mixtures were added to each well of 24-well plates ( sumilon , tokyo , japan ) containing subconfluent crfk / cslam , and the plate was incubated at 37c for 1 hr , washed twice with dmem without fcs and overlaid with dmem containing 0.8% agarose and 7% fcs . cells were fixed with 5% buffered formaldehyde for 1 hr , and agarose layers were removed . after staining with crystal violet , plaques were counted . vn titer was expressed as the highest dilution of serum that reduced plaques by more than 75% in comparison with that of control wells without serum . animal samples : between 2006 and 2012 , a total of 1,686 serum samples were collected from wild animals captured in wakayama prefecture , under permission from the governor . wild animals included 1,261 raccoons ( procyon lotor ) , 317 raccoon dogs ( nyctereutes procyonoides ) , 23 badgers ( meles meles ) , one weasel ( mustela itatsi ) , one japanese marten ( martes melampus ) , three korean yellow weasel ( mustela sibirica ) , one red fox ( vulpes vulpes ) , six sika deer ( cervus nippon ) , 72 wild boars ( sus scrofa ) and one alley cat ( felis silvestris catus ) . all serum samples were heat inactivated at 56c for 30 min and stored at 20c until use . tissue swabs and fecal samples were collected from two raccoon dogs and one fox for virus isolation . virus isolation : swabs and fecal samples were dissolved in 2 ml dmem containing antibiotics , 200 u / ml penicillin and 200 g / ml streptomycin , and then centrifuged at 2,000 g for 15 min at 4c . tissue samples from dead animals were homogenized in a nine - fold volume of dmem containing antibiotics and then centrifuged at 2,000 g for 15 min at 4c . the supernatants were passed through 0.45 m filters ( millipore , bedford , ma , u.s.a . ) and then inoculated onto a72/cslam cells . sequence analysis of hemagglutinin ( h ) gene : rna was extracted from virus - infected cells or tissue samples using qiagen rneasy mini kit ( qiagen , hilden , germany ) or qiagen viral rna mini kit ( qiagen ) , respectively , and reverse - transcription ( rt ) was performed with random 9-mer primer using takara rna la pcrtm kit ( amv ) ver.1.1 ( takara , otsu , japan ) at 30c for 10 min , 42c for 30 min and 70c for 15 min . h gene was amplified using primers , cdv - hr ( 5-aga tgg acc tca ggg tat ag-3 ) and cdv - hf ( 5-aac tta ggg ctc agg tag tc-3 ) , at 94c for 2 min and 30 cycles consisting of denaturation at 94c for 30 sec , annealing at 60c for 30 sec and extension at 72c for 3 min , followed by a final extension at 72c for 15 min . the amplified products were purified using qiaquick pcr purification kit ( qiagen ) , and nucleotide sequences were directly determined using 3130 genetic analyzer ( applied biosystem , carlsbad , ca , u.s.a . ) . the nucleotide sequences were deposited in the dna data bank of japan ( ddbj , table 4 ) . homology search and phylogenetic analysis : homologies among strains were analyzed using the genetyx ver.8 ( genetyx corporation , tokyo , japan ) , and phylogenetic trees were constructed by the neighbor - joining method using mega5.0 software on the basis of the amino acid pairwise distance . preparation of antigens : elisa antigen was prepared ; kdk-1- or mock - infected vero cells were lysed with lysis buffer , tsa ( 2 mm tris - hcl ph 8.0 , 140 mm sodium chloride and 0.025% sodium azide ) containing 1% triton x-100 and 0.5% sodium deoxycholate at 4c for 1 hr and then centrifuged at 20,630 g for 30 min . elisa using anti - dog igg or igm antibody as a secondary antibody : antigens were diluted to 5 g / ml with an adsorption buffer ( 0.05 m carbonate bicarbonate buffer , ph 9.6 ) , and 100 l was added per well into 96-well microplates ( maxisorp ; nunc , roskilde , denmark ) . after incubation at 37c for 2 hr , plates were placed at 4c overnight . the wells were washed three times with phosphate - buffered saline ( pbs ) and then incubated with 100 l per well of 0.1% bovine serum albumin ( bsa , fraction v ; sigma , st . after three washes with pbs containing 0.05% tween20 ( pbs - t ) , sera diluted with dilution buffer and pbs - t containing 10% fcs were added to duplicate wells , and plates were incubated at 37c for 30 min . next , wells were washed 3 times with pbs - t and incubated with 100 l per well of peroxidase - conjugated sheep anti - dog igg or goat anti - dog igm antibody ( bethyl , montgomery , tx , u.s.a . ) diluted with dilution buffer at 37c for 30 min . following washing three times with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad , hercules , ca , u.s.a . ) was added to each well . after incubation at room temperature for 30 min , the enzymatic reaction was stopped by adding 100 l of 2% oxalic acid to each well . the absorbance was measured by a spectrophotometer ( bio - rad ) , with a 415-nm filter . all results were subtracted by the value of control wells containing extract from mock - infected cells . elisa using protein a / g as a secondary antibody : the antigen was diluted to 5 g / ml with an adsorption buffer , and 100 l was added per well into 96-well microplates . after incubation at 37c for 2 hr , plates were placed at 4c overnight . the wells were washed 3 times with pbs and then incubated with 100 l per well of 1% block ace ( dainippon pharmaceutical , osaka , japan ) in pbs at 37c for 30 min . after three washes with pbs - t , 100 l of diluted sera were added to duplicate wells , and plates were incubated at 37c for 30 min . next , the wells were washed three times with pbs - t and incubated with 100 l per well of peroxidase conjugated purified recomb protein a / g ( thermo fischer scientific , rockford , il , u.s.a . ) diluted with pbs - t containing 0.4% block ace at 37c for 30 min . following three washes with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad ) was added to each well . after incubation at room temperature for 30 min , the enzymatic reaction was stopped by adding 100 l of 2% oxalic acid to each well . the absorbance was measured by a spectrophotometer ( bio - rad ) , with a 415-nm filter . all results were subtracted by the value of control wells containing extract from mock - infected cells . sera from dogs experimentally infected with cdv : three dogs ( beagle , female , 3 months old ; narc , japan ) were orally , intranasally and ocularly inoculated with 10 ml of viral solution containing 10 plaque - forming units ( pfu ) of cdv kochio1a . cdv kochio1a was isolated from a dead masked palm civet from kochi prefecture , japan , in 2008 . blood samples were collected from the cephalic vein under anesthesia with ketamine on days 0 , 3 , 6 , 9 , 12 , 15 , 21 and 27 post challenge . vn test : vn test to kdk-1 was performed by 75% plaque - reduction neutralization test ( prnt75 ) using our established cell line , crfk / cslam . to determine vn titers , sera were diluted to 1:5 and then serially diluted 2-fold with dmem containing 2% fcs . diluted sera were mixed with equal volumes of virus solution containing 100 pfu of kdk-1 , followed by incubation at 37c for 1 hr . then , 50 l of mixtures were added to each well of 24-well plates ( sumilon , tokyo , japan ) containing subconfluent crfk / cslam , and the plate was incubated at 37c for 1 hr , washed twice with dmem without fcs and overlaid with dmem containing 0.8% agarose and 7% fcs . cells were fixed with 5% buffered formaldehyde for 1 hr , and agarose layers were removed . after staining with crystal violet , plaques were counted . vn titer was expressed as the highest dilution of serum that reduced plaques by more than 75% in comparison with that of control wells without serum . animal samples : between 2006 and 2012 , a total of 1,686 serum samples were collected from wild animals captured in wakayama prefecture , under permission from the governor . wild animals included 1,261 raccoons ( procyon lotor ) , 317 raccoon dogs ( nyctereutes procyonoides ) , 23 badgers ( meles meles ) , one weasel ( mustela itatsi ) , one japanese marten ( martes melampus ) , three korean yellow weasel ( mustela sibirica ) , one red fox ( vulpes vulpes ) , six sika deer ( cervus nippon ) , 72 wild boars ( sus scrofa ) and one alley cat ( felis silvestris catus ) . all serum samples were heat inactivated at 56c for 30 min and stored at 20c until use . tissue swabs and fecal samples were collected from two raccoon dogs and one fox for virus isolation . virus isolation : swabs and fecal samples were dissolved in 2 ml dmem containing antibiotics , 200 u / ml penicillin and 200 g / ml streptomycin , and then centrifuged at 2,000 g for 15 min at 4c . tissue samples from dead animals were homogenized in a nine - fold volume of dmem containing antibiotics and then centrifuged at 2,000 g for 15 min at 4c . the supernatants were passed through 0.45 m filters ( millipore , bedford , ma , u.s.a . ) and then inoculated onto a72/cslam cells . sequence analysis of hemagglutinin ( h ) gene : rna was extracted from virus - infected cells or tissue samples using qiagen rneasy mini kit ( qiagen , hilden , germany ) or qiagen viral rna mini kit ( qiagen ) , respectively , and reverse - transcription ( rt ) was performed with random 9-mer primer using takara rna la pcrtm kit ( amv ) ver.1.1 ( takara , otsu , japan ) at 30c for 10 min , 42c for 30 min and 70c for 15 min . h gene was amplified using primers , cdv - hr ( 5-aga tgg acc tca ggg tat ag-3 ) and cdv - hf ( 5-aac tta ggg ctc agg tag tc-3 ) , at 94c for 2 min and 30 cycles consisting of denaturation at 94c for 30 sec , annealing at 60c for 30 sec and extension at 72c for 3 min , followed by a final extension at 72c for 15 min . the amplified products were purified using qiaquick pcr purification kit ( qiagen ) , and nucleotide sequences were directly determined using 3130 genetic analyzer ( applied biosystem , carlsbad , ca , u.s.a . ) . the nucleotide sequences were deposited in the dna data bank of japan ( ddbj , table 4 ) . homology search and phylogenetic analysis : homologies among strains were analyzed using the genetyx ver.8 ( genetyx corporation , tokyo , japan ) , and phylogenetic trees were constructed by the neighbor - joining method using mega5.0 software on the basis of the amino acid pairwise distance . although the vn test has been the standard assay to detect the cdv - specific antibodies in serum , it requires a large volume of serum , a special facility ( biosafety level-2 ) and a high level of technical skill . in contrast , elisa does not use live cdv and requires only a small amount of sample , and the procedure is simple . therefore , to detect antibodies against cdv in many mammalians , we established a new elisa using peroxidase - conjugated protein a / g ( thermo scientific , waltham , ma , u.s.a . ) as a second antibody and block ace ( dainippon pharmaceutical , osaka , japan ) as antibody blocking and dilution agent . at first , an elisa using protein a / g was performed with sera collected from dogs experimentally infected with cdv . this study showed that elisa and vn titer results were almost similar ( fig . three dogs ( nos.13 ) were experimentally infected with cdv kochio1a , and sera were sequentially collected . dog sera were diluted to 1:100 , and anti - dog igg ( open circle ) , igm ( open square ) and protein a / g ( black circle ) were used as secondary antibodies . for the vn test , sera were two - fold diluted , and prnt75 was performed ( vertical bar ) . ) . elisa using either anti - igg or anti - igm antibody showed different results ; igg antibody gradually increased from day 1227 , and igm antibody peaked on day 15 after infection . on the other hand , the anti - cdv antibody detected by protein a / g peaked on day 15 and then increased from day 2127 . a mixture of both igg- and igm - antibodies was detected by protein a / g . three dogs ( nos.13 ) were experimentally infected with cdv kochio1a , and sera were sequentially collected . dog sera were diluted to 1:100 , and anti - dog igg ( open circle ) , igm ( open square ) and protein a / g ( black circle ) were used as secondary antibodies . for the vn test , sera were two - fold diluted , and prnt75 was performed ( vertical bar ) . to confirm whether elisa using protein a / g is reliable for many mammalian species , results were compared with those obtained by the vn test . the results of elisa were correlated with those of the vn tests in raccoons and raccoon dogs ( fig . values in elisa and titers of virus - neutralizing antibody in raccoons and raccoon dogs . 803 and 326 sera of raccoons ( a ) and raccoon dogs ( b ) , respectively , were analyzed by elisa and prnt75 . ) . in 660 raccoons , which were cdv negative by the vn test , average and standard deviation ( s.d . ) of od values were 0.051 and 0.086 , respectively . in 268 raccoon dogs , which were cdv negative by the vn test , average and s.d . of od values were 0.097 and 0.131 , respectively . was applied , and the cut - off values for elisa were 0.309 and 0.491 for raccoons and raccoon dogs , respectively . in comparison with the result of vn test , specificity and sensitivity of this elisa for raccoons were 98.3% and 73.9% , respectively , and those for raccoon dogs were 98.9% and 79.3% , respectively . values in elisa and titers of virus - neutralizing antibody in raccoons and raccoon dogs . 803 and 326 sera of raccoons ( a ) and raccoon dogs ( b ) , respectively , were analyzed by elisa and prnt75 . in a serological survey of cdv infection in raccoons , raccoon dogs and other wild animals , cut - off values of elisa were tentatively determined to be 0.309 , 0.491 and 0.5 , respectively . the results indicated that 10.3% of raccoons , 13.2% of raccoon dogs , 18% of wild boars , 9% of badgers , one marten , one siberian weasel and 2 sika deer were sero - positive for cdv ( tables 1table 1.seroprevalence of cdv infection in raccoonsplaces2006200720082009201020112012totaltanabeno . of examined animals6924741291087098572% of positive animals163287.84.611511.4ryujinno . of examined animals000244414% of positive animals---50005021other townsno . of examined animals61158130140164166675% of positive animals17182414.65.04.96.69.2totalno of examined animals75351322612522382681261% of positive animals34926.511.54.86.76.710.3 , 2table 2.seroprevalence of cdv infection in raccoon dogsplaces200720082009201020112012totaltanabeno . of examined animals1414162262119% of positive animals0500693120.2ryujinno . of examined animals0023191052% of positive animals--013117024other townsno . of examined animals2122621445146% of positive animals0056704.1totalno . of examined353810945117317% of positive animals04038.3922.213.2 , 3table 3.seroprevalence of cdv infection in other wild animalsanimals2007200820092012totalno . of examined animals% of cdv - positive animalsno . of examined animals% of cdv - positive animalsno . of examined animals% of cdv - positive animalsbadger(meles meles)250215239weasel(mustela itatsi)100 - 10marten(martes melampus)11000 - 1100siberian weasel(mustela sibirica coreana)110020333fox(vulpes vulpes)100 - 10sika deer(cervus nippon)54010633wild boar(sus scrofa)41273167218alley cat(felis silvestris catus)0 - 1010 ) stocks of hemolytic sera collected from raccoons in 2006 were not suitable for the vn test . elisa could only detect the cdv - specific antibody from 2 raccoons ( 2.7% ) from the 2006 sample set ( table 1 ) . to examine whether these old hemolytic sera were suitable for elisa , further elisas the results obtained indicated that many of the sera samples were positive for the jev antibody ( data not shown ) . we previously reported the cdv epidemic among wild mammalians around tanabe city in wakayama prefecture in 20072008 using the vn test . in this study , we detected the anti - cdv antibodies by elisa using protein a / g as described above ( tables 1 and 2 , fig . 3fig . . the positive rate of cdv infection in raccoons ( solid circle ) and raccoon dogs ( open circle ) is plotted . ) . in 2006 , most raccoons did not possess the anti - cdv antibody ( 1.4% ) , but 62.5% of raccoons possessed the anti - cdv antibody in 2007 during the cdv epidemic . after the epidemic , 28.4% in 2008 , 7.8% in 2009 , 4.6% in 2010 , 11.4% in 2011 and 5.1% in 2012 were cdv positive ( table 1 , fig . the age of raccoons ( calculated by the rings of teeth ) indicated that many cdv - positive raccoons captured between 2009 and 2012 were born before the epidemic ( date not shown ) . change in seroprevalence of cdv infection among raccoons and raccoon dogs in tanabe city . the positive rate of cdv infection in raccoons ( solid circle ) and raccoon dogs ( open circle ) is plotted . on the other hand , 0% , 6.3% and 9.1% of raccoon dogs in tanabe city had anti - cdv antibodies in 2009 , 2010 and 2011 , respectively ( table 2 ) . however , in 2012 , 19 raccoon dogs ( 30.6% ) were infected with cdv ( fig . 3 ) , and the virus was also isolated from one diseased raccoon dog in tanabe city ( table 4table 4.viruses isolated or detected from wild animals in wakayama prefecturestrainanimal speciesdate of deathsexweight ( kg)virus isolationaccession no.(month / day / year)w729b / rd/070416raccoon dog4/16/20072.4+ab605891w812b / rd/080131raccoon dog1/31/20082.4+ab605890wakayama / fox/101125fox11/24/20105lc007974wakayama / rd/110407raccoon dog4/6/20113.1lc007975wakayama / rd/120927raccoon dog9/26/20123+lc007976a ) these viruses were previously reported ( kameo et al . ) . ) . tanabe city is surrounded by several other towns , including ryujin . before the cdv epidemic , one raccoon ( 16.7% ) was cdv positive , but the number of seropositives was less than 10% after 2010 . in raccoon dogs in ryujin , the seroprevalence of cdv infection was approximately 10% during 20092011 , although seven raccoon dogs ( 70% ) became positive for cdv infection in 2012 ( table 2 ) . two cdv genomes were detected by rt - pcr , one from a dead fox and the other from a dead raccoon dog , in 2010 and 2011 , respectively ( table 4 ) . furthermore , one cdv virus was isolated from a dead raccoon dog in 2012 ( table 4 ) . phylogenetic analysis of the predicted h proteins was performed to elucidate the evolutionary relationships among cdv . we report that cdv in this area formed one cluster independent of animal species ( fig . phylogenetic analysis was performed using a total of 607 amino acid positions with the mega5 program . accession numbers of the sequences are bak79007 ( yamaguchi / rd/091204 ) , yamaguchi / rd/091216 ) , bak79006 ( yamaguchi / wt/100311 ) , baa19586 ( yanaka ) , baa19584 ( ueno ) , baa19585 ( hamamatsu ) , bag41887 ( pr780-lu ) , baa84209 ( kdk-1 ) , baa33740 ( tanu96 ) , bab39167 ( hm-3 ) , baa84208 ( 98 - 002 ) , bab39166 ( 26d ) , aad49703 ( a75/17 ) , caa90879 ( black panther a92 - 6 ) , caa87691 ( american dog ) , caa59359 ( 5804/han90 ) , aam11476 ( dog turkey ) , aaq05829 ( dk91a ) , cab01252 ( 2544 ) , caa87688 ( danish mink ) , caa59358 ( german ferret ) , caa87689 ( greenlandic dog ) , caa59357 ( pdv-2 ) , aag15490 ( snyder hill ) , caa84626 ( convac ) and aak54669 ( onderstepoort ) . phylogenetic analysis was performed using a total of 607 amino acid positions with the mega5 program . accession numbers of the sequences are bak79007 ( yamaguchi / rd/091204 ) , yamaguchi / rd/091216 ) , bak79006 ( yamaguchi / wt/100311 ) , baa19586 ( yanaka ) , baa19584 ( ueno ) , baa19585 ( hamamatsu ) , bag41887 ( pr780-lu ) , baa84209 ( kdk-1 ) , baa33740 ( tanu96 ) , bab39167 ( hm-3 ) , baa84208 ( 98 - 002 ) , bab39166 ( 26d ) , aad49703 ( a75/17 ) , caa90879 ( black panther a92 - 6 ) , caa87691 ( american dog ) , caa59359 ( 5804/han90 ) , aam11476 ( dog turkey ) , aaq05829 ( dk91a ) , cab01252 ( 2544 ) , caa87688 ( danish mink ) , caa59358 ( german ferret ) , caa87689 ( greenlandic dog ) , caa59357 ( pdv-2 ) , aag15490 ( snyder hill ) , caa84626 ( convac ) and aak54669 ( onderstepoort ) . nucleotide sequences of haku00 and haku06 were reported by hirama et al . ( 2004 ) . in this study , an elisa using horseradish peroxidase - conjugated protein a / g was developed to detect the cdv antibody in various mammalian species . comparing the vn test and elisa , it was confirmed that the established elisa was available for at least dogs , raccoons and raccoon dogs . we obtained 75 hemolytic blood samples that were collected from raccoons in 2006 and stored at 20c . because these hemolytic blood samples were not available for the vn test the results indicated that 2 raccoons ( 2.7% ) were cdv positive . to examine whether antibodies in the hemolytic blood samples were damaged , elisa for jev was also performed , because many raccoons in this area were seropositive for jev . the results showed that many raccoons were positive for jev , indicating that antibodies in these hemolytic blood samples function well enough for elisa . the cdv epidemic among wild mammalians in and around tanabe city in wakayama prefecture in 20072008 resulted in a widespread cdv infection of many species , particularly raccoons . in this study , the seroprevalence of cdv infection before and after the cdv epidemic was examined . in tanabe city , 62.5% and 28.4% of raccoons became positive in 2007 and 2008 , respectively , but less than 10% of raccoons were positive for cdv in 2006 and after 2009 . in addition , the age of raccoons ( calculated by the rings of teeth ) suggested that cdv - positive raccoons after 2009 were born before or during the cdv epidemic . these results indicated that most raccoons were infected with cdv during this epidemic and probably not after this epidemic . since raccoons are considered as a natural reservoir of cdv in the u.s.a . [ 7 , 12 , 20 , 22 ] , raccoons may also spread cdv to other animals in japan . in 20072008 , very few raccoon dogs were captured in wakayama prefecture , suggesting that cdv may have spread among raccoon dogs and killed many of them because of their high sensitivity to cdv . many dead raccoon dogs were found during this epidemic . although the prevalence of cdv - positive raccoon dogs was less than 10% after the epidemic , 22.2% of raccoon dogs became positive for cdv in 2012 . in particular , 70.0% and 30.6% of raccoon dogs were positive for cdv in ryujin and tanabe cities , respectively . in addition , cdv was detected in two raccoon dogs in 2011 and 2012 . in particular , these results indicated that cdv was maintained among a population of raccoon dogs , and a small epidemic without transmission to raccoons occurred in tanabe and ryujin cities in 2012 . in this study , it seems likely that raccoon dogs play a crucial role in maintenance of cdv in this area , and the epidemic in 2007 was enhanced by transmission of cdv to raccoons , resulting in many wild animals , including the sika deer and wild boar , being infected with cdv . raccoons were introduced from north america , and the population is rapidly increasing in japan .

in 20072008 , a canine distemper virus ( cdv ) epidemic occurred among wild animals in wakayama prefecture , japan , and many mammals , including the wild boar and deer , were infected . in this study , cdv prevalence among wild animals was surveyed before and after the epidemic . at first , an enzyme - linked immunosorbent assay ( elisa ) with horseradish peroxidase - conjugated protein a / g was established to detect cdv antibodies in many mammalian species . this established elisa was available for testing dogs , raccoons and raccoon dogs as well as virus - neutralization test . next , a serological survey of wild mammalians was conducted , and it was indicated that many wild mammalians , particularly raccoons , were infected with cdv during the epidemic , but few were infected before and after the epidemic . on the other hand , many raccoon dogs died during the epidemic , but cdv remained prevalent in the remaining population , and a small epidemic occurred in raccoon dogs in 20122013 . these results indicated that the epidemic of 20072008 may have been intensified by transmission to raccoons .

MATERIALS AND METHODS ELISA RESULTS DISCUSSION

virus : cdv kdk-1 strain ( genotype asia-1 ) was isolated from a diseased dog in kagoshima , japan , in 1991 using vero cells and propagated in a72/cslam cells after six passages in vero cells . next , wells were washed 3 times with pbs - t and incubated with 100 l per well of peroxidase - conjugated sheep anti - dog igg or goat anti - dog igm antibody ( bethyl , montgomery , tx , u.s.a . ) following washing three times with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad , hercules , ca , u.s.a . ) elisa using protein a / g as a secondary antibody : the antigen was diluted to 5 g / ml with an adsorption buffer , and 100 l was added per well into 96-well microplates . next , the wells were washed three times with pbs - t and incubated with 100 l per well of peroxidase conjugated purified recomb protein a / g ( thermo fischer scientific , rockford , il , u.s.a . ) following three washes with pbs - t , 100 l of horseradish peroxidase substrate kit ( bio - rad ) was added to each well . sera from dogs experimentally infected with cdv : three dogs ( beagle , female , 3 months old ; narc , japan ) were orally , intranasally and ocularly inoculated with 10 ml of viral solution containing 10 plaque - forming units ( pfu ) of cdv kochio1a . cdv kochio1a was isolated from a dead masked palm civet from kochi prefecture , japan , in 2008 . vn test : vn test to kdk-1 was performed by 75% plaque - reduction neutralization test ( prnt75 ) using our established cell line , crfk / cslam . diluted sera were mixed with equal volumes of virus solution containing 100 pfu of kdk-1 , followed by incubation at 37c for 1 then , 50 l of mixtures were added to each well of 24-well plates ( sumilon , tokyo , japan ) containing subconfluent crfk / cslam , and the plate was incubated at 37c for 1 hr , washed twice with dmem without fcs and overlaid with dmem containing 0.8% agarose and 7% fcs . animal samples : between 2006 and 2012 , a total of 1,686 serum samples were collected from wild animals captured in wakayama prefecture , under permission from the governor . wild animals included 1,261 raccoons ( procyon lotor ) , 317 raccoon dogs ( nyctereutes procyonoides ) , 23 badgers ( meles meles ) , one weasel ( mustela itatsi ) , one japanese marten ( martes melampus ) , three korean yellow weasel ( mustela sibirica ) , one red fox ( vulpes vulpes ) , six sika deer ( cervus nippon ) , 72 wild boars ( sus scrofa ) and one alley cat ( felis silvestris catus ) . tissue swabs and fecal samples were collected from two raccoon dogs and one fox for virus isolation . sequence analysis of hemagglutinin ( h ) gene : rna was extracted from virus - infected cells or tissue samples using qiagen rneasy mini kit ( qiagen , hilden , germany ) or qiagen viral rna mini kit ( qiagen ) , respectively , and reverse - transcription ( rt ) was performed with random 9-mer primer using takara rna la pcrtm kit ( amv ) ver.1.1 ( takara , otsu , japan ) at 30c for 10 min , 42c for 30 min and 70c for 15 min . the amplified products were purified using qiaquick pcr purification kit ( qiagen ) , and nucleotide sequences were directly determined using 3130 genetic analyzer ( applied biosystem , carlsbad , ca , u.s.a . ) homology search and phylogenetic analysis : homologies among strains were analyzed using the genetyx ver.8 ( genetyx corporation , tokyo , japan ) , and phylogenetic trees were constructed by the neighbor - joining method using mega5.0 software on the basis of the amino acid pairwise distance . next , wells were washed 3 times with pbs - t and incubated with 100 l per well of peroxidase - conjugated sheep anti - dog igg or goat anti - dog igm antibody ( bethyl , montgomery , tx , u.s.a . ) elisa using protein a / g as a secondary antibody : the antigen was diluted to 5 g / ml with an adsorption buffer , and 100 l was added per well into 96-well microplates . next , the wells were washed three times with pbs - t and incubated with 100 l per well of peroxidase conjugated purified recomb protein a / g ( thermo fischer scientific , rockford , il , u.s.a . ) sera from dogs experimentally infected with cdv : three dogs ( beagle , female , 3 months old ; narc , japan ) were orally , intranasally and ocularly inoculated with 10 ml of viral solution containing 10 plaque - forming units ( pfu ) of cdv kochio1a . cdv kochio1a was isolated from a dead masked palm civet from kochi prefecture , japan , in 2008 . then , 50 l of mixtures were added to each well of 24-well plates ( sumilon , tokyo , japan ) containing subconfluent crfk / cslam , and the plate was incubated at 37c for 1 hr , washed twice with dmem without fcs and overlaid with dmem containing 0.8% agarose and 7% fcs . cells were fixed with 5% buffered formaldehyde for 1 hr , and agarose layers were removed . animal samples : between 2006 and 2012 , a total of 1,686 serum samples were collected from wild animals captured in wakayama prefecture , under permission from the governor . wild animals included 1,261 raccoons ( procyon lotor ) , 317 raccoon dogs ( nyctereutes procyonoides ) , 23 badgers ( meles meles ) , one weasel ( mustela itatsi ) , one japanese marten ( martes melampus ) , three korean yellow weasel ( mustela sibirica ) , one red fox ( vulpes vulpes ) , six sika deer ( cervus nippon ) , 72 wild boars ( sus scrofa ) and one alley cat ( felis silvestris catus ) . sequence analysis of hemagglutinin ( h ) gene : rna was extracted from virus - infected cells or tissue samples using qiagen rneasy mini kit ( qiagen , hilden , germany ) or qiagen viral rna mini kit ( qiagen ) , respectively , and reverse - transcription ( rt ) was performed with random 9-mer primer using takara rna la pcrtm kit ( amv ) ver.1.1 ( takara , otsu , japan ) at 30c for 10 min , 42c for 30 min and 70c for 15 min . h gene was amplified using primers , cdv - hr ( 5-aga tgg acc tca ggg tat ag-3 ) and cdv - hf ( 5-aac tta ggg ctc agg tag tc-3 ) , at 94c for 2 min and 30 cycles consisting of denaturation at 94c for 30 sec , annealing at 60c for 30 sec and extension at 72c for 3 min , followed by a final extension at 72c for 15 min . the nucleotide sequences were deposited in the dna data bank of japan ( ddbj , table 4 ) . homology search and phylogenetic analysis : homologies among strains were analyzed using the genetyx ver.8 ( genetyx corporation , tokyo , japan ) , and phylogenetic trees were constructed by the neighbor - joining method using mega5.0 software on the basis of the amino acid pairwise distance . although the vn test has been the standard assay to detect the cdv - specific antibodies in serum , it requires a large volume of serum , a special facility ( biosafety level-2 ) and a high level of technical skill . in contrast , elisa does not use live cdv and requires only a small amount of sample , and the procedure is simple . therefore , to detect antibodies against cdv in many mammalians , we established a new elisa using peroxidase - conjugated protein a / g ( thermo scientific , waltham , ma , u.s.a . ) as a second antibody and block ace ( dainippon pharmaceutical , osaka , japan ) as antibody blocking and dilution agent . at first , an elisa using protein a / g was performed with sera collected from dogs experimentally infected with cdv . this study showed that elisa and vn titer results were almost similar ( fig . three dogs ( nos.13 ) were experimentally infected with cdv kochio1a , and sera were sequentially collected . dog sera were diluted to 1:100 , and anti - dog igg ( open circle ) , igm ( open square ) and protein a / g ( black circle ) were used as secondary antibodies . elisa using either anti - igg or anti - igm antibody showed different results ; igg antibody gradually increased from day 1227 , and igm antibody peaked on day 15 after infection . on the other hand , the anti - cdv antibody detected by protein a / g peaked on day 15 and then increased from day 2127 . a mixture of both igg- and igm - antibodies was detected by protein a / g . three dogs ( nos.13 ) were experimentally infected with cdv kochio1a , and sera were sequentially collected . dog sera were diluted to 1:100 , and anti - dog igg ( open circle ) , igm ( open square ) and protein a / g ( black circle ) were used as secondary antibodies . to confirm whether elisa using protein a / g is reliable for many mammalian species , results were compared with those obtained by the vn test . the results of elisa were correlated with those of the vn tests in raccoons and raccoon dogs ( fig . values in elisa and titers of virus - neutralizing antibody in raccoons and raccoon dogs . 803 and 326 sera of raccoons ( a ) and raccoon dogs ( b ) , respectively , were analyzed by elisa and prnt75 . ) in 268 raccoon dogs , which were cdv negative by the vn test , average and s.d . was applied , and the cut - off values for elisa were 0.309 and 0.491 for raccoons and raccoon dogs , respectively . in comparison with the result of vn test , specificity and sensitivity of this elisa for raccoons were 98.3% and 73.9% , respectively , and those for raccoon dogs were 98.9% and 79.3% , respectively . values in elisa and titers of virus - neutralizing antibody in raccoons and raccoon dogs . 803 and 326 sera of raccoons ( a ) and raccoon dogs ( b ) , respectively , were analyzed by elisa and prnt75 . in a serological survey of cdv infection in raccoons , raccoon dogs and other wild animals , cut - off values of elisa were tentatively determined to be 0.309 , 0.491 and 0.5 , respectively . the results indicated that 10.3% of raccoons , 13.2% of raccoon dogs , 18% of wild boars , 9% of badgers , one marten , one siberian weasel and 2 sika deer were sero - positive for cdv ( tables 1table 1.seroprevalence of cdv infection in raccoonsplaces2006200720082009201020112012totaltanabeno . to examine whether these old hemolytic sera were suitable for elisa , further elisas the results obtained indicated that many of the sera samples were positive for the jev antibody ( data not shown ) . we previously reported the cdv epidemic among wild mammalians around tanabe city in wakayama prefecture in 20072008 using the vn test . in this study , we detected the anti - cdv antibodies by elisa using protein a / g as described above ( tables 1 and 2 , fig . the positive rate of cdv infection in raccoons ( solid circle ) and raccoon dogs ( open circle ) is plotted . ) in 2006 , most raccoons did not possess the anti - cdv antibody ( 1.4% ) , but 62.5% of raccoons possessed the anti - cdv antibody in 2007 during the cdv epidemic . after the epidemic , 28.4% in 2008 , 7.8% in 2009 , 4.6% in 2010 , 11.4% in 2011 and 5.1% in 2012 were cdv positive ( table 1 , fig . the age of raccoons ( calculated by the rings of teeth ) indicated that many cdv - positive raccoons captured between 2009 and 2012 were born before the epidemic ( date not shown ) . change in seroprevalence of cdv infection among raccoons and raccoon dogs in tanabe city . the positive rate of cdv infection in raccoons ( solid circle ) and raccoon dogs ( open circle ) is plotted . on the other hand , 0% , 6.3% and 9.1% of raccoon dogs in tanabe city had anti - cdv antibodies in 2009 , 2010 and 2011 , respectively ( table 2 ) . however , in 2012 , 19 raccoon dogs ( 30.6% ) were infected with cdv ( fig . 3 ) , and the virus was also isolated from one diseased raccoon dog in tanabe city ( table 4table 4.viruses isolated or detected from wild animals in wakayama prefecturestrainanimal speciesdate of deathsexweight ( kg)virus isolationaccession no. tanabe city is surrounded by several other towns , including ryujin . before the cdv epidemic , one raccoon ( 16.7% ) was cdv positive , but the number of seropositives was less than 10% after 2010 . in raccoon dogs in ryujin , the seroprevalence of cdv infection was approximately 10% during 20092011 , although seven raccoon dogs ( 70% ) became positive for cdv infection in 2012 ( table 2 ) . two cdv genomes were detected by rt - pcr , one from a dead fox and the other from a dead raccoon dog , in 2010 and 2011 , respectively ( table 4 ) . in this study , an elisa using horseradish peroxidase - conjugated protein a / g was developed to detect the cdv antibody in various mammalian species . comparing the vn test and elisa , it was confirmed that the established elisa was available for at least dogs , raccoons and raccoon dogs . because these hemolytic blood samples were not available for the vn test the results indicated that 2 raccoons ( 2.7% ) were cdv positive . to examine whether antibodies in the hemolytic blood samples were damaged , elisa for jev was also performed , because many raccoons in this area were seropositive for jev . the results showed that many raccoons were positive for jev , indicating that antibodies in these hemolytic blood samples function well enough for elisa . the cdv epidemic among wild mammalians in and around tanabe city in wakayama prefecture in 20072008 resulted in a widespread cdv infection of many species , particularly raccoons . in this study , the seroprevalence of cdv infection before and after the cdv epidemic was examined . in tanabe city , 62.5% and 28.4% of raccoons became positive in 2007 and 2008 , respectively , but less than 10% of raccoons were positive for cdv in 2006 and after 2009 . these results indicated that most raccoons were infected with cdv during this epidemic and probably not after this epidemic . [ 7 , 12 , 20 , 22 ] , raccoons may also spread cdv to other animals in japan . in 20072008 , very few raccoon dogs were captured in wakayama prefecture , suggesting that cdv may have spread among raccoon dogs and killed many of them because of their high sensitivity to cdv . although the prevalence of cdv - positive raccoon dogs was less than 10% after the epidemic , 22.2% of raccoon dogs became positive for cdv in 2012 . in addition , cdv was detected in two raccoon dogs in 2011 and 2012 . in particular , these results indicated that cdv was maintained among a population of raccoon dogs , and a small epidemic without transmission to raccoons occurred in tanabe and ryujin cities in 2012 . in this study , it seems likely that raccoon dogs play a crucial role in maintenance of cdv in this area , and the epidemic in 2007 was enhanced by transmission of cdv to raccoons , resulting in many wild animals , including the sika deer and wild boar , being infected with cdv .

the vertebrate retina receives efferent inputs from different parts of the central nervous system but we still do not understand how these regulate visual processing ( ramon y cajal , 1894 ; reprant et al . , 1989 ) . in teleosts , the main source of retinopetal fibers is the terminal nerve ( tn ) , which receives dense afferents from the olfactory bulb and in turn projects gnrh- and fmrfamide - containing fibers to the retina ( springer , 1983 ; zucker and dowling , 1987 ; demski , 1993 ; yamamoto and ito , 2000 ; reprant et al . , 2007 ) . the tn is tonically active , with a firing frequency that changes according to the physiological conditions of the animal , including arousal , motivational state , hormonal milieu , and glutamatergic inputs from various sensory systems ( abe and oka , 2006 ; wang et al . , 2011 ) . together , the pathways linking the olfactory bulb to the retina through the tn are known as the olfacto - retinal circuit ( orc ) . behavioral assays examining visual threshold have shown that stimulation of the olfactory bulb by food - related amino acids induces an increase in luminance sensitivity through activation of the orc ( maaswinkel and li , 2003 ; behrens and wagner , 2004 ; li and maaswinkel , 2007 ) . it has been suggested that an olfactory stimulus alters the processing of visual signals by decreasing the concentration of dopamine in the retina ( huang et al . , 2005 ) . the sole source of dopamine in the retina of teleosts is a specialized class of amacrine cell , the interplexiform cells ( ipcs ) , which are the target of the tn ( umino and dowling , 1991 ) . li and dowling ( 2000a ) have shown that zebrafish affected by the night blindness b mutation ( nbb ) , which provokes a progressive reduction in the number of ipcs , exhibit a 23 log unit decrease in luminance sensitivity and a profound loss of signals derived from rods . dopamine ( da ) released from ipcs has a number of actions on the retinal circuit , which together act to enhance cone - mediated signals under bright conditions . in the outer retina , dopamine decreases electrical coupling between rods and cones ( ribelayga et al . , 2008 ) , while inhibiting voltage - gated calcium currents in rods and boosting calcium currents in cones ( stella and thoreson , 2000 ) . dopamine also inhibits electrical coupling between horizontal cells and increases their sensitivity to glutamate , resulting in less powerful negative feedback to cones ( knapp and dowling , 1987 ; devries and schwartz , 1989 ; mcmahon , 1994 ) . in the inner retina , actions on bipolar cells and retinal ganglion cells ( rgcs ) have also been reported , but their roles in altering retinal processing under different lighting conditions are not clearly established ( jensen and daw , 1984 ; jensen , 1992 ; heidelberger and matthews , 1994 ; li and dowling , 2000b ; ribelayga et al . , 2002 ) . how might the actions of dopamine underlie the modulation of retinal processing by an olfactory stimulus ? one of the difficulties in studying a multisensory circuit is the need to conduct experiments in vivo in order to maintain the link between the different sensory systems . in this study , we take advantage of zebrafish expressing genetically encoded calcium reporters in the synaptic terminals of bipolar cells or dendrites of rgcs ( dreosti et al . , 2009 ; odermatt et al . , these fish allow the visual signal to be monitored as it is transmitted to the inner retina and rgcs providing the output from this circuit . by imaging signals through all layers of the inner retina , we have observed activity at the origins of the on and off channels that encode a change in light intensity with signals of opposite polarity ( schiller et al . , 1986 ) . here , we demonstrate that an olfactory stimulus reduces the gain but increases the sensitivity with which off bipolar cells transmit signals encoding luminance and contrast . pharmacological manipulations in vivo demonstrated that olfactory stimuli regulate the presynaptic calcium signal of bipolar cells by reducing the activity of d1 dopamine receptors , and electrophysiology in isolated bipolar cells demonstrated that activation of endogenous dopamine receptors potentiates voltage - dependent calcium channels that control synaptic transmission . together , these results indicate that the orc acts through the neuromodulator dopamine to regulate synaptic transmission through the off channel in the retina . transmission of the visual signal through the on and off pathways in the retina was assessed in transgenic zebrafish expressing sygcamp2 under the ribeye promoter ( dreosti et al . , 2009 ) , allowing synaptic activity to be monitored across the population of bipolar cells projecting to all layers of the inner plexiform layer ( ipl ) ( figure 1a ) . first , we investigated the effects of olfactory stimulation on the response to changes in the luminance of full - field stimuli . all measurements were carried out between 9:00 and 11:00 a.m. , when behavioral experiments have demonstrated that the orc is most effective in modulating visual sensitivity ( maaswinkel and li , 2003 ) . figure 1b shows responses of an individual off terminal to light steps of different intensity , before ( dark red ) and after ( light red ) the addition of 1 mm methionine to the medium surrounding the fish . in off terminals , there was a decrease in the maximum amplitude of the response , which we term a decrease in gain . additionally , off terminals began to respond at lower intensities , which we term an increase in sensitivity . in contrast , the large majority of on bipolar cells were unaffected by olfactory stimulation , and an individual example is shown in figure 1c . collected results from 84 off terminals from seven fish are shown in figures 1d1 g . methionine reduced the response to the brightest step of light by an average of 36.6% 8.2% ( p < 0.0001 ) , and this effect was evident across the population of off terminals ( figure 1e ) . in parallel ( figure 1f ) , methionine increased the luminance sensitivity of off terminals by 0.5 log units , assessed by the lowest light level eliciting responses > 3 sd of the baseline noise . a similar shift could be assessed by fitting the intensity - response measurements with a hill function ( figure 1 g ) and estimating i1/2 , the intensity generating a half - maximal response ( experimental procedures ) . the increase in luminance sensitivity observed at the synaptic output of bipolar cells was quantitatively similar to the increase observed previously in ganglion cell recordings and behaviorally following olfactory stimulation ( maaswinkel and li , 2003 ; huang et al . , 2005 ) . the actions of methionine were almost exclusively on the off pathway . the large majority of on synapses in a population of 116 were not affected , either in terms of response amplitude or sensitivity ( figures 1h1k ) . the exception was a small but distinct fraction of terminals ( 9% ) , in which the amplitude of the presynaptic calcium signal was increased by a factor of at least three ( arrow in figure 1i and described further in figures s1a and s1b available online ) . this subset of on terminals was not distinguishable from others in terms of size or position in the ipl and is therefore unlikely to reflect a difference between cone - driven and mixed rod - cone bipolar cells . no significant changes in on or off responses were observed over the same time window in the absence of methionine . although behavioral experiments showed olfactory stimulation to be ineffective in the afternoon ( maaswinkel and li , 2003 ) , we found that methionine administration produced a qualitatively similar but significantly smaller modulation of responses through the off pathway tested between 12:00 and 3:30 p.m. ( figures s1c s1f ) . many retinal neurons signal fluctuations in light intensity at frequencies up to 20 hz . to test the effects of an olfactory stimulus on the signaling of temporal contrast , we modulated full - field stimuli at 5 hz and measured the sygcamp2 signal ( 5 fish , n = 122 terminals ) . figure 2 shows that the most obvious effect of an increase in contrast was a steady offset in the sygcamp2 signal , reflecting a net accumulation of calcium . a change in the sygcamp2 signal in the absence of a change in the mean the relation between the amplitude of the steady sygcamp2 signal ( a ) and contrast ( c ) is shown in figure 2b : it could be described by a simple power function of the form a = k c , with = 1.8 0.1 under control conditions . stimulation with methionine reduced the amplitude of the rectifying response at all contrasts above 20% ( figure 2a ) . furthermore , methionine increased the exponent of the power function to 2.9 0.2 ( p < 0.0001 ) . this renders off terminals more sensitive to higher contrasts at the expense of lower contrasts , e.g. , a change in temporal contrast from 80% to 90% caused a change of 23.5% f / f0 in control versus 40% f / f0 in methionine . olfactory stimulation did not , however , affect the responses of on bipolar cells ( figures 2c and 2d ) . we also observed a small subset of on bipolar cells in which the dc presynaptic calcium levels were reduced by stimulation at 5 hz , and these were also unaffected by application of methionine ( figures s2a and s2b ) . the inhibition of presynaptic calcium signals by olfactory stimulation was also a function of stimulus frequency . we tested the responses of off terminals to frequencies between 0.2 and 25 hz at 90% contrast ( 6 fish , n = 96 terminals ) and found that methionine reduced the amplitude of the sygcamp2 signal elicited by stimuli below 10 hz . again together , the results in figures 1 , 2 , and s2 indicate that olfactory stimulation alters the processing of visual signals in the retina by two distinct actions on off bipolar cells : a suppression of the presynaptic calcium signal , resulting in a reduction in gain and an increase in luminance sensitivity . the large majority of on bipolar cells were unaffected by this form of cross - modal regulation . having observed an action of olfactory stimulation on the visual signal as it is transmitted by bipolar cells , we investigated how far the responses of postsynaptic ganglion cells were also affected . to monitor signals across large populations of neurons in vivo , we made a line of zebrafish expressing the calcium reporter gcamp3.5 under the eno2 promoter , which drives expression in rgcs ( bai et al . step changes in luminance were a relatively ineffective stimulus for rgcs , so we examined the effects of an olfactory stimulus on full - field stimuli modulated at 5 hz . the advantage of this in vivo imaging approach over electrophysiology is that it allows stimulation of the olfactory system while observing activity across a large population of rgcs . responses from rgc dendrites were classified as off ( figures 3b and 3c ) , on ( figures 3d and 3e ) , or on - off ( figures 3f and 3 g ) , according to the responses to steps of light ( figure s3 ) . methionine induced a reduction in gain of off and on - off rgcs at contrasts of 50% and above , without an appreciable effect on on rgcs . these results are consistent with the reduced gain of responses to contrast observed in off bipolar cell terminals , but not on , following application of methionine ( figure 2 ) . they also confirm that the actions of the orc are evident in the retinal output , as previously demonstrated by maaswinkel and li ( 2003 ) and huang et al . existing evidence suggests that a key signal is dopamine released by ipcs ( umino and dowling , 1991 ; huang et al . , 2005 ) . to investigate how dopaminergic signaling might be involved in modulating synaptic activity of bipolar cells , we injected agonists or antagonists of dopamine receptors into the anterior chamber of one eye of a fish , with a parallel sham injection into the other eye acting as a control . the first manipulation was to activate dopamine receptors by injecting the agonist [ 3h ] 2-amino-6,7-dihydroxy 1,2,3,4-tetrahydronapthalene ( adtn ) at an estimated concentration of 0.2 m ( see experimental procedures ) . in off terminals , adtn increased the amplitude of sygcamp2 responses to all but the brightest lights and luminance sensitivity ( i1/2 ) increased by a factor of 420 ( figures 4a and 4b ; n = 92 terminals ) . in on terminals , adtn increased the amplitude of the sygcamp2 response to bright lights by 108% and increased luminance sensitivity by a factor of 15 ( figures 4c and 4d ) . strong activation of dopamine receptors therefore potentiated presynaptic calcium signals in both on and off bipolar cells , an effect opposite to an olfactory stimulus . further , application of methionine in the presence of 0.2 m adtn no longer depressed signals through off bipolar cells ( figures 4a and 4b ) . both these observations are consistent with the idea that an olfactory stimulus modulates retinal function by decreasing dopamine release . the second manipulation was to antagonize the action of endogenous dopamine by injection of 100 nm sch 23390 ( 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol ) , a selective antagonist of dopamine d1 receptors ( mora - ferrer and neumeyer , 1996 ; bourne , 2001 ) . sch 23390 injection resulted in a complete impairment of luminance signaling through off bipolar cells ( figures 5a and 5b ) . in contrast , the maximum amplitude of the response in on bipolar cells was not significantly affected , although the light sensitivity ( i1/2 ) was increased by a factor of 3.8 ( figures 5c and 5d ) . antagonizing d1 receptors therefore caused effects qualitatively similar to an olfactory stimulus : a selective decrease in the gain of signaling through the off pathway ( cf . , we used the antagonist sulpiride at a concentration of 2 m ( lin and yazulla , 1994 ; mora - ferrer and gangluff , 2000 ) . first , the sensitivity increased sufficiently to reduce threshold by 2 log units , likely reflecting the potentiation of rod inputs ( ribelayga et al . , 2008 ) . second , the luminance - response relation did not simply rise monotonically , but instead passed through a maximum ( figures 5e and 5f ) . despite these changes in circuit function , the maximum response to luminance was reduced by 29% 5.6% when methionine was applied after injection of sulpiride , an effect similar to that of olfactory stimulation under control conditions ( figures 5e and 5 g ) . sulpiride also increased sensitivity of signals through on bipolar cells ( 0.85 log units ) , but methionine had no effect on the amplitude of these responses ( figures 5h5j ) . an olfactory stimulus therefore continued to cause a selective reduction of responses through the off pathway when activation of d2 receptors was blocked . together , the results in figures 4 and 5 indicate that cross - modal regulation of retinal function depends primarily on the activity of d1 receptors . to test further the idea that activation of the orc acts by decreasing dopamine level in the retina , we attempted to prevent these changes without interfering with the activity of dopamine receptors . our strategy was to inject vanoxerine ( gbr 12909 ; 2 m ) , a potent and specific blocker of the transporters involved in dopamine reuptake from extracellular space and into secretory vesicles ( reith et al . vanoxerine administration has been reported to result in a small but steady increase in extracellular dopamine concentration , followed by a persistent clamp once both uptake and release are blocked ( rothman et al . , 1991 ; lima et al . , 1994 ; reith et al . , 1994 ; schlicker et al . first , the luminance sensitivity of off terminals was increased by a factor of 26 ( figures 6a and 6b ) and of on terminals by a factor of 2 ( figures 6d and 6e ) . notably , these increases in sensitivity were much smaller than those caused by the dopamine receptor agonist adtn ( figures 6b and 6e ) , indicating that increases in dopamine levels were relatively small and not sufficient to saturate dopamine receptors . the second action of vanoxerine was to prevent the application of methionine from modulating luminance signaling through off bipolar cells ( figures 6a and 6c ) , consistent with the idea that this modulation occurs through changes in dopamine levels . the manipulations of dopamine receptors and transporters shown in figures 4 , 5 , and 6 support the idea that olfactory stimulation modulates synaptic transmission from off bipolar cells by reducing dopamine levels and d1 dopamine receptor activity . what are the cellular mechanisms by which dopamine modulates the visual signal transmitted to the inner retina ? in the outer retina of fish and mammals , dopamine acts through d1 receptors to uncouple horizontal cells providing negative feedback to the synaptic terminals of photoreceptors ( dowling , 1991 ) , but this seems an unlikely mechanism for the selective modulation of transmission through off bipolar cells given that these diverge from the on pathway downstream of photoreceptor output ( schiller et al . , 1986 ) . we therefore investigated the possibility that dopamine might also act directly on bipolar cells to modulate synaptic calcium signals . mixed rod - cone ( mb1 ) bipolar cells from the retina of goldfish were isolated for electrophysiological recording ( burrone and lagnado , 1997 ) . in these neurons , voltage - dependent calcium channels are l - type and localized to the synaptic terminal ( burrone and lagnado , 1997 ) . in current - clamp configuration , using a standard intracellular solution , addition of 10 m dopamine depolarized bipolar cells by an average of 30.7 1.5 mv , indicating activation of a net inward current ( n = 9 ; figures 7a and 7b ) . the depolarization was completely reversed by blocking voltage - dependent ca channels with 100 m cadmium ( catterall et al . , 2003 ) , indicating that dopamine potentiates the calcium conductance ( n = 6 ) . the mb1 bipolar cell stands out in a preparation of dissociated retinal neurons because of its large terminal . of the bipolar cells with small terminals , off outnumber on by 3:1 ( odermatt et al . we also made recordings from the cell bodies of bipolar cell with small terminals , and in all three cases dopamine caused a depolarization of 15 mv . it therefore seems very likely that dopamine also acts to enhance calcium currents in off bipolar cells . heidelberger and matthews ( 1994 ) also observed that dopamine potentiated calcium influx in all morphological types of bipolar cell that they tested . to investigate the actions of dopamine on the calcium conductance more directly , we made voltage - clamp recordings using an intracellular solution designed to block potassium channels . figure 7c shows the calcium currents elicited by voltage steps from 70 mv to 40 mv and 20 mv in a single cell , and figure 7d shows an example of the current - voltage relation around the threshold for activation of the calcium current , approximately 43 mv ( burrone and lagnado , 1997 ) . to quantify changes in the calcium conductance over a number of cells , we measured the amplitude of the tail current 0.5 ms after a voltage step returning to 70 mv ( dashed red line in figure 7e ) . averaged conductance - voltage ( g - v ) relations before and after addition of 10 m dopamine are shown in figure 7f , with conductance values normalized to the maximum in the absence of dopamine ( n = 6 cells ) . the g - v relation could be described by a boltzmann function ( see experimental procedures ) . addition of 10 m dopamine increased gmax by 44% 11% and shifted v1/2 from 14.2 0.4 mv to 16.5 0.4 mv ( figure 7f , p = 0.002 ) . the 2.3 mv shift in v1/2 to lower membrane potentials is significant in the context of the voltage signals that bipolar cells generate in response to light ( baden et al . , 2011 ) , which are just a few millivolts in amplitude and span the voltage range at which l - type calcium channels begin to activate . around this threshold , dopamine potentiated presynaptic calcium currents by a factor averaging 1.9 ( figure 7f ) . these results demonstrate that dopamine can act directly on bipolar cells to increase the magnitude of the presynaptic ca current that controls transmission of the visual signal . it seems likely that this action makes a significant contribution to the profound increase in the gain of luminance signals observed in vivo in the presence of the dopamine receptor agonist adtn ( figure 4 ) , as well as the decrease in gain in the presence of the antagonist sch 23390 ( figure 5 ) . if an olfactory stimulus acts to lower dopamine levels and therefore inhibits activation of presynaptic calcium channels , one might expect to observe a decrease in the basal calcium concentration in bipolar cells in darkness , with this effect being most obvious in off cells resting at more depolarized potentials . we therefore compared resting sygcamp2 signals in bc terminals before and after the bath application of methionine ( 233 on and 211 off from nine fish ; figure s4 ) . methionine induced a statistically significant reduction in sygcamp2 fluorescence in off terminals ( median = 10.9% , p < 0.01 ) but not on ( median = 0.2% , not significant ) , providing further support for the idea that inhibition of presynaptic calcium channels is one of the mechanisms by which an olfactory stimulus reduces the gain of signaling through off bipolar cells . the vertebrate retina receives centrifugal input from a variety of brain regions , depending on the species ( behrens and wagner , 2004 ) . the olfacto - retinal circuit in fish is a good example of such cross - modal interactions and provides the opportunity to investigate the cellular mechanisms that regulate processing in the early visual system . by monitoring calcium signals in vivo , we find that an olfactory stimulus reduces the gain with which changes in luminance or temporal contrast are transmitted through the off pathway , while also increasing sensitivity at lower light levels ( figures 1 , 2 , and 3 ) . the results demonstrate that the calcium signal controlling neurotransmission from bipolar cells is a key site for regulating the flow of the visual information . the observed modulation of presynaptic calcium responses is likely to contribute to the increase in luminance sensitivity observed behaviorally when the orc circuit is activated ( maaswinkel and li , 2003 ; huang et al . , 2005 ) . the chemical signal coordinating these changes in retinal performance has been suggested to be a reduction in dopamine release . strong evidence for this idea is provided by the demonstration that a blocker of dopamine release and reuptake suppresses the change in synaptic gain and sensitivity normally caused by an olfactory stimulus ( figure 6 ) . manipulations of dopamine receptor activity in vivo are also consistent with this mechanism ( figures 4 , 5 , and 6 ) and , in particular , for a key role of d1 receptors ( figures 5b and 5d ) . finally , we demonstrate that dopamine regulates the activity of voltage - dependent calcium channels in the synaptic terminals of bipolar cells , providing a direct mechanism for regulating the gain of the visual signal ( figure 7 ) . of course , these results do not rule out the possibility that there are other sites at which orc also regulates the retinal circuit . an overview of changes in the amplitude of the calcium signal through on and off bipolar cell terminals is shown in figure 8 . the response is quantified as the relative change in sygcamp2 fluorescence caused by a bright step of light applied from darkness , and the various experimental conditions are ordered according to the expected level of dopamine activity , with the measurement in 100 nm of the d1 dopamine receptor antagonist sch 23390 at one extreme and in 200 nm of the agonist adtn at the other . this comparison reveals a fundamental difference in the sensitivity of the on and off pathways to changes in retinal dopamine levels . under control conditions , luminance signaling through the off pathway is operating at its maximum gain ( i.e. , similar to that measured in adtn ) , whereas signaling through the on pathway is operating at its minimum gain ( measured in sch 23390 ) . thus , although an olfactory stimulus that results in decreased dopamine levels may be expected to decrease the gain of signals through the off pathway ( figures 1 and 8a ) , it is not expected to suppress synaptic calcium signals in on bipolar cells ( figures 1 and 8b ) . the mechanism for the differential effects of dopamine on the on and off pathways is still not clear . on and off bipolar cells both express d1 receptors but not d2 ( fan and yazulla , 2005 ; yu and li , 2005 ) . d1 receptors act through gs proteins which couple to adenylyl cyclase to increase camp and direct activation of adenylyl cyclase by forskolin also increases bipolar ca responses ( heidelberger and matthews , 1994 ) . a possible explanation for the contrasting effect in on versus off could be differential sensitivity of the cav channels to camp that may reflect which cav channels underlie the response ( pan et al . , 2001 ; logiudice et al . , an alternative possibility is that the on and off channels are regulated by a second neuromodulator , which interacts with dopamine pathways . for instance , iuvone and gan ( 1995 ) have demonstrated that activation of mt2 melatonin receptors antagonizes signaling through d1 dopamine receptors in bipolar cells by inhibiting camp synthesis through a gi protein , and wiechmann and sherry ( 2012 ) have found that mt2 melatonin receptors are localized to off but not on bipolar cells in xenopus laevis . the fast decrease in melatonin concentration that occurs after dawn might therefore act to enhance selectively the sensitivity of off bipolar cells to variations in dopamine levels . we did see a small population of on bipolar cell terminals ( 9% ) strongly potentiated by olfactory stimulation ( figures 1i , s1a , and s1b ) . might this reflect differences in the mechanism by which glutamate released from photoreceptors act on different types of on bipolar cells ? in zebrafish , some on bipolar cells respond through metabotropic glutamate receptors and others through a glutamate transporter with a large chloride conductance ( connaughton and nelson , 2000 ; nelson and connaughton , 2004 ) . although the former mechanism predominates in mixed rod - cone bipolar cells with large terminals , the latter occurs in cone bipolar cells with smaller terminals . we tested , therefore , if there was any relationship between the size of on terminals and their response to methionine , but did not find any ; i.e. , the size distribution of on terminals responding to methionine was very similar to those that did not , both varying between 0.6 m and 5 m in radius . we also investigated whether there might be any relation between the location of on terminals within the ipl and their response to methionine and again there was not . as a consequence , at present we do not have elements to consider this as a separate subpopulation of on bipolar cells . our results are consistent with the hypothesis that odor stimulation reduces the conductance and shifts the v1/2 of cav channels in bipolar terminals , with dopamine being the key mediator . this mechanism is able to explain several of the observed effects of olfactory stimulation on the transmission of visual information through bipolar cells . for example , the decreased conductance and shift in the activation of the calcium channels will lower ca influx for small depolarizations such as low contrasts , nevertheless larger depolarizations , such as high contrasts , will still be effective . this manifests in a more nonlinear contrast response function ( figure 2b ) with greater sensitivity for higher contrasts . the decrease in the ca channel maximum conductance also explains the lower gain seen at maximum luminance ( figure 1d ) . this highlights the presynaptic terminal of bipolar cells as a key site for regulating the transmission of visual signals through the retina . as well as the dramatic gain reduction as the expected effects of reduced dopamine will shift the cav activation to more depolarized potentials , it is unlikely to explain the increased luminance sensitivity . however , d1 receptors do enhance glutamate - gated ionic channels in off bipolar cells ( maguire and werblin , 1994 ) . when d1 receptors are activated , ionotropic glutamate receptors generate enhanced current that will result in off bipolar cells being less sensitive to small decreases in glutamate concentration ; a similar phenomenon has been described in horizontal cells ( knapp and dowling , 1987 ) . the olfacto - retinal circuit endows the vertebrate visual system with the ability to quickly reduce the gain and increase the sensitivity of the retina in the presence of food , independently of changes in mean luminance . a behavior that is likely to be related to this process has recently been described by stephenson et al . ( 2011 ) , who found that zebrafish show a preference for darker areas in their environment when background levels of light are low , and brighter areas when background light levels are high . an olfactory stimulus applied in low background would then mimick the effects of light adaptation by encouraging fish to explore brighter areas . the reduction in gain of bipolar cell synapses transmitting the visual signal to the inner retina ( figure 1 ) , as well as the increase in sensitivity to high contrast ( figure 2 ) , is likely to be one of the mechanisms by which an olfactory stimulus allows the visual system of the zebrafish to operate in brighter areas . in the future , it will be interesting to investigate the behavioral consequences of a selective decrease in gain of the off pathway . certainly it would be expected to help the retina avoid saturation under bright conditions , but then so would a decrease in gain through the on pathway . a possible explanation for the selective control of the off pathway might lie in the recent study of ratliff et al . ( 2010 ) who asked why off rgcs are so much more numerous than ons in most retinas ( including zebrafish ) . they found that natural scenes contain an excess of negative spatial contrasts over positive , leading to the suggestion that the excess of off rgcs is a structural adaptation of the retina to the excess of darkness in natural scenes . in zebrafish , off bipolar cells outnumber ons by a ratio of 3:1 ( odermatt et al . , 2012 ) , so it may be that a decrease in the gain of the off pathway is the most important adaptation required to process negative contrasts at higher mean light levels . another possible explanation could be linked to the differential role proposed by burgess et al . ( 2010 ) for the two systems , being the on pathway mainly involved in appetitive behaviors and the off pathway more implicated in escape responses . in this perspective , the observed food odor - induced inhibition of the off would suppress escape responses , thus favoring appetitive behaviors . the re - tuning of retinal processing by a food - related olfactory stimulus is likely to be relevant to different aspects of zebrafish behavior , but especially hunting and prey - capture . the observed increase in the gain of the on channel relative to the off is expected to make the retina more sensitive to regions of positive contrast , such as bright spots appearing when sunlight reflects off small prey . bright spots are an effective stimulus for eliciting prey - capture behavior in a virtual reality assay ( bianco et al . , 2011 ) , and this may provide an experimental context in which to study the behavioral consequences of olfactory - visual integration . all procedures were carried out according to the uk animals ( scientific procedures ) act 1986 and approved by the uk home office . we made transgenic zebrafish ( danio rerio ) expressing the synaptically localized fluorescent calcium reporter sygcamp2.0 under the ribeye - a promoter , as in dreosti et al . ( 2012 ) , or the calcium reporter gcamp3.5 under the eno2 promoter , as in bai et al . sygcamp2 and gcamp3.5 zebrafish were kept at a 14:10 hr light : dark cycle and bred naturally . larvae were grown in 200 m 1-phenyl-2-thiourea ( sigma ) from 28 hr postfertilization to inhibit melanin formation ( karlsson et al . , 2001 ) . whole zebrafish larvae ( 811 days postfertilization [ dpf ] ) were immobilized in 2.5% low melting point agarose ( biogene ) on a glass coverslip and submersed in e2 embryo medium ( nusslein - volhard and dahm , 2001 ) . bipolar cell terminals were imaged in vivo using a custom - built two - photon microscope equipped with a mode - locked chameleon titanium - sapphire laser tuned to 915 nm ( coherent ) with an olympus lumplanfi 40 water immersion objective ( n.a . emitted fluorescence was captured through both the objective and a substage oil condenser , filtered through a hq 520/60 m-2p gfp emission filter ( chroma technology ) and detected by a set of photo - multiplier tubes ( hamamatsu ) . scanning and image acquisition were controlled under scanimage v.3.6 software ( pologruto et al . , full - field light stimuli were delivered by amber leds ( luxeon ) , 590 nm band - passed 10 nm , and controlled in igor pro 4.01 ( wavemetrics ) and time locked to image acquisition . bipolar cell terminal responses were analyzed in terms of light sensitivity ( irradiance ) , contrast sensitivity , and frequency sensitivity . luminance ( irradiance ) sensitivity was assessed by stimulating the dark - adapted fish with a series of flashes ( 4 3 s flashes at 6 s intervals ) at nine different light intensities , ranging between 11 pw / mm and 110 nw / mm with 0.5 log unit steps . maximum light intensity , 110 nw / mm , is equivalent to 3.3 10 photons / mm s. contrast sensitivity was assessed by stimulating the dark - adapted fish with a series of 10 s light oscillations at 5 hz around a constant light level ( 55 nw / mm ) at 10 different levels of contrast , ranging from 10% to 100% of the constant light level . finally , frequency sensitivity was assessed by stimulating the dark - adapted fish with a series of 10 s light oscillations around a constant light level ( 55 nw / mm ) at 90% contrast at 14 different frequencies , ranging from 0.2 to 25 hz . image sequences were acquired at 10 hz ( 256 100 pixels per frame , 1 ms per line ) for the irradiance and contrast experiments and at 40 hz ( 256 25 pixels per frame , 1 ms per line ) for frequency experiments . the stimulation of the olfactory bulb was obtained by bath application of the amino acid methionine ( sigma ) 1 mm , as in maaswinkel and li ( 2003 ) . to manipulate dopamine signaling in the retina we injected neuroactive drugs into the eye . final concentrations of the drugs were calculated by diluting the injected concentration into the free volume of the eye . the volume of a typical 9 dpf old zebrafish eye was assessed by three - dimensional reconstruction of the eye chamber and the lens through two - photon microscopy scanning . the final volume was estimated as the difference between the total eye volume and the volume of the lens core . we calculated a total free volume of 500 m . given a typical injected volume of 10 l , the final dilution factor can be approximated to 1:50 . dopamine receptors were activated by injection of the long - lasting dopamine receptor ligand [ 3h ] 2-amino-6,7-dihydroxy 1,2,3,4-tetrahydronapthalene ( adtn ) ( sigma ) 10 m , as in li and dowling ( 2000b ) . dopamine action on postsynaptic targets was prevented by injection of the strong dopamine d1 receptor antagonist sch 23390 ( sigma ) 2 nm , as in huang et al . ( 2005 ) or the selective dopamine d2 receptor antagonist sulpiride ( sigma ) , as in lin and yazulla ( 1994 ) and mora - ferrer and gangluff ( 2000 ) . finally , the level of dopamine in the circuit was frozen by injection of the dopamine release and reuptake inhibitor vanoxerine ( santa cruz biotechnology ) 2 m , as in schlicker et al . preprocessing was carried out in image j ( national institutes of health ) and consisted of stack registration and kalman stack filter denoising ( filter gain = 0.6 ) . regions of interest ( roi ) extraction , background subtraction , and brightness normalization ( f / f0 ) were performed in igor pro 6.2 and facilitated by sarfia analysis routines ( dorostkar et al . , 2010 ) . the detection of active roi in the ipl was based on the thresholding of the laplacian transform of the two - photon recordings . in this way , responding bipolar cell terminals and active areas of the ganglion cell dendrites were identified in ribeye::sygcamp2 and eno2::gcamp3.5 fish , respectively . the responses to light of bipolar cell terminals and retinal ganglion cell dendrites were characterized according to their response amplitude , i.e. , the variation in fluorescence during stimulation in comparison to baseline ( f / f0 ) . responses to light were plotted in full , as in figure 1b , left , or in stimulus versus amplitude plots ( e.g. , figure 1b , right ) . in the case of traces representing single terminals ( e.g. , figure 1b ) , the error curve ( gray shadow in figure 1b ) represents the sem of the four trials employed to assess the terminal responsiveness ( see stimulation protocols ) . in the case of traces representing whole populations of terminals ( e.g. , figure 1d ) , the error curve represents the standard error of all the responses employed to generate the final average . as described in the stimulation protocols section , a stimulus could be light intensity , contrast , or frequency . intensity versus amplitude plots were obtained by averaging amplitude values over 300 ms long time windows around the maximum response occurring during the stimulation time ( e.g. , figure 1b , right ) . contrast versus amplitude and frequency versus amplitude plots were obtained by averaging amplitude values over the whole stimulation period ( e.g. , figures 2b and s2d , respectively ) . the intensity versus amplitude plots were fitted with hill curves , in the form a = i /i + i1/2 , a being the response amplitude , i the stimulation intensity , h the hill coefficient , and i1/2 the sensitivity at half maximum , i.e. , the stimulation intensity that elicits half of the maximum response . i1/2 has been used as a metric for the sensitivity of each intensity versus amplitude curve . contrast versus amplitude plots were fitted with power functions , in the form a = k c being a the response amplitude , k a constant , c the stimulation contrast , and the power exponent . the sensitivity shift induced by olfactory stimulation for each individual terminal ( e.g. , figure 1f ) was measured by comparing the values of the lowest light intensity eliciting a statistically significant response before and after methionine administration . the statistical significance of a response was assessed by comparing ( t test ) the average calcium level during light stimulation with a threshold defined as three times the sd of a baseline epoch . the effect of drugs or of olfactory stimulation has been also described in terms of percentage variation in the amplitude of response to the maximum irradiance stimulus ( e.g. , figure 1e ) . goldfish ( carassius auratus ) were dark - adapted for 1 hr and killed by decapitation followed immediately by destruction of the brain and spinal cord under schedule 1 of the uk animals ( scientific procedures ) act 1986 . depolarizing bipolar cells were isolated from the retina of goldfish by enzymatic digestion , using methods described by burrone and lagnado ( 1997 ) . the standard ringer solution contained the following : 110 mm nacl , 2.5 mm cacl2 , 2.5 mm kcl , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ( 260 mosmol l-1 , ph 7.3 ) . the solution in the patch pipette to record voltage membrane in current - clamp experiments contained : 110 mm k - gluconate , 4 mm mgcl2 , 3 mm na2atp , 1 mm na2gtp , 0.5 mm egta , 20 mm hepes , and 10 mm na - phosphocreatine ( 260 mosmol l-1 , ph 7.2 ) . to isolate ca channel currents , the intracellular solution contained 110 mm cs - gluconate , 4 mm mgcl2 , 3 mm na2atp , 1 mm na2gtp , 10 mm tetraethylammonium chloride , 20 mm hepes , 0.5 mm egta , and 10 mm na - phosphocreatine ( 260 mosmol l-1 , ph 7.2 ) . room temperature solutions were superfused via a fast perfusion system ( vc8-s ; ala scientific ) . patch electrodes with 57 m tip resistance were pulled from fire - polished borosilicate glass capillary tubes using a micropipette puller ( sutter instrument ) . voltage - clamp and current - clamp recordings were made in synaptic terminals . in voltage - clamp experiments , the membrane potential was held at 60 mv , and stimuli were delivered by stepping the membrane potential to 10 mv . to construct g / v plots the tail current amplitude measured 0.5 ms after returning to 70 mv was plotted against the preceding voltage step . the voltage dependence of activation was determined from normalized conductance versus voltage curves , which were fitted according to the boltzmann function : g=gmax1+exp(vv1/2k),where g is the normalized conductance , v1/2 is the membrane potential at which activation is half - maximal , and k is the slope factor . signals were recorded using an axopatch 200a amplifier ( molecular devices ) , interfaced with an itc-16 ( heka ) and controlled with pulse control 4.3 running under igor pro 5 ( wavemetrics ) .

summarycross - modal regulation of visual performance by olfactory stimuli begins in the retina , where dopaminergic interneurons receive projections from the olfactory bulb . however , we do not understand how olfactory stimuli alter the processing of visual signals within the retina . we investigated this question by in vivo imaging activity in transgenic zebrafish expressing sygcamp2 in bipolar cell terminals and gcamp3.5 in ganglion cells . the food - related amino acid methionine reduced the gain and increased sensitivity of responses to luminance and contrast transmitted through off bipolar cells but not on . the effects of olfactory stimulus were blocked by inhibiting dopamine uptake and release . activation of dopamine receptors increased the gain of synaptic transmission in vivo and potentiated synaptic calcium currents in isolated bipolar cells . these results indicate that olfactory stimuli alter the sensitivity of the retina through the dopaminergic regulation of presynaptic calcium channels that control the gain of synaptic transmission through off bipolar cells .

Introduction Results Discussion Experimental Procedures

the vertebrate retina receives efferent inputs from different parts of the central nervous system but we still do not understand how these regulate visual processing ( ramon y cajal , 1894 ; reprant et al . in teleosts , the main source of retinopetal fibers is the terminal nerve ( tn ) , which receives dense afferents from the olfactory bulb and in turn projects gnrh- and fmrfamide - containing fibers to the retina ( springer , 1983 ; zucker and dowling , 1987 ; demski , 1993 ; yamamoto and ito , 2000 ; reprant et al . together , the pathways linking the olfactory bulb to the retina through the tn are known as the olfacto - retinal circuit ( orc ) . behavioral assays examining visual threshold have shown that stimulation of the olfactory bulb by food - related amino acids induces an increase in luminance sensitivity through activation of the orc ( maaswinkel and li , 2003 ; behrens and wagner , 2004 ; li and maaswinkel , 2007 ) . it has been suggested that an olfactory stimulus alters the processing of visual signals by decreasing the concentration of dopamine in the retina ( huang et al . the sole source of dopamine in the retina of teleosts is a specialized class of amacrine cell , the interplexiform cells ( ipcs ) , which are the target of the tn ( umino and dowling , 1991 ) . in the inner retina , actions on bipolar cells and retinal ganglion cells ( rgcs ) have also been reported , but their roles in altering retinal processing under different lighting conditions are not clearly established ( jensen and daw , 1984 ; jensen , 1992 ; heidelberger and matthews , 1994 ; li and dowling , 2000b ; ribelayga et al . how might the actions of dopamine underlie the modulation of retinal processing by an olfactory stimulus ? in this study , we take advantage of zebrafish expressing genetically encoded calcium reporters in the synaptic terminals of bipolar cells or dendrites of rgcs ( dreosti et al . by imaging signals through all layers of the inner retina , we have observed activity at the origins of the on and off channels that encode a change in light intensity with signals of opposite polarity ( schiller et al . here , we demonstrate that an olfactory stimulus reduces the gain but increases the sensitivity with which off bipolar cells transmit signals encoding luminance and contrast . pharmacological manipulations in vivo demonstrated that olfactory stimuli regulate the presynaptic calcium signal of bipolar cells by reducing the activity of d1 dopamine receptors , and electrophysiology in isolated bipolar cells demonstrated that activation of endogenous dopamine receptors potentiates voltage - dependent calcium channels that control synaptic transmission . together , these results indicate that the orc acts through the neuromodulator dopamine to regulate synaptic transmission through the off channel in the retina . transmission of the visual signal through the on and off pathways in the retina was assessed in transgenic zebrafish expressing sygcamp2 under the ribeye promoter ( dreosti et al . first , we investigated the effects of olfactory stimulation on the response to changes in the luminance of full - field stimuli . in off terminals , there was a decrease in the maximum amplitude of the response , which we term a decrease in gain . in parallel ( figure 1f ) , methionine increased the luminance sensitivity of off terminals by 0.5 log units , assessed by the lowest light level eliciting responses > 3 sd of the baseline noise . the increase in luminance sensitivity observed at the synaptic output of bipolar cells was quantitatively similar to the increase observed previously in ganglion cell recordings and behaviorally following olfactory stimulation ( maaswinkel and li , 2003 ; huang et al . the exception was a small but distinct fraction of terminals ( 9% ) , in which the amplitude of the presynaptic calcium signal was increased by a factor of at least three ( arrow in figure 1i and described further in figures s1a and s1b available online ) . although behavioral experiments showed olfactory stimulation to be ineffective in the afternoon ( maaswinkel and li , 2003 ) , we found that methionine administration produced a qualitatively similar but significantly smaller modulation of responses through the off pathway tested between 12:00 and 3:30 p.m. ( figures s1c s1f ) . to test the effects of an olfactory stimulus on the signaling of temporal contrast , we modulated full - field stimuli at 5 hz and measured the sygcamp2 signal ( 5 fish , n = 122 terminals ) . a change in the sygcamp2 signal in the absence of a change in the mean the relation between the amplitude of the steady sygcamp2 signal ( a ) and contrast ( c ) is shown in figure 2b : it could be described by a simple power function of the form a = k c , with = 1.8 0.1 under control conditions . stimulation with methionine reduced the amplitude of the rectifying response at all contrasts above 20% ( figure 2a ) . olfactory stimulation did not , however , affect the responses of on bipolar cells ( figures 2c and 2d ) . we also observed a small subset of on bipolar cells in which the dc presynaptic calcium levels were reduced by stimulation at 5 hz , and these were also unaffected by application of methionine ( figures s2a and s2b ) . the inhibition of presynaptic calcium signals by olfactory stimulation was also a function of stimulus frequency . we tested the responses of off terminals to frequencies between 0.2 and 25 hz at 90% contrast ( 6 fish , n = 96 terminals ) and found that methionine reduced the amplitude of the sygcamp2 signal elicited by stimuli below 10 hz . again together , the results in figures 1 , 2 , and s2 indicate that olfactory stimulation alters the processing of visual signals in the retina by two distinct actions on off bipolar cells : a suppression of the presynaptic calcium signal , resulting in a reduction in gain and an increase in luminance sensitivity . the large majority of on bipolar cells were unaffected by this form of cross - modal regulation . having observed an action of olfactory stimulation on the visual signal as it is transmitted by bipolar cells , we investigated how far the responses of postsynaptic ganglion cells were also affected . to monitor signals across large populations of neurons in vivo , we made a line of zebrafish expressing the calcium reporter gcamp3.5 under the eno2 promoter , which drives expression in rgcs ( bai et al . step changes in luminance were a relatively ineffective stimulus for rgcs , so we examined the effects of an olfactory stimulus on full - field stimuli modulated at 5 hz . the advantage of this in vivo imaging approach over electrophysiology is that it allows stimulation of the olfactory system while observing activity across a large population of rgcs . these results are consistent with the reduced gain of responses to contrast observed in off bipolar cell terminals , but not on , following application of methionine ( figure 2 ) . they also confirm that the actions of the orc are evident in the retinal output , as previously demonstrated by maaswinkel and li ( 2003 ) and huang et al . to investigate how dopaminergic signaling might be involved in modulating synaptic activity of bipolar cells , we injected agonists or antagonists of dopamine receptors into the anterior chamber of one eye of a fish , with a parallel sham injection into the other eye acting as a control . in off terminals , adtn increased the amplitude of sygcamp2 responses to all but the brightest lights and luminance sensitivity ( i1/2 ) increased by a factor of 420 ( figures 4a and 4b ; n = 92 terminals ) . in on terminals , adtn increased the amplitude of the sygcamp2 response to bright lights by 108% and increased luminance sensitivity by a factor of 15 ( figures 4c and 4d ) . strong activation of dopamine receptors therefore potentiated presynaptic calcium signals in both on and off bipolar cells , an effect opposite to an olfactory stimulus . further , application of methionine in the presence of 0.2 m adtn no longer depressed signals through off bipolar cells ( figures 4a and 4b ) . sch 23390 injection resulted in a complete impairment of luminance signaling through off bipolar cells ( figures 5a and 5b ) . in contrast , the maximum amplitude of the response in on bipolar cells was not significantly affected , although the light sensitivity ( i1/2 ) was increased by a factor of 3.8 ( figures 5c and 5d ) . antagonizing d1 receptors therefore caused effects qualitatively similar to an olfactory stimulus : a selective decrease in the gain of signaling through the off pathway ( cf . sulpiride also increased sensitivity of signals through on bipolar cells ( 0.85 log units ) , but methionine had no effect on the amplitude of these responses ( figures 5h5j ) . an olfactory stimulus therefore continued to cause a selective reduction of responses through the off pathway when activation of d2 receptors was blocked . together , the results in figures 4 and 5 indicate that cross - modal regulation of retinal function depends primarily on the activity of d1 receptors . to test further the idea that activation of the orc acts by decreasing dopamine level in the retina , we attempted to prevent these changes without interfering with the activity of dopamine receptors . the second action of vanoxerine was to prevent the application of methionine from modulating luminance signaling through off bipolar cells ( figures 6a and 6c ) , consistent with the idea that this modulation occurs through changes in dopamine levels . the manipulations of dopamine receptors and transporters shown in figures 4 , 5 , and 6 support the idea that olfactory stimulation modulates synaptic transmission from off bipolar cells by reducing dopamine levels and d1 dopamine receptor activity . in the outer retina of fish and mammals , dopamine acts through d1 receptors to uncouple horizontal cells providing negative feedback to the synaptic terminals of photoreceptors ( dowling , 1991 ) , but this seems an unlikely mechanism for the selective modulation of transmission through off bipolar cells given that these diverge from the on pathway downstream of photoreceptor output ( schiller et al . mixed rod - cone ( mb1 ) bipolar cells from the retina of goldfish were isolated for electrophysiological recording ( burrone and lagnado , 1997 ) . we also made recordings from the cell bodies of bipolar cell with small terminals , and in all three cases dopamine caused a depolarization of 15 mv . it therefore seems very likely that dopamine also acts to enhance calcium currents in off bipolar cells . to investigate the actions of dopamine on the calcium conductance more directly , we made voltage - clamp recordings using an intracellular solution designed to block potassium channels . figure 7c shows the calcium currents elicited by voltage steps from 70 mv to 40 mv and 20 mv in a single cell , and figure 7d shows an example of the current - voltage relation around the threshold for activation of the calcium current , approximately 43 mv ( burrone and lagnado , 1997 ) . to quantify changes in the calcium conductance over a number of cells , we measured the amplitude of the tail current 0.5 ms after a voltage step returning to 70 mv ( dashed red line in figure 7e ) . the 2.3 mv shift in v1/2 to lower membrane potentials is significant in the context of the voltage signals that bipolar cells generate in response to light ( baden et al . around this threshold , dopamine potentiated presynaptic calcium currents by a factor averaging 1.9 ( figure 7f ) . these results demonstrate that dopamine can act directly on bipolar cells to increase the magnitude of the presynaptic ca current that controls transmission of the visual signal . it seems likely that this action makes a significant contribution to the profound increase in the gain of luminance signals observed in vivo in the presence of the dopamine receptor agonist adtn ( figure 4 ) , as well as the decrease in gain in the presence of the antagonist sch 23390 ( figure 5 ) . if an olfactory stimulus acts to lower dopamine levels and therefore inhibits activation of presynaptic calcium channels , one might expect to observe a decrease in the basal calcium concentration in bipolar cells in darkness , with this effect being most obvious in off cells resting at more depolarized potentials . methionine induced a statistically significant reduction in sygcamp2 fluorescence in off terminals ( median = 10.9% , p < 0.01 ) but not on ( median = 0.2% , not significant ) , providing further support for the idea that inhibition of presynaptic calcium channels is one of the mechanisms by which an olfactory stimulus reduces the gain of signaling through off bipolar cells . by monitoring calcium signals in vivo , we find that an olfactory stimulus reduces the gain with which changes in luminance or temporal contrast are transmitted through the off pathway , while also increasing sensitivity at lower light levels ( figures 1 , 2 , and 3 ) . the results demonstrate that the calcium signal controlling neurotransmission from bipolar cells is a key site for regulating the flow of the visual information . strong evidence for this idea is provided by the demonstration that a blocker of dopamine release and reuptake suppresses the change in synaptic gain and sensitivity normally caused by an olfactory stimulus ( figure 6 ) . manipulations of dopamine receptor activity in vivo are also consistent with this mechanism ( figures 4 , 5 , and 6 ) and , in particular , for a key role of d1 receptors ( figures 5b and 5d ) . finally , we demonstrate that dopamine regulates the activity of voltage - dependent calcium channels in the synaptic terminals of bipolar cells , providing a direct mechanism for regulating the gain of the visual signal ( figure 7 ) . an overview of changes in the amplitude of the calcium signal through on and off bipolar cell terminals is shown in figure 8 . this comparison reveals a fundamental difference in the sensitivity of the on and off pathways to changes in retinal dopamine levels . thus , although an olfactory stimulus that results in decreased dopamine levels may be expected to decrease the gain of signals through the off pathway ( figures 1 and 8a ) , it is not expected to suppress synaptic calcium signals in on bipolar cells ( figures 1 and 8b ) . the mechanism for the differential effects of dopamine on the on and off pathways is still not clear . on and off bipolar cells both express d1 receptors but not d2 ( fan and yazulla , 2005 ; yu and li , 2005 ) . a possible explanation for the contrasting effect in on versus off could be differential sensitivity of the cav channels to camp that may reflect which cav channels underlie the response ( pan et al . for instance , iuvone and gan ( 1995 ) have demonstrated that activation of mt2 melatonin receptors antagonizes signaling through d1 dopamine receptors in bipolar cells by inhibiting camp synthesis through a gi protein , and wiechmann and sherry ( 2012 ) have found that mt2 melatonin receptors are localized to off but not on bipolar cells in xenopus laevis . the fast decrease in melatonin concentration that occurs after dawn might therefore act to enhance selectively the sensitivity of off bipolar cells to variations in dopamine levels . we did see a small population of on bipolar cell terminals ( 9% ) strongly potentiated by olfactory stimulation ( figures 1i , s1a , and s1b ) . might this reflect differences in the mechanism by which glutamate released from photoreceptors act on different types of on bipolar cells ? as a consequence , at present we do not have elements to consider this as a separate subpopulation of on bipolar cells . this mechanism is able to explain several of the observed effects of olfactory stimulation on the transmission of visual information through bipolar cells . for example , the decreased conductance and shift in the activation of the calcium channels will lower ca influx for small depolarizations such as low contrasts , nevertheless larger depolarizations , such as high contrasts , will still be effective . this highlights the presynaptic terminal of bipolar cells as a key site for regulating the transmission of visual signals through the retina . however , d1 receptors do enhance glutamate - gated ionic channels in off bipolar cells ( maguire and werblin , 1994 ) . the olfacto - retinal circuit endows the vertebrate visual system with the ability to quickly reduce the gain and increase the sensitivity of the retina in the presence of food , independently of changes in mean luminance . an olfactory stimulus applied in low background would then mimick the effects of light adaptation by encouraging fish to explore brighter areas . the reduction in gain of bipolar cell synapses transmitting the visual signal to the inner retina ( figure 1 ) , as well as the increase in sensitivity to high contrast ( figure 2 ) , is likely to be one of the mechanisms by which an olfactory stimulus allows the visual system of the zebrafish to operate in brighter areas . in the future , it will be interesting to investigate the behavioral consequences of a selective decrease in gain of the off pathway . certainly it would be expected to help the retina avoid saturation under bright conditions , but then so would a decrease in gain through the on pathway . in zebrafish , off bipolar cells outnumber ons by a ratio of 3:1 ( odermatt et al . , 2012 ) , so it may be that a decrease in the gain of the off pathway is the most important adaptation required to process negative contrasts at higher mean light levels . the re - tuning of retinal processing by a food - related olfactory stimulus is likely to be relevant to different aspects of zebrafish behavior , but especially hunting and prey - capture . the observed increase in the gain of the on channel relative to the off is expected to make the retina more sensitive to regions of positive contrast , such as bright spots appearing when sunlight reflects off small prey . bipolar cell terminals were imaged in vivo using a custom - built two - photon microscope equipped with a mode - locked chameleon titanium - sapphire laser tuned to 915 nm ( coherent ) with an olympus lumplanfi 40 water immersion objective ( n.a . the stimulation of the olfactory bulb was obtained by bath application of the amino acid methionine ( sigma ) 1 mm , as in maaswinkel and li ( 2003 ) . to manipulate dopamine signaling in the retina we injected neuroactive drugs into the eye . finally , the level of dopamine in the circuit was frozen by injection of the dopamine release and reuptake inhibitor vanoxerine ( santa cruz biotechnology ) 2 m , as in schlicker et al . the detection of active roi in the ipl was based on the thresholding of the laplacian transform of the two - photon recordings . in this way , responding bipolar cell terminals and active areas of the ganglion cell dendrites were identified in ribeye::sygcamp2 and eno2::gcamp3.5 fish , respectively . the responses to light of bipolar cell terminals and retinal ganglion cell dendrites were characterized according to their response amplitude , i.e. the sensitivity shift induced by olfactory stimulation for each individual terminal ( e.g. depolarizing bipolar cells were isolated from the retina of goldfish by enzymatic digestion , using methods described by burrone and lagnado ( 1997 ) .

cell line and chemicals : to investigate the potential effects of the selective cox-2 inhibitor der and cytotoxic anthracycline dox against canine mammary carcinoma cells , the cmt - u27 cell line was chosen , as it has high proliferative and anti - apoptotic potential . the canine mammary carcinoma cell line ( cmt - u27 ) was kindly supplied by prof . this cell line was derived from a primary tumor ( infiltrating ductal carcinoma ) , and when inoculated in the fat mammary pad of female nude mice , it metastasized to the lymph nodes , lungs , liver and heart . polyclonal anti - bcl-2 ( sc-492 ) , antibody , a secondary antibody kit and 3,3-diaminobenzidine ( dab ) ( sc-2018 ) were purchased from santa cruz biotechnology ( dallas , tx , u.s.a . ) . unless otherwise indicated , all reagents were purchased from sigma - aldrich ( st . cell culture : cells were cultured in dulbecco s modified eagle s medium ( dmem - f12 ) supplemented with 10% fetal bovine serum , 100 iuml penicillin g , 100 gml streptomycin and 2.5 gml amphotericin b in an atmosphere of 37c in 5% co2 , and the cells were harvested at approximately 8090% confluence using 0.25% trypsin - edta solution . dox and der were dissolved in dmem - f12 and sterile dimethyl sulfoxide ( dmso ) , respectively , and further serial dilutions for both drugs were made with dmem - f12 . the final dmso concentration did not exceed 0.1% ( and had no effect on cell growth ) in any experimental group , and this condition was used as a control in each experiment ( all groups comprised 0.1% dmso ) . cell viability assay : to determine dox and der concentrations that would be used in combination studies , an mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) tetrazolium reduction assay was performed on cmt - u27 cells . for this purpose , cells were seeded at 1 10/well in a final volume of 100 l medium in 96-well flat - bottomed tissue culture plates , in triplicate , and incubated in a humidified atmosphere at 37c under 5% co2 and 95% air to allow cell adhesion . after 24 hr incubation , the medium was removed , and cells were treated with various concentrations of dox ( 0.1 , 1 , 10 , 50 and 100 m ) and der ( 50 , 100 and 250 m ) for 72 hr . the concentrations for dox were chosen on the basis of previous reports about the effects of this drug on the in vitro viability of canine mammary tumor cells . cell viability , based on mitochondrial dehydrogenase activity , was determined by colorimetric assay using a mtt cell proliferation kit ( roche applied science , mannheim , germany ) in accordance with the instruction manual . the optical density of each well at 550 nm against a reference wavelength of 650 nm was measured using a microplate reader ( elx800 , biotek instruments , winooski , vt , u.s.a . ) . cell viability was calculated as follows : viability ( % ) = ( absorbance of the treated wells)/(absorbance of the control wells ) 100 . the dose - response curves were plotted for each drug , and the concentration of drug required for 50% inhibition of cell viability ( ic50 ) was determined graphically . subsequently , we tested 0.9 m ( ic50 ) and 0.09 m ( 1/10 ic50 ) of dox with 50 , 100 and 250 m of der in combination to learn whether der could enhance the antiproliferative effects of dox in cmt - u27 cells . for this purpose , the cells were treated with dox and der for 72 hr , and the antiproliferative effects of the combined agents were evaluated according to the mtt assay . drug interaction analysis : the nature of the interaction between dox ( 0.9 m and 0.09 m ) and der ( 50 , 100 and 250 m ) was determined by calculating the ratio index ( ri ) , which was initially described by kern et al . and later modified by romanelli et al . . the ri is calculated as the ratio of expected cell survival ( sexp , defined as the product of the viability observed with drug a alone and the survival observed with drug b alone ) to the observed cell survival ( sobs ) for the combination of a and b ( ri = sexp / sobs ) . type of interaction was defined as follows : ri1.5 , synergistic ; ri<1.5 to > 0.5 , additive ; and ri0.5 , antagonistic . this method was selected , because treatment with der had little effect on cell viability , which meant that other methods , such as the median effect principle and isobologram methods , were not suitable . apoptosis assay : to determine the mechanism of interaction between der and dox , an apoptosis assay was performed . flow cytometric analyses of phosphatidylserine exposure were quantitatively performed using an annexin v - fluorescein isothiocyanate ( fitc ) apoptosis detection kit ( bd biosciences , san jose , ca , u.s.a . ) . the method is based on the binding of annexin v to phosphatidylserine that is translocated from the inner membrane leaflet to the outer layer in cells undergoing apoptosis . the cells were cultured at a density of 1 10/ml in 24-well flat bottom microtiter plates ( jet biofil , seoul , korea ) and cultivated in a medium as described above . after 24 hr , the medium was replaced with fresh medium containing dox ( 0.9 m ) with or without der ( 50250 m ) . the cells were trypsinized 72 hr after treatment , washed twice each with ice - cold phosphate - buffered saline ( pbs ) consisting of 0.01 m phosphate buffer , 0.0027 m potassium chloride and 0.137 m sodium chloride and then resuspended in 100 l binding buffer ( 0.1 m hepes / naoh ( ph 7.4 ) , 1.4 m nacl , 25 mm cacl2 ) , additive supplemented with 5 l of fitc - annexin v and 5 l of propidium iodide ( pi ) . the cell suspension was gently vortexed and incubated for 15 min at room temperature in the dark . following incubation , 400 l of binding buffer was added to each tube , and then , the cell suspension was analyzed within 1 hr on a facscan flow cytometer ( bd biosciences ) using the standard optics for detecting fl1 ( fitc ) and fl3 ( pi ) . data were analyzed with the cellquest winmdi software ( bd biosciences , san jose , ca , u.s.a . ) . cell cycle analysis : the effects of dox ( 0.9 and 0.09 m ) with or without der ( 50250 m ) on the cmt - u27 cell cycle were evaluated by flow cytometry using a coulter dna prep reagents kit ( beckman coulter , high wycombe , buckinghamshire , u.k . ) . the cells were cultured at a density of 1 10/ml in 24-well flat bottom microtiter plates and cultivated and treated as described for an apoptosis assay . after the 72 hr treatment , the floating and adherent cells were combined for the analyses . cells were washed with pbs , and the cell suspensions were resuspended in 100 l of pbs . the dna content of the stained cells was immediately analyzed using a facscan flow cytometer ( bd biosciences ) . the percentages of cells in the g0/g1 phase , s phase and g2/m phase were calculated using the cellquest winmdi software ( bd biosciences ) . prostaglandin e2 ( pge2 assay ) : in order to elucidate whether the antiproliferative effect was mediated via the cox pathway , we studied the effect of exogenous addition of pge2 ( 15 g / ml ) on the in vitro antiproliferative activity of der alone and in combination with dox against cmt - u27 cells . the cells were seeded at 1 10/well in 100 l of medium in 96-well plates and incubated overnight . subsequently , dox ( 0.9 and 0.09 m ) was added to the plate in the presence or absence of pge2 ( 15 g / ml ) . similarly , der ( 50250 m ) in combination with dox ( 0.9 and 0.09 m ) was added in the presence or absence of pge2 ( 15 g / ml ) and incubated for 72 hr , and the antiproliferative activity was assessed by mtt assay . immunocytochemistry : in order to examine the protein expression of the apoptosis marker bcl-2 in cmt - u27 cells , sterilized coverslips were placed on the bottom of a 24-well plate , and the cells were seeded at a density of 1 10 cells / ml and incubated overnight . after incubation , cells were treated with 50250 m der alone or in combination with 0.9 m and 0.09 m dox for 72 hr . at the end of treatment , the cells were rinsed with pbs and fixed with cold methanol for 10 min . after fixation , the cells were washed with pbs ( ph 7.4 , 0.1 m ) for 5 min , and the endogenous peroxidase activity was inactivated by incubation with 0.3% h2o2 in methanol for 10 min . then , the cells were washed three times with pbs and incubated for 10 min with a protein blocking agent to block nonspecific immunolabeling . afterwards , cells were incubated with polyclonal anti - bcl-2 ( sc-492 ) antibody at a dilution of 1:200 at room temperature for 90 min . after extensive washing in pbs , the cover slips were incubated with a secondary antibody kit ( sc-2018 ) containing a biotinylated secondary antibody and avidin - peroxidase for 20 min , at room temperature . finally , cells were rinsed with pbs and incubated with dab complexes according to the manufacturer s protocol and then counterstained with mayer s hematoxylin and mounted onto glass slides . the cells were observed with a light microscope ( olympus bx50 , olympus , tokyo , japan ) . intensity of immunolabeling was assessed by examination of 10 representative high - power fields ( 400 ) . the number of immunoreactive cells was assessed semiquantitatively . for staining density , specimens were classified as negative , + ( < 10% positive cells ) , + + ( 1050% positive cells ) and + + + ( > 50% positive cells ) . results were considered inconclusive when there were insufficient neoplastic cells available for analysis . statistical analysis : samples were assayed at least three times for each determination , and results were expressed as the mean se . the statistical differences between the treatments and the control were tested by one - way analysis of variance ( anova ) followed by the student s t - test using the graphpad instat software ( graphpad software , san diego , ca , u.s.a . ) . a difference in means with a p - value of 0.05 or less antiproliferative effects of der and dox combinations on cmt - u27 cells : the antiproliferative effects of der and dox combinations on cmt - u27 cells were evaluated by mtt assay . the antiproliferative effects of der and dox as single agents against cmt - u27 cells were shown in our previous studies [ 2 , 3 ] . those studies demonstrated that der even at concentration as high as 250 m had weak growth inhibitory activity in the cells ( with growth inhibition of 16.49% compared with the control ) . by contrast , cmt - u27 cells responded to dox sensitively , with an obvious dose - dependent antiproliferative effect and ic50 of approximately 0.9 m . subsequently , to determine whether der enhances the anti - proliferative effect of dox on cmt - u27 cells , three doses of der ( 50 , 100 and 250 m ) were used in combination with dox ( 0.9 and 0.09 m ) . as shown in fig . 1.antiproliferative effects of doxorubicin and deracoxib combinations in the cmt - u27 cell line . cells were treated with the indicated doses of doxorubicin and deracoxib , and cell viability was assayed 72 hr after treatment . data are expressed as mean percentage of cell viabilities standard error ( se ) . * * p<0.01 compared with the doxorubicin - treated group . , der potentiated the inhibitory effect of dox ( 0.9 m ) on cmt - u27 cells . the influence of der on the effect of dox ( 0.9 m ) appeared to be moderately dose dependent , and the strongest inhibition , approaching 71% , was observed with the combination of dox at 0.9 m and der at 250 m . also , the cox inhibitor at the concentrations used enhanced the antiproliferative activity of dox in cmt - u27 cells , which decreased ( 2.89- to 3.71-fold ) the ic50 value from 0.876 m ( dox at 0.9 m ) to 0.303 m , 0.245 m and 0.236 m ( 0.9 m in combination with 50 , 100 and 250 m der ) , respectively . antiproliferative effects of doxorubicin and deracoxib combinations in the cmt - u27 cell line . cells were treated with the indicated doses of doxorubicin and deracoxib , and cell viability was assayed 72 hr after treatment . data are expressed as mean percentage of cell viabilities standard error ( se ) . * * p<0.01 compared with the doxorubicin - treated group . the nature of the interaction between dox and der was analyzed using the ri , which quantitatively measures the interaction of two drugs . the combinations of dox at 0.9 m and der at 50250 m exhibited synergistic activity against cmt - u27 cells ( table 1table 1.type of interaction between doxorubicin and deracoxib in cmt - u27 cellscombination of dox with derri valuedox ( 0.9 m)dox+ der ( 50 m)2.24 ( synergistic)dox+ der ( 100 m)1.98 ( synergistic)dox+ der ( 250 m)2.33 ( synergistic)dox ( 0.09 m)dox+ der ( 50 m)1.15 ( additive)dox+ der ( 100 m)1.13 ( additive)dox+ der ( 250 m)1.08 ( additive)dox , doxorubicin ; der , deracoxib ; ri , ratio index . type of interaction : ri1.5 , synergistic ; ri<1.5 to > 0.5 , additive ; ri0.5 , antagonistic interaction . ) . the synergistic effect was most prominent when 0.9 m dox was combined with 250 m der ( ri=2.33 ) . on the other hand , the combinations of a low dose ( 0.09 m ) of dox with der ( 50250 m ) resulted in additive interaction ( ri=1.081.15 ) dox , doxorubicin ; der , deracoxib ; ri , ratio index . type of interaction : ri1.5 , synergistic ; ri<1.5 to > 0.5 , additive ; ri0.5 , antagonistic interaction . effects of der and dox combinations on apoptosis in cmt - u27 cells : the apoptotic effects of der as a single agent against cmt - u27 cells were shown in our previous study . to explore the mechanisms of synergistic effects of dox and der combinations , an apoptosis assay was performed using specific concentrations of dox ( 0.9 m ) and der ( 50250 m ) that can not lead to toxicity . treatment with dox ( 0.9 m ) alone induced 36.22% apoptosis in cmt - u27 cells . the combined treatment of dox and der ( 100 m and 250 m ) significantly augmented apoptosis induction in cmt - u27 cells ( sum of early and late apoptotic cells ; 48.91% and 49.58% , respectively ) , and approximately 1.35- and 1.37-fold increases , respectively , in apoptotic response were observed as compared with that caused by dox . treatment with dox ( 0.09 m ) alone induced 24.02% apoptosis in cmt - u27 cells . the combined treatment of dox ( at 0.09 m ) and der ( at 50 m , 100 m and 250 m ) induced 27.77% , 35% and 37.69% apoptosis , respectively , in cmt - u27 cells in terms of the sum of early and late apoptotic cells ( fig . 2.effects of doxorubicin and deracoxib combination treatment on apoptosis of cmt - u27 cells . ( a ) the number of viable , early apoptotic , late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the cmt - u27 cell line . ( b ) representative cytograms of the cmt - u27 cell line double stained with annexin v - fitc and propidium iodide ( pi ) . the numbers written on histograms represent the sum of early and late apoptotic cells ( % ) . ) . effects of doxorubicin and deracoxib combination treatment on apoptosis of cmt - u27 cells . ( a ) the number of viable , early apoptotic , late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the cmt - u27 cell line . ( b ) representative cytograms of the cmt - u27 cell line double stained with annexin v - fitc and propidium iodide ( pi ) . the numbers written on histograms represent the sum of early and late apoptotic cells ( % ) . effects of dox and der combinations on the cell cycle distribution of cmt - u27 cells : to confirm the mechanism of such synergistic effects of dox and der combinations , we also examined the effects of their combinations on cell cycle parameters under the same experimental conditions . as shown in table 2table 2.effects of doxorubicin and deracoxib combination treatment for 72 hr on cell cycle kinetics of cmt - u27 cellsdrugsconcentrationg0/g1 ( % ) s ( % ) g2/m ( % ) control-54.51 2.6829.05 1.6816.44 1.24dox0.9 m56.6 4.2434.83 2.538.51 1.14dox + der0.9 m + 50 m84.49 3.6015.30 2.080.21 0.04dox + der0.9 m + 100 m84.96 1.4514.83 2.650.21 0.03dox + der0.9 m + 250 m91.34 3.647.16 1.321.49 0.18dox0.09 m86.93 4.9912.92 1.720.14 0.04dox + der0.09 m + 50 m89.58 5.4610.30 1.060.12 0.03dox + der0.09 m + 100 m89.13 4.4910.84 1.500.20 0.04dox + der0.09 m + 250 m89.79 2.8510.1 1.460.10 0.02each value represents the mean se of three experiments . , the percentage of cells in the g0/g1 phase and s phase of the cell cycle increased with dox ( 0.9 m ) treatment as a single agent , while the proportion of cells in the g2/m phase decreased compared with the control . the combined treatments of dox and der altered the cell cycle profile in cmt - u27 cells . the most remarkable change in cell cycle progression was seen with the combination of 0.9 m dox and 250 m der . this combination led to a further increase in the g0/g1 phase ( from 56.6 to 91.34% ) with a concomitant decrease in the s phase ( from 34.83 to 7.16 ) compared with dox alone ( 0.9 m ) . effects of supplementation of pge2 on growth inhibitory activity of dox enhanced by der in cmt - u27 cells : the effects of supplementation of pge2 on the growth inhibitory activity of dox enhanced by der in cmt - u27 cells may be due to cox-2 inhibition . to examine this , we determined whether the cox-2 end product pge2 could reverse the observed effects . therefore , exogenous pge2 ( 15 m ) was added to the medium in order to take into account the fact that some pge2 may degrade or be internalized into cells . as shown in fig . 3.effects of supplementation of pge2 on enhancement of growth inhibitory activity of doxorubicin caused by deracoxib in cmt - u27 cells . cells were treated with or without deracoxib ( 50 - 250 m ) in the absence or presence of pge2 ( 1 or 5 m ) and 0.9 m doxorubicin ( a ) or 0.09 m doxorubicin ( b ) alone or in combination for 72 hr before determination of cell viability by mtt assay . pge2 at all tested concentrations failed to reverse the enhancing effect of der on the growth inhibitory activity of dox . effects of supplementation of pge2 on enhancement of growth inhibitory activity of doxorubicin caused by deracoxib in cmt - u27 cells . cells were treated with or without deracoxib ( 50 - 250 m ) in the absence or presence of pge2 ( 1 or 5 m ) and 0.9 m doxorubicin ( a ) or 0.09 m doxorubicin ( b ) alone or in combination for 72 hr before determination of cell viability by mtt assay . effects of dox and der alone or in combination on bcl-2 expression in cmt u27 cells : to determine the apoptotic pathway activated by der , we examined the apoptosis - related target bcl-2 in cmt - u27 cells . immunocytochemical staining revealed that increasing the concentrations of der inhibited expression of the anti - apoptotic protein bcl-2 ( fig . a ) deracoxib 50 m , moderate immunopositivity ; b ) deracoxib 250 m , slight immunopositivity ; c ) doxorubicin 0.09 m + deracoxib 50 m , strong immunopositivity ; d ) doxorubicin 0.9 m + deracoxib 250 m slight immunopositivity ; e ) control cells , very strong immunopositivity ; f ) negative control cells , no reaction . these effects were more pronounced in the combinations of dox ( at 0.9 m ) with der ( at 100 m and 250 m ) ( table 3table 3.effects of deracoxib or doxorubicin alone or in combination on bcl-2 expression in cmt - u27 cellsdrugsconcentrationbcl-2control-+++dox0.9 m++der50 m++der100 m+der250 m+dox + der0.9 m + 50 m++dox + der0.9 m + 100 m+dox + der0.9 m + 250 m+dox0.09 m+++dox + der0.09 m + 50 m+++dox + der0.09 m + 100 m++dox + der0.09 m + 250 m++dox , doxorubicin ; der , deracoxib . results are categorized as negative , + ( < 10% positive cells ) , + + ( 1050% positive cells ) and + + + ( > 50% positive cells ) . ) . however , the combinations of dox at 0.09 m with der ( at 100 m and 250 m ) decreased the expression of bcl-2 slightly . a ) deracoxib 50 m , moderate immunopositivity ; b ) deracoxib 250 m , slight immunopositivity ; c ) doxorubicin 0.09 m + deracoxib 50 m , strong immunopositivity ; d ) doxorubicin 0.9 m + deracoxib 250 m slight immunopositivity ; e ) control cells , very strong immunopositivity ; f ) negative control cells , no reaction . results are categorized as negative , + ( < 10% positive cells ) , + + ( 1050% positive cells ) and + + + ( > 50% positive cells ) . despite many advances in the field of cancer therapeutics , canine malignant mammary tumors continue to be a leading cause of death in dogs , which is in part due to the failure of chemotherapy . clearly , new therapeutic modalities are needed to both improve the outcome of dogs suffering from the tumors and to reduce the long - term toxicities associated with the current standard of treatment . drug development strategies include the evaluation of new drug combinations that may have improved efficacy compared with single agents . by treating a tumor with a combination of agents that employ different mechanisms of action and have different spectra of normal tissue toxicity , the overall response can be enhanced without an increase in toxicity [ 35 , 37 ] . besides enhanced cytotoxicity , combinations of chemotherapeutic agents may minimize or delay the induction of drug resistance [ 10 , 21 ] . furthermore , drug combinations that use lower doses of individual agents may improve selectivity and reduce the severity of undesired side effects of chemotherapy . selective cox-2 inhibitors exert antitumor effects on tumor cells directly via inhibition of cell proliferation , induction of apoptosis and reduction of cell motility and adhesion and also have anti - angiogenic activity by suppressing tumor angiogenesis [ 36 , 56 , 69 , 72 ] . these anticancer properties make it worthwhile to examine the possible benefit of combining selective cox-2 inhibitors with conventional anticancer therapies , such as chemotherapy . related studies have shown that selective cox-2 inhibitors in particular strengthen the effectiveness of chemotherapy treatment in various types of human tumors , including breast tumors [ 5 , 9 , 56 , 69 ] . similarly , during the last decade , various preclinical and clinical trials in the field of veterinary oncology have been conducted to study the use of cox-2 selective inhibitors in combination with other agents in early and advanced cancers and have been shown to improve treatment outcome in dogs with transitional cell carcinoma and osteosarcoma [ 34 , 70 ] . however , to our knowledge , there is no published report demonstrating the therapeutic efficiency of selective cox-2 inhibitors with conventional anti - cancer agents in canine mammary tumor . based on the encouraging results from cox-2 inhibitors as chemotherapeutic and chemopreventive agents in the treatment of several types of human and canine malignancy including mammary tumor [ 2 , 14 ] , we evaluated the potential of a selective cox-2 inhibitor ( der ) in potentiating the antitumor activity of a conventional cytotoxic agent ( dox ) in vitro in canine mammary carcinoma cells ( cmt - u27 ) . for this purpose , we preferred der , a highly selective canine cox-2 inhibitor accepted as safe and well - tolerated in dogs , and dox , a cytotoxic anthracycline antibiotic commonly used in veterinary clinical treatments for various cancers . der is widely used in veterinary medicine for the control of pain and inflammation associated with osteoarthritis and orthopedic surgery in dogs . recently , it has been reported that this drug might be a useful alternative for the prevention and/or treatment of some cancer types in dogs [ 34 , 54 ] . similarly , in our previous investigation , we proved that der had a clear antiproliferative and apoptotic effect on canine mammary carcinoma cells in vitro . these effects have only been observed at high concentrations ( 2501,000 m ) of der as well as seen in other nsaids ( e.g. , indomethacin , meloxicam ) , which are 10- to 1,000-fold those required to inhibit pg synthesis or cox enzymes [ 38 , 51 , 63 ] . the clinical importance of the direct cytotoxicity observed in vitro is presently unknown , as it is not known what plasma concentrations of der would be necessary to achieve effective concentrations ( 250 m ) intratumorally or what duration of drug exposure is necessary to induce the observed cytotoxicity . however , it is known that plasma concentrations of der can reach as much as 20 m when der is administrated at a dose of 4 mg / kg daily ( i.e. the dosage used for postoperative orthopedic pain ) . it is also accepted that use of the drug at higher than approved dosages , especially long - term use , can lead to an increased risk of toxicity . on the other hand , it has been reported that the concentrations of nsaids , extensively bound to proteins , in the acidic environment of inflammation ( such as in a tumor ) relating to high protein content might be higher than in plasma [ 28 , 70 ] . similarly , tumor cells are able to produce angiogenic proteins like the vascular endothelial growth factor ( vegf ) , which could lead to a preferential accumulation of nsaids , so it is possible that the concentrations in the tumor cells are higher than in plasma . it has been reported that vegf and a beta - galactoside - binding protein ( galectin-3 , an important mediator of vegf ) are highly expressed in cmt - u27 cells . based on this information , we used the lowest possible concentrations ( 50 m , 100 m and 250 m ) of der , which are likely the most practical for clinical use , in our combination studies . dox is a cytotoxic anthracycline antibiotic that is commonly used in both veterinary and human cancer chemotherapy protocols [ 40 , 62 ] . this medication has antitumor activity against a wide variety of carcinomas including mammary tumors , however , its utility is limited due to acute and chronic toxicities , such as myelosuppression , immunosuppression and dose - cumulative cardiotoxicity [ 11 , 66 ] . therefore , it is important to use this drug in in vitro therapeutic tests with the purpose of searching for a new methodology for combination protocols with nontoxic drugs , as it could enable the dose of dox to be lowered due to its good antitumor activity , which is mainly observed in the metastatic mammary tumor . our previous study indicated that a broad concentration range ( 0.1100 m ) of dox inhibited canine mammary carcinoma cell proliferation in vitro . the ic50 of 876 nm determined for dox in cmt - u27 cells was within the range of clinically relevant concentrations , as dogs that were treated with 1 mg / kg dox achieved plasma concentrations of 0.7 g / ml ( 1.2 m ) 5 min after intravenous administration . the concentrations of dox for a desirable pharmacological effect in canine mammary tumor cell lines in vitro were reported to be 280 nm and 840 nm [ 58 , 70 ] . the ic50 value for cmt - u27 cells is higher than reported for the same compound in different cmt cells . we suggest that dox is less potent in cmt - u27 cells than other mammary tumor cell types . cmt - u27 cells have a high growth rate and anti - apoptotic potential associated with enhanced expression of genes involved in the ca signaling pathway and growth hormone cellular pathway . the high anti - apoptotic potential of these cells has been shown to be related to elevated expression abr , which interacts with the tumor protein p53 and tmd1 genes , which are involved in drug resistance in tumor cells . prior studies have also demonstrated that cmt - u27 cells express high levels of anti - apoptotic bcl-2 protein , which is linked to resistance to several therapeutic modalities [ 30 , 68 ] . these data confirm our finding that highly metastatic cmt - u27 cells have low sensitivity to dox . therefore , we concluded that cmt - u27 cells could be an in vitro model for canine mammary cancer refractory to dox chemotherapy . in this study , we tested whether a cox-2 inhibitor drug could restore the response of a chemotherapeutic agent in canine mammary cancer . our results demonstrated that der enhanced the cytotoxic action of dox at 0.9 m in cmt - u27 cells in a dose dependent manner , which decreased the ic50 value from 0.876 m ( dox at 0.9 m ) to 0.303 m , 0.245 m and 0.236 m ( 0.9 m in combination with 50 , 100 and 250 m der ) , respectively . these results suggested that der sensitized cmt - u27 cells to the action of dox . the addition of der to 0.9 m dox , even at concentrations that did not affect cell viability , achieved at least the same level of cytotoxicity as an approximately 3- to 3.5-fold higher dose of dox alone in the cell line . in contrast , der exerted no effect on the action of dox at 0.09 m in the cell line . these results suggest that the enhancement of cytotoxicity in combination treatment of dox with der depends on the dox chemotherapy dose and that the addition of der ( 50250 m ) to dox ( 0.9 m ) therapy may be beneficial to reduce the chemotherapy dose necessary to achieve a cytotoxic response in canine mammary cancer . to determine the nature of the interaction between dox and der , we used the method of kern et al . this method is accepted as the only correct method to evaluate the type of interaction between two drugs when one or both has a low cytotoxic effect or no dose - response curve . interaction analysis of the data showed that the combinations of dox at 0.9 m with der produced synergism in the cmt - u27 cell line , with a ratio index ranging from 1.98 to 2.33 . dox at a low concentration ( 0.09 m ) with der exhibited interaction in an additive manner . our results are consistent with the work of van wijngaarden et al . , who demonstrated that celecoxib enhanced dox - induced cytotoxicity in mda - mb231 breast cancer cells . the induction of cox-2 and its associated production of pge2 from arachidonic acid are thought to play a role in the initiation and maintenance of cancer cell survival and growth . the downstream product of the cox-2 catalyzed metabolism of arachidonic acid , pge2 , has been shown to promote the growth of breast carcinoma cells and reverse the growth inhibitory effect of the selective cox-2 inhibitor celecoxib in breast cancer mcf-7 cells . in our study , we found that exogenous addition of pge2 ( 15 g / ml ) did not reverse the antiproliferative effect of der in combination with dox . these results suggest that growth inhibition in cmt - u27 cells by dox and its combination with der are not directly associated with their ability to suppress pge2 production . ( 1998 ) , who showed that exogenous pge2 did not reverse the synergistic cytotoxicity of dox with indomethacin against a dlkp cell line . similarly , pge2 and the prostaglandin precursor arachidonic acid were shown to not reverse the growth inhibitory effects of the cox-2-selective inhibitor sc236 with dox on hkesc-1 and hkesc-2 cells . these observations suggest that cox - independent mechanisms are responsible for the in vitro growth inhibitory effects of these compounds . the synergistic and additive effects on cmt - u27 cells elicited by treatment with dox ( 0.9 and 0.09 m , respectively ) and der ( especially at the concentrations of 100 and 250 m ) are associated with a marked increase in apoptosis . our results demonstrated that der ( 100 and 250 m ) enhanced the apoptotic activity of dox at 0.9 and 0.09 m in the cells , which increased the apoptotic index from 36.22% to 48.91% and 49.58% and from 24.02% to 35% and 37.69% , respectively . also , our results show that dox alone caused late apoptosis rather than early apoptosis . however , the early apoptotic cell number was significantly increased by the addition of der ( 250 m ) . we suggest that the increase in the percentage of early apoptotic cells could be related to der . der may cause cell membrane loss and therefore allow dox to more easily penetrate into cells , and it may sensitize mammary cancer cells to dox by activating the apoptotic program . also , the increases in apoptotic activity according to dox concentration support this suggestion . in support of these possibilities , some reports have [ 44 , 65 ] suggested that nsaids , as disordering agents on membranes , might interfere with the lateral heterogeneity of membranes , disrupting the organization and function of microdomains , which are involved in the regulation of protein location and signaling pathways . alteration of membrane properties , such as perturbations of membrane fluidity by anti - inflammatory agents , has been recently described and has been indicated as an additional mechanism by which anti - inflammatory agents have pharmacological effects relating to anticancer activity in some publications [ 32 , 65 ] . in this regard , previous studies have demonstrated that cox-2 inhibitors ( e.g. , celecoxib , indomethacin , nimesulide ) augmented chemotherapeutic drug - induced apoptosis by downregulation of anti - apoptotic mediators , such as bcl-2 , in breast , prostate and osteosarcoma cells [ 13 , 38 , 69 ] . to define the mechanistic role of der in apoptosis induction , we examined bcl-2 expression , which is known to promote cell survival and has been correlated with the development of dox resistance , in cmt - u27 cells . we showed that der ( 100 and 250 m ) induced downregulation of bcl-2 at 0.9 m dox more strongly than at 0.09 m dox in cmt - u27 cells . we concluded that der may be altered the sensitivity of cmt - u27 cells to dox - induced apoptosis by downregulating bcl-2 expression . other possible functions of der relating to anti - apoptotic genes need to be further investigated . in additional studies identifying the mechanism of observed synergistic and additive effects in treatment with dox and der , we found that der potentiated dox - caused g0/g1 arrest in cell cycle progression , while dox - induced cell cycle arrest in the g0/g1 phase , as well as induction of cell death , has been demonstrated in breast cancer mcf-7 cells . we showed that a low concentration of dox ( 0.09 m ) induced higher g0/g1 arrest compared with a high concentration of dox ( 0.9 m ) . the current results indicated that cell cycle regulation by dox occurs differentially according to the concentrations applied . this effect could result from the increased penetration of dox into cmt - u27 cells caused by der . on the other hand , the percentages of cmt - u27 cells arrested at the g0/g1 phase are inversely proportional to the dox concentrations , and this could arise from the density of the viable cells observed with both dox treatments , because high concentrations of dox lead to necrosis . this is the first study to demonstrate that dox and dox with der induced cell cycle arrest in cmt - u27 cells , and the molecular mechanism of accumulation of cells in the g0/g1 phase induced by der in the presence of dox is unknown . we suggest that the increase in the percentage of cmt - u27 cells in the g0/g1 phase may be related to the accompanying changes in the level of proteins regulating the cell cycle and the decline of cell proliferation activity . in conclusion , we elucidated that der enhanced the antiproliferative effect of dox in conjunction with induction of apoptosis by modulation of bcl-2 expression and changes in the cell cycle of the cmt - u27 cell line . although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed for der and dox combinations require further investigations , the results suggest that the synergistic effect of dox and der combinations in cmt therapy may be achieved at relatively lower doses of dox with lesser side effects .

cyclooxygenase ( cox ) inhibitors have been shown to exert anti - angiogenic and anti - tumor activities on many types of malignant tumors . these anticancer properties make it worthwhile to examine the possible benefit of combining cox inhibitors with other anti - cancer agents . in the present study , we evaluated the potential of deracoxib ( der ) in potentiating antitumor activity of doxorubicin ( dox ) in canine mammary carcinoma cells ( cmt - u27 ) . der ( 50250 m ) enhanced the antiproliferative activity of dox by reducing the ic50 ( approximately 3- to 3.5 fold ) . interaction analysis of the data showed that combinations of dox at 0.9 m with der ( 100250 m ) produced synergism in the cmt - u27 cell line , with a ratio index ranging from 1.98 to 2.33 . in additional studies identifying the mechanism of observed synergistic effect , we found that der strongly potentiated dox - caused g0/g1 arrest in cell cycle progression . also , der ( 100250 m ) augmented apoptosis induction with approximately 1.35- and 1.37- fold increases in apoptotic response caused by dox in the cells . der enhanced the antiproliferative effect of dox in conjunction with induction of apoptosis by modulation of bcl-2 expression and changes in the cell cycle of the cmt - u27 cell line . although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed with der and dox combinations require further investigations , the results suggest that the synergistic effect of dox and der combinations in cmt therapy may be achieved at relatively lower doses of dox with lesser side effects . therefore , combining der with dox may prove beneficial in the clinical treatment of canine mammary cancer .

MATERIALS AND METHODS RESULTS DISCUSSION

cell line and chemicals : to investigate the potential effects of the selective cox-2 inhibitor der and cytotoxic anthracycline dox against canine mammary carcinoma cells , the cmt - u27 cell line was chosen , as it has high proliferative and anti - apoptotic potential . the canine mammary carcinoma cell line ( cmt - u27 ) was kindly supplied by prof . subsequently , we tested 0.9 m ( ic50 ) and 0.09 m ( 1/10 ic50 ) of dox with 50 , 100 and 250 m of der in combination to learn whether der could enhance the antiproliferative effects of dox in cmt - u27 cells . for this purpose , the cells were treated with dox and der for 72 hr , and the antiproliferative effects of the combined agents were evaluated according to the mtt assay . drug interaction analysis : the nature of the interaction between dox ( 0.9 m and 0.09 m ) and der ( 50 , 100 and 250 m ) was determined by calculating the ratio index ( ri ) , which was initially described by kern et al . after 24 hr , the medium was replaced with fresh medium containing dox ( 0.9 m ) with or without der ( 50250 m ) . cell cycle analysis : the effects of dox ( 0.9 and 0.09 m ) with or without der ( 50250 m ) on the cmt - u27 cell cycle were evaluated by flow cytometry using a coulter dna prep reagents kit ( beckman coulter , high wycombe , buckinghamshire , u.k . ) prostaglandin e2 ( pge2 assay ) : in order to elucidate whether the antiproliferative effect was mediated via the cox pathway , we studied the effect of exogenous addition of pge2 ( 15 g / ml ) on the in vitro antiproliferative activity of der alone and in combination with dox against cmt - u27 cells . similarly , der ( 50250 m ) in combination with dox ( 0.9 and 0.09 m ) was added in the presence or absence of pge2 ( 15 g / ml ) and incubated for 72 hr , and the antiproliferative activity was assessed by mtt assay . immunocytochemistry : in order to examine the protein expression of the apoptosis marker bcl-2 in cmt - u27 cells , sterilized coverslips were placed on the bottom of a 24-well plate , and the cells were seeded at a density of 1 10 cells / ml and incubated overnight . a difference in means with a p - value of 0.05 or less antiproliferative effects of der and dox combinations on cmt - u27 cells : the antiproliferative effects of der and dox combinations on cmt - u27 cells were evaluated by mtt assay . by contrast , cmt - u27 cells responded to dox sensitively , with an obvious dose - dependent antiproliferative effect and ic50 of approximately 0.9 m . subsequently , to determine whether der enhances the anti - proliferative effect of dox on cmt - u27 cells , three doses of der ( 50 , 100 and 250 m ) were used in combination with dox ( 0.9 and 0.09 m ) . 1.antiproliferative effects of doxorubicin and deracoxib combinations in the cmt - u27 cell line . , der potentiated the inhibitory effect of dox ( 0.9 m ) on cmt - u27 cells . the influence of der on the effect of dox ( 0.9 m ) appeared to be moderately dose dependent , and the strongest inhibition , approaching 71% , was observed with the combination of dox at 0.9 m and der at 250 m . also , the cox inhibitor at the concentrations used enhanced the antiproliferative activity of dox in cmt - u27 cells , which decreased ( 2.89- to 3.71-fold ) the ic50 value from 0.876 m ( dox at 0.9 m ) to 0.303 m , 0.245 m and 0.236 m ( 0.9 m in combination with 50 , 100 and 250 m der ) , respectively . antiproliferative effects of doxorubicin and deracoxib combinations in the cmt - u27 cell line . the combinations of dox at 0.9 m and der at 50250 m exhibited synergistic activity against cmt - u27 cells ( table 1table 1.type of interaction between doxorubicin and deracoxib in cmt - u27 cellscombination of dox with derri valuedox ( 0.9 m)dox+ der ( 50 m)2.24 ( synergistic)dox+ der ( 100 m)1.98 ( synergistic)dox+ der ( 250 m)2.33 ( synergistic)dox ( 0.09 m)dox+ der ( 50 m)1.15 ( additive)dox+ der ( 100 m)1.13 ( additive)dox+ der ( 250 m)1.08 ( additive)dox , doxorubicin ; der , deracoxib ; ri , ratio index . on the other hand , the combinations of a low dose ( 0.09 m ) of dox with der ( 50250 m ) resulted in additive interaction ( ri=1.081.15 ) dox , doxorubicin ; der , deracoxib ; ri , ratio index . effects of der and dox combinations on apoptosis in cmt - u27 cells : the apoptotic effects of der as a single agent against cmt - u27 cells were shown in our previous study . to explore the mechanisms of synergistic effects of dox and der combinations , an apoptosis assay was performed using specific concentrations of dox ( 0.9 m ) and der ( 50250 m ) that can not lead to toxicity . treatment with dox ( 0.9 m ) alone induced 36.22% apoptosis in cmt - u27 cells . the combined treatment of dox and der ( 100 m and 250 m ) significantly augmented apoptosis induction in cmt - u27 cells ( sum of early and late apoptotic cells ; 48.91% and 49.58% , respectively ) , and approximately 1.35- and 1.37-fold increases , respectively , in apoptotic response were observed as compared with that caused by dox . the combined treatment of dox ( at 0.09 m ) and der ( at 50 m , 100 m and 250 m ) induced 27.77% , 35% and 37.69% apoptosis , respectively , in cmt - u27 cells in terms of the sum of early and late apoptotic cells ( fig . ( a ) the number of viable , early apoptotic , late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the cmt - u27 cell line . ( b ) representative cytograms of the cmt - u27 cell line double stained with annexin v - fitc and propidium iodide ( pi ) . ( a ) the number of viable , early apoptotic , late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the cmt - u27 cell line . ( b ) representative cytograms of the cmt - u27 cell line double stained with annexin v - fitc and propidium iodide ( pi ) . effects of dox and der combinations on the cell cycle distribution of cmt - u27 cells : to confirm the mechanism of such synergistic effects of dox and der combinations , we also examined the effects of their combinations on cell cycle parameters under the same experimental conditions . , the percentage of cells in the g0/g1 phase and s phase of the cell cycle increased with dox ( 0.9 m ) treatment as a single agent , while the proportion of cells in the g2/m phase decreased compared with the control . the combined treatments of dox and der altered the cell cycle profile in cmt - u27 cells . the most remarkable change in cell cycle progression was seen with the combination of 0.9 m dox and 250 m der . this combination led to a further increase in the g0/g1 phase ( from 56.6 to 91.34% ) with a concomitant decrease in the s phase ( from 34.83 to 7.16 ) compared with dox alone ( 0.9 m ) . effects of supplementation of pge2 on growth inhibitory activity of dox enhanced by der in cmt - u27 cells : the effects of supplementation of pge2 on the growth inhibitory activity of dox enhanced by der in cmt - u27 cells may be due to cox-2 inhibition . 3.effects of supplementation of pge2 on enhancement of growth inhibitory activity of doxorubicin caused by deracoxib in cmt - u27 cells . cells were treated with or without deracoxib ( 50 - 250 m ) in the absence or presence of pge2 ( 1 or 5 m ) and 0.9 m doxorubicin ( a ) or 0.09 m doxorubicin ( b ) alone or in combination for 72 hr before determination of cell viability by mtt assay . effects of supplementation of pge2 on enhancement of growth inhibitory activity of doxorubicin caused by deracoxib in cmt - u27 cells . cells were treated with or without deracoxib ( 50 - 250 m ) in the absence or presence of pge2 ( 1 or 5 m ) and 0.9 m doxorubicin ( a ) or 0.09 m doxorubicin ( b ) alone or in combination for 72 hr before determination of cell viability by mtt assay . effects of dox and der alone or in combination on bcl-2 expression in cmt u27 cells : to determine the apoptotic pathway activated by der , we examined the apoptosis - related target bcl-2 in cmt - u27 cells . these effects were more pronounced in the combinations of dox ( at 0.9 m ) with der ( at 100 m and 250 m ) ( table 3table 3.effects of deracoxib or doxorubicin alone or in combination on bcl-2 expression in cmt - u27 cellsdrugsconcentrationbcl-2control-+++dox0.9 m++der50 m++der100 m+der250 m+dox + der0.9 m + 50 m++dox + der0.9 m + 100 m+dox + der0.9 m + 250 m+dox0.09 m+++dox + der0.09 m + 50 m+++dox + der0.09 m + 100 m++dox + der0.09 m + 250 m++dox , doxorubicin ; der , deracoxib . however , the combinations of dox at 0.09 m with der ( at 100 m and 250 m ) decreased the expression of bcl-2 slightly . these anticancer properties make it worthwhile to examine the possible benefit of combining selective cox-2 inhibitors with conventional anticancer therapies , such as chemotherapy . similarly , during the last decade , various preclinical and clinical trials in the field of veterinary oncology have been conducted to study the use of cox-2 selective inhibitors in combination with other agents in early and advanced cancers and have been shown to improve treatment outcome in dogs with transitional cell carcinoma and osteosarcoma [ 34 , 70 ] . however , to our knowledge , there is no published report demonstrating the therapeutic efficiency of selective cox-2 inhibitors with conventional anti - cancer agents in canine mammary tumor . based on the encouraging results from cox-2 inhibitors as chemotherapeutic and chemopreventive agents in the treatment of several types of human and canine malignancy including mammary tumor [ 2 , 14 ] , we evaluated the potential of a selective cox-2 inhibitor ( der ) in potentiating the antitumor activity of a conventional cytotoxic agent ( dox ) in vitro in canine mammary carcinoma cells ( cmt - u27 ) . the ic50 of 876 nm determined for dox in cmt - u27 cells was within the range of clinically relevant concentrations , as dogs that were treated with 1 mg / kg dox achieved plasma concentrations of 0.7 g / ml ( 1.2 m ) 5 min after intravenous administration . cmt - u27 cells have a high growth rate and anti - apoptotic potential associated with enhanced expression of genes involved in the ca signaling pathway and growth hormone cellular pathway . therefore , we concluded that cmt - u27 cells could be an in vitro model for canine mammary cancer refractory to dox chemotherapy . our results demonstrated that der enhanced the cytotoxic action of dox at 0.9 m in cmt - u27 cells in a dose dependent manner , which decreased the ic50 value from 0.876 m ( dox at 0.9 m ) to 0.303 m , 0.245 m and 0.236 m ( 0.9 m in combination with 50 , 100 and 250 m der ) , respectively . the addition of der to 0.9 m dox , even at concentrations that did not affect cell viability , achieved at least the same level of cytotoxicity as an approximately 3- to 3.5-fold higher dose of dox alone in the cell line . in contrast , der exerted no effect on the action of dox at 0.09 m in the cell line . these results suggest that the enhancement of cytotoxicity in combination treatment of dox with der depends on the dox chemotherapy dose and that the addition of der ( 50250 m ) to dox ( 0.9 m ) therapy may be beneficial to reduce the chemotherapy dose necessary to achieve a cytotoxic response in canine mammary cancer . interaction analysis of the data showed that the combinations of dox at 0.9 m with der produced synergism in the cmt - u27 cell line , with a ratio index ranging from 1.98 to 2.33 . in our study , we found that exogenous addition of pge2 ( 15 g / ml ) did not reverse the antiproliferative effect of der in combination with dox . these results suggest that growth inhibition in cmt - u27 cells by dox and its combination with der are not directly associated with their ability to suppress pge2 production . the synergistic and additive effects on cmt - u27 cells elicited by treatment with dox ( 0.9 and 0.09 m , respectively ) and der ( especially at the concentrations of 100 and 250 m ) are associated with a marked increase in apoptosis . our results demonstrated that der ( 100 and 250 m ) enhanced the apoptotic activity of dox at 0.9 and 0.09 m in the cells , which increased the apoptotic index from 36.22% to 48.91% and 49.58% and from 24.02% to 35% and 37.69% , respectively . to define the mechanistic role of der in apoptosis induction , we examined bcl-2 expression , which is known to promote cell survival and has been correlated with the development of dox resistance , in cmt - u27 cells . we showed that der ( 100 and 250 m ) induced downregulation of bcl-2 at 0.9 m dox more strongly than at 0.09 m dox in cmt - u27 cells . we concluded that der may be altered the sensitivity of cmt - u27 cells to dox - induced apoptosis by downregulating bcl-2 expression . in additional studies identifying the mechanism of observed synergistic and additive effects in treatment with dox and der , we found that der potentiated dox - caused g0/g1 arrest in cell cycle progression , while dox - induced cell cycle arrest in the g0/g1 phase , as well as induction of cell death , has been demonstrated in breast cancer mcf-7 cells . we showed that a low concentration of dox ( 0.09 m ) induced higher g0/g1 arrest compared with a high concentration of dox ( 0.9 m ) . on the other hand , the percentages of cmt - u27 cells arrested at the g0/g1 phase are inversely proportional to the dox concentrations , and this could arise from the density of the viable cells observed with both dox treatments , because high concentrations of dox lead to necrosis . this is the first study to demonstrate that dox and dox with der induced cell cycle arrest in cmt - u27 cells , and the molecular mechanism of accumulation of cells in the g0/g1 phase induced by der in the presence of dox is unknown . we suggest that the increase in the percentage of cmt - u27 cells in the g0/g1 phase may be related to the accompanying changes in the level of proteins regulating the cell cycle and the decline of cell proliferation activity . in conclusion , we elucidated that der enhanced the antiproliferative effect of dox in conjunction with induction of apoptosis by modulation of bcl-2 expression and changes in the cell cycle of the cmt - u27 cell line . although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed for der and dox combinations require further investigations , the results suggest that the synergistic effect of dox and der combinations in cmt therapy may be achieved at relatively lower doses of dox with lesser side effects .

in caenorhabditis elegans , attenuation of the insulin / igf-1 signaling ( iis ) pathway during adulthood leads to significant changes in worm physiology , including extended life - span , increased innate immunity and stress - resistance , and extensively altered metabolism . these phenotypic adaptations rely , for the most part , on the activation and nuclear translocation of the forkhead box o ( foxo ) transcription factor daf-16 , which triggers an extensive genetic program that is normally associated with an alternate developmental arrest state called the dauer diapause . in mammals , disruption of the insulin receptor in the brain is associated with expression of endocrine neuropeptides that stimulate the uptake of food , leading to obesity and symptoms of type ii diabetes along with reduced reproductive competence . in this respect , iis in c. elegans appears remarkably similar to mammalian insulin signaling in that the nematode nervous system ( together with the intestine ) was identified as a central regulator of daf-2-mediated energy metabolism and longevity . although disruption of iis in nematodes does not lead to any clear pathological conditions , the conservation of this pathway in the regulation of fat and carbohydrate metabolism as well as in life - span determination from nematodes to mammals is at least striking . therefore , a thorough understanding of daf-2-specific metabolism and its regulation , including species - specific peculiarities , may lead to the discovery of novel metabolic pathways involved in insulin - dependent pathologies or other fundamental processes such as aging in humans . whole - genome transcript profiling of daf-2 mutants and/or dauers , using either microarray or sage analysis , has proven to be successful at identifying differentially expressed gene sets dependent on daf-16 , some of which are involved in energy metabolism . however , reported transcript profiles , especially with regard to energy metabolism , were sometimes inconsistent between studies , complicating the formulation of straightforward statements about iis - mediated metabolism . moreover , the poor correlation between mrna transcript levels and actual protein abundances necessitates great caution when drawing conclusions based solely on interpreting transcript profiles . proteomics analysis by dong et al . of daf-2(e1370 ) mutants has identified a limited set of enzymes in carbohydrate metabolism ( glycolysis / gluconeogenesis ) as well as the tca and glyoxylate cycles that are differentially expressed in daf-2 mutants and showed that these enzymes are important determinants of nematode life span . recently , several groups have published the first metabolite profiles of the daf-2 mutant , revealing significant shifts in carbohydrate , amino acid , and lipid metabolism , several of which were not apparent from previously published transcriptomics studies . although these incentives contribute significantly to the identification of altered metabolic pathways upon impaired iis , an integrated understanding of the metabolic restructuring and its impact on worm physiology and longevity is still lacking . using lc ms / ms - based proteomics analysis , we found that activation of daf-16 in the nucleus results in over - representation of many enzymes of major , often reciprocally regulated , metabolic pathways involved in the synthesis and utilization of internal carbohydrate ( glycolysis and gluconeogenesis ) , lipid stores ( lipogenesis and lipolysis ) , amino acid metabolism , and respiration ( aerobic and anaerobic ) . in the following sections , we integrate our proteomic data set together with a selection of enzymatic activity assays , available literature , and metabolomics data in an attempt to reconstruct and resolve the intricacies of altered daf-2 metabolism . the following strains were used in this study : nlga154 glp-4(bn2ts)i;daf-2(e1370)iii ( long - lived ) and ga153 glp-4(bn2ts)i daf-16(mgdf50)i;daf-2(e1370)iii ( reference strain ) , which were kindly provided by david gems at the university college of london . the glp-4(bn2 ) background allele results in germline - deficient worms when grown at the nonpermissive temperature of 24 c . this allows for focusing on the proteome of aging somatic cells , excluding the gonadal and complex embryonic material . and tekipe and alballay demonstrated that no increase in lifespan results from the presence of the glp-4(bn2 ) allele in otherwise wild - type or daf-16;daf-2 mutants grown on live escherichia coli . however , glp-4(bn2 ) causes a small but significant daf-16-dependent extension of lifespan of daf-2 mutants and nematodes grown on killed e. coli . previous ms proteome profiling of glp-4(bn2 ) after n metabolic labeling revealed only modest changes compared to the n2 wild - type . the rnai - hypersensitive nl2099 rrf-3(pk1426 ) ii strain was used for rnai lifespan analyses . the initiation and culturing of large age - synchronized populations have been described in depuydt et al . to prevent glp-4daf-16;daf-2 animals from becoming dauer larvae , worms were grown at 16 c until the third larval stage ( l3 ) and were then shifted to 24 c for the remainder of the experiment . although shifting temperature at the l3 stage rendered glp-4;daf-2 animals sterile , we noticed that by day 2 of adulthood glp-4daf-16;daf-2 mutants exhibited gonads with incomplete morphogenesis , yet they were able to produce 24 eggs per animal . therefore , we opted to add 5-fluoro-2-deoxyuridine ( fudr , 75 m fc . , acros organics ) to all mass cultures in all biological replicates in order to maintain complete sterility . we note that the use of fudr in light of recent reports warrants caution , especially with regard to insulin / igf-1 mutants . however , we argue that our data reflect bona fide metabolic changes in response to the daf-2 mutation . our results are in line and corroborate with the reported metabolomics findings by fuchs et al . who used both the daf-2(e1370 ) and daf-2(m41 ) alleles without the use of fudr in their cultures . also , the daf-2(e1370 ) proteomic fingerprint made by dong et al . in the absence of fudr identified a limited number of differentially expressed enzymes in carbohydrate , fatty acid , and amino acid metabolism whose expression pattern was confirmed in our study . a wealth of transcriptomics data is available that show extensive changes in metabolic gene expression upon daf-16 activation in the absence of fudr . although there is poor concordance between these studies , our data correspond reasonably well with reported microarray profiles ( but it clashes with sage - generated transcript profiles ) . we also note that the fudr concentration used by the davies study is very high ( 400 m , compared to the more - than - sufficient 75 m used in our study ) , and the dose - dependency of fudr was not been taken into account . this 400 m concentration , in our hands , is borderline toxic for c. elegans . for all experiments , samples were collected at day 2 of adulthood and freed from dead animals , debris , and bacteria through percoll ( sigma - aldrich ) washing and flotation on a 60% w / w sucrose solution . animals were immediately flash frozen in liquid nitrogen and stored at 80 c . for comparing the metabolic profile between long - lived glp-4(bn2);daf-2(e1370 ) and reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm strains , the accurate mass and time ( amt ) lc ms proteomics data set was used , which was published earlier by our group in depuydt et al . and is freely available through peptideatlas at http://www.peptideatlas.org/pass/pass00308 ( see supporting information file 1 for details ) . the same criteria as in depuydt et al . were used to determine relative protein abundances , with the exception of the following proteins that included redundant peptides arising from different expression isoforms of the same gene : tps-1 , r11a5.4 , w05g11.6 , t22f3.3 , mdh-1 , h24k24.3 , gpd-1,-2,-3,-4 , gpi-1 , and enol-1 . proteomics data was visualized with mev ( multiexperiment viewer ) , part of the tm4 microarray software suite . pavlidis template matching ( ptm ) was used to rank proteins conforming to an expression pattern of interest . the ptm algorithm allows a data set to be searched for proteins of which the abundance profile matches a user - defined template profile , which is based on the pearson correlation between the template and the proteins in the data set.the kyoto encyclopedia of genes and genomes ( kegg ) pathway and uniprot knowledgebase were used in conjunction with the process of reconstructing the c. elegans metabolic network . gene set - enrichment analysis ( gsea ) was used to determine whether metabolic pathways show statistically significant differential expression as described in detail in subramanian et al . in short , a ranked list of proteins is generated according to their differential abundance level between two experimental groups . an enrichment score ( es ) test statistic is calculated that reflects the degree to which a defined set of proteins is overrepresented at one of the extremes of the entire ranked list . positive es values correspond with enrichment for enzymes ranked to have higher expression levels in daf-2 mutants , whereas negative es values correspond with enrichment for enzymes ranked to have lower expression levels . gene sets eligible for gsea are reported with their nominal p value and false discovery rate ( fdr ) , calculated from 1000 permutations of the experimental group labels . the nominal p value estimates the statistical significance of the es for a single set . the fdr is the estimated probability that an enriched set represents a false positive finding . subramanian et al . have suggested a fdr cutoff of 25% as appropriate to signify biologically significant results . the gsea - p software used for gsea can be found at the broad institute s web site http://www.broadinstitute.org/gsea/index.jsp . elegans nematodes were fixed , and transverse sections were taken according to fonderie et al . electron microscopy was performed using a jeol jem 1010 ( jeol , tokyo , japan ) operating at 60 kv . digitizing of images was performed using a ditabis system ( pforzheim , germany ) . determination of relative fat content in individual worms by oil red o staining was performed according to the protocol described in orourke et al . briefly , age - synchronized worms were sampled at different ages and washed in s - buffer ( 0.05 m k2hpo4 , 0.05 m kh2po4 , and 100 mm nacl , ph 7.4 ) . nematodes were fixed in mrwb - buffer ( 160 mm kcl , 40 mm nacl , 14 mm na2egta , 1 mm spermidine - hcl , 0.4 mm spermine , 30 mm na - pipes , ph 7.4 , and 0.2% -mercaptoethanol ) containing 1% paraformaldehyde ( pfa ) for 1 h at room temperature with gentle stirring . nematodes were then washed with s - buffer ( ph 7.4 ) to remove pfa and incubated in 60% isopropanol for 15 min at room temperature to dehydrate . isopropanol was removed , and samples were stained overnight in 60% oil red o stain ( 0.5 g/100 ml isopropanol stock solution ) with gentle stirring at room temperature . after the dye was removed by washing in s - buffer containing 0.01% triton , animals were mounted and observed with a reichert - jung polyvar light microscope and imaged with an olympus camedia c-5050 digital camera . corel photopaint ( corel corporation , ottawa , canada ) and fiji were used for image processing and analysis . in short , the red channel of rgb images was converted to 8-bit grayscale , and the resulting pixel values were inverted . the density per worm surface was then determined by calculating the integrated density for each worm , normalized to the total worm surface . equal amounts of worms were flash frozen in liquid nitrogen and stored at 80 c . worms were homogenized by bead beating ( glass beads 0.2480.318 m ) in 50 mm sodium / potassium phosphate buffer , ph 7.0 , at 5000 strokes / min for 30 s. chaps was added at a 1% final concentration to the resulting homogenate , and the mixture was bead - beated again for another 30 s. the mixture was kept on ice for 15 min and centrifuged at 20 000 rcf for 8 min at 4 c . the enzymatic activities were assayed spectrophotometrically against the appropriate blanks at 25 c in microtiter plate wells using an infinite m200 multiplate reader ( tecan , mnnedorf , switzerland ) . isocitrate lyase , pyruvate kinase , aconitase , and phosphenolpyruvate carboxykinase activities were assayed as described previously by castelein et al . and references therein . 6-phosphogluconic dehydrogenase activity was assayed according to the method of bergmeyer et al . following the manufacturer s instructions ( sigma no . p4553 ) , with the exception that only half of the described concentrations were used for nadp and 6-phosphogluconate . fumarase activity was assayed following the protocol of racker , with the exception that only 8 mm sodium malate was used ( final concentration ) instead of the 50 mm in racker s work . activity of 3-hydroxyacyl - coa dehydrogenase , catalyzing the conversion of s - acetoacetyl - coa to -hydroxybutyryl - coa with consequent nadh consumption , was monitored according to the method by lynen et al . all enzyme activities were scaled to the protein concentration of the worm extracts , estimated by the bicinchoninic acid protein assay kit ( pierce , rockford , il , usa ) . functional annotation clustering of the proteomics data set showed a significant enrichment of proteins involved in intermediary metabolism among long - lived glp-4(bn2);daf-2(e1370 ) compared to reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm strains ( throughout the text , we will refer to both strains as daf-2 and daf-16;daf-2 , respectively ) . during culturing of the worms , because the use of fudr in c. elegans aging studies warrants caution , we discuss this issue in more detail in the materials and methods ( c. elegans culturing and sampling ) . table 1 shows the result of functional annotation clustering for entries in the kegg database category , which deals mainly with annotation of intermediary metabolism genes . by searching the uniprot protein knowledgebase and on the basis of the available literature , we were able to reconstruct many of the pathways in c. elegans intermediary metabolism from our data set , which is the focus of this article . we detected increased protein abundance of many glycolytic enzymes in daf-2 mutants , including hexokinase ( h25p06.1 ) and phosphofructokinase ( y71h10a.1 ) , two major regulatory sites of glycolysis that catalyze irreversible reactions ( figure 1 ) . the only exception occurs in the final ( irreversible ) step in glycolysis catalyzed by the pyruvate kinase enzymes pyk-1 and pyk-2 , which remain unaltered in daf-2 mutants . we also find increased protein expression of enzymes in gluconeogenesis : fructose-1,6-bisphosphatase ( fbp-1 ) , pyruvate carboxylase ( pyc-1 ) , and two isoforms of phosphoenolpyruvate carboxykinase ( pepck ) , r11a5.4 and w05g11.6 . pepck constitutes a key activator of gluconeogenesis , and elevated levels of pepck in daf-2 mutants confirm earlier reports on pepck transcript and protein levels in insulin / igf-1 signaling - defective mutants . simultaneous activation of glycolysis and gluconeogenesis would lead to a futile cycle , generating heat at the cost of atp hydrolysis . this seems unlikely because daf-2 mutants have lower mass - specific thermogenesis and both pathways are reciprocally regulated . to investigate the relative activities of glycolysis / gluconeogenesis , we measured in vitro enzymatic activities of pepck ( gluconeogenesis ) and pyruvate kinase ( glycolysis ) ( figure 1 ) . surprisingly , in stark contrast with the more than doubled pepck enzyme levels , relative pepck enzymatic activity in daf-2 mutants was only 50% compared to the daf-2;daf-16 reference , indicating that gluconeogenesis in young adult daf-2 mutants is repressed . in contrast , pyruvate kinase activity remains unchanged in the daf-2 mutant , suggesting normal glycolysis activity . ( left ) heat map of proteins that are part of carbohydrate and fermentative metabolism ( base-2 logarithmic scale ) . red and green colors indicate a relative increase and decrease in protein content for a particular protein , respectively ( row ) . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . proteins are grouped according to whether protein profiles comply with a preset template as defined by the pavlidis template ( pt ) matching algorithm ( see materials and methods and ref ( 51 ) ) . green and red pt bars indicate lower and higher protein levels among strains , respectively . gray pt bars indicate protein profiles that do not match any obvious expression pattern ( see materials and methods for details ) . gene set enrichment analysis ( gsea ) allows for the assessment of whether a group of proteins ( e.g. , the set of genes that are part of carbohydrate metabolism in our data set ) shows statistically significant differential expression as a whole ( see materials and methods and ref ( 54 ) for more details ) . es , enrichment score from gene set enrichment analysis of the metabolic pathway ; p , nominal p value of the es ; fdr , false discovery rate . the color of protein names matches their expression profile ( green , down ; red , up ; and black , not significant ) . green arrows depict the possible route of malate production via the reverse action of phosphoenolpyruvate carboxykinase and malate dehydrogenase ( see results and discussion ) . inset bar graphs represent relative enzyme activity of phosphoenolpyruvate carboxykinase ( pepck ) and pyruvate kinase ( pk ) . ( 1 ) glycogen synthase , ( 2 ) glycogen phosphorylase , ( 3 ) hexokinase / glucokinase , ( 4 ) phosphohexoseisomerase , ( 5 ) phosphofructokinase , ( 6 ) fructose-1,6-bisphosphate , ( 7 ) aldolse , ( 8) triose phosphate isomerase , ( 9 ) glyceraldehyde-3-phosphate dehydrogenase , ( 10 ) phosphoglycerate kinase , ( 11 ) phosphoglycerate mutase , ( 12 ) enolase , ( 13 ) pyruvate kinase , ( 14 ) pyruvate dehydrogenase complex ( e1 , pyruvate dehydrogenase ; e2 , dihydrolipyl transacetylase ; and e3 , dihydrolipoyl dehydrogenase ) , ( 15 ) pyruvate carboxylase , ( 16 ) mitochondrial / cytosolic malate dehydrogenase , ( 17 ) malate--ketoglutarate antiporter ( malate shuttle ) , ( 18 ) phosphoenolpyruvate carboxykinase , ( 19 ) phosphoglycerate dehydrogenase , ( 20 ) lactate dehydrogenase , ( 21 ) alcohol dehydrogenase , ( 22 ) aldehyde dehydrogenase , ( 23 ) acetyl - coa synthetase , and ( 24 ) trehalose-6-phosphate synthase . glu-6p , glucose-6-phosphate ; fru-6p , fructose-6-phosphate ; fru-1,6-bp , fructose 1,6-bisphosphate ; dhap , dihydroxyacetone phosphate ; g3p , glyceraldehyde-3-phosphate ; 3pg , 3-phosphoglycerate ; pep , phosphoenolpyruvate ; and ooa , oxaloacetate . we also found significantly elevated protein levels of both glycogen synthase-1 ( gsy-1/y46g5a.31 , glycogenesis ) and the orthologue of human glycogen phosphorylase ( t22f3.3 , catalyzing the first step in glycogenolysis ) in daf-2 adults ( figure 1 ) . ultrastructural and biochemical studies showed daf-2 mutants and dauers store significantly more glycogen in their intestinal and hypodermal cells compared to wild - type nematodes . electron micrographs taken from our samples also confirmed the presence of high amounts of glycogen in hypodermal cells and body - wall muscles of daf-2 mutants ( figure 2 ) , consistent with increased glycogen synthase activity . the fermentation of stored glycogen constitutes a major source of energy during anoxia / hypoxia and is determinate for nematode survival under these conditions . consistently , the increased survival of daf-2 mutants during anoxic insult is dependent on its high glycogen levels . in addition , in dauer larvae , high glycogen reserves likely serve to maintain their high degree of motility during the first few weeks after dauer formation , which is in agreement with the expression of glycogen phosphorylase in c. elegans muscles . we found significantly increased protein levels of tps-1 , one of two c. elegans trehalose-6-phosphate synthase genes responsible for trehalose biosynthesis , consistent with increased trehalose levels in iis - defective c. elegans mutants and dauers and daf-16-dependent transcription regulation . importantly , tps-1 and tps-2 were found to be required , in part , for daf-2 longevity . besides its role in carbohydrate storage and transport , trehalose also has extensive cytoprotective functions , most probably by stabilizing the proteome and lipid membranes under various stress conditions , including cold , heat , dehydration , hypoxic , and oxidative insult . elevated levels of trehalose are associated with life - span extension in c. elegans(74 ) and also render daf-2 dauers resistant to extreme desiccation . transmission electron microscopy images of transversal midbody cross sections of day 2 adult nematodes . ( left ) daf-2 mutants exhibit relatively large amounts of glycogen ( delineated in green ) and fat droplets ( orange arrowhead ) stored inside hypodermal and intestinal cells compared to the reference strain . daf-2 mutants also show a smaller cross - sectional diameter compared to the daf-16;daf-2 reference . quantitative determination of worm length , diameter , and volume of both daf-2 and daf-2;daf-16 worms is shown in supporting information file 2 . ( right ) high - magnification image ( 25 000 ) of a small section of daf-2 striated muscle tissue showing the tight organization of glycogen and mitochondria close to the myofilament lattice . our data show a significant upregulation in the expression of three enzymes predicted to be part of the pentose phosphate pathway , including gspd-1 , the c. elegans orthologue of glucose-6-phosphate dehydrogenase ( g6pdh ) , which catalyzes the first step in the pentose phosphate pathway and converts nadp into nadph ( figure 3 ) . however , we found no significant difference in the activity of this pathway by measuring the enzymatic activity of 6-phosphogluconate dehydrogenase ( figure 3 ) . nonetheless , temporal activation of this pathway could be involved in the increased oxidative stress resistance of the daf-2 mutant because the rerouting of carbohydrate intermediates from glycolysis to the pentose phosphate shunt has been shown to be part of a coordinated response to oxidative stress by maintaining high cytoplasmatic nadph / nadp ratios in c. elegans , drosophila , and mammals . ( right ) heat map ( base-2 logarithmic scale ) and ( left ) schematic overview of proteins that are part of the pentose phosphate pathway . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; * * * , p < 0.0005 . ( 1 ) glucose-6-phosphate degydrogenase , ( 2 ) gluconolactone hydrolase , ( 3 ) 6-phosphogluconate dehydrogenase , ( 4 ) ribulose-5-phosphate-3-epimerase , ( 5 ) ribose-5-phosphate - ketoisomerase , ( 6 ) transketolase , ( 7 ) transaldolase , and ( 8) ribokinase . inset bar graph represents relative enzyme activity of 6-phosphogluconate dehydrogenase ( 6pgdh ) . a strong upregulation was found of several putative alcohol dehydrogenases and aldehyde dehydrogenases involved in alcohol metabolism ( figure 1 ) . alcohol dehydrogenases include the predicted sorbitol dehydrogenases sodh-1 and its homologue r04b5.5 , the ribitol - dehydrogenase domain containing protein f20g2.2 . the actual substrate of these alcohol dehydrogenases is unknown at present , but sodh-1 appears capable of metabolizing ethanol . sodh-1 is known to be activated by daf-16 , and higher mrna levels of sodh-1 were also found in long - lived worms cultured axenically . furthermore , sodh-1 was the most upregulated ( 1200% ) spot in 2d - dige gels from staphylococcus aureus - treated worms but not in aeromonas hydrophila - treated worms . therefore , the biological role of this enzyme may reach further than fermentative metabolism . indeed , some alcohol dehydrogenase enzymes are also capable of neutralizing cytotoxic aldehydes originating from lipid peroxidation , including 4-hydroxynonenal ( 4-hne ) . alh-1 is an orthologue of mitochondrial aldehyde dehydrogenase 2 in humans that possibly catalyzes the conversion of acetaldehyde , resulting from the oxidation of ethanol , into acetate . although we found strongly increased abundance of alh-1 in daf-2 mutants , metabolomic profiles of daf-2 mutants indicate a significant drop in acetate content in iis mutants . alh-1 is also involved in the detoxification of toxic aldehyde byproducts of alcohol metabolism or lipid peroxidation . more specifically , c. elegans alh-1 was shown capable to oxidize 4-hne further to the lesser reactive 4-hna ( 4-hydroxynon-2-enoic acid ) , similar to mammalian aldehyde dehydrogenase . on the basis of these reports , the activation of alcohol - fermenting enzymes in daf-2 mutants may well be part of a coordinated stress response system that boosts the nematodes resistance to toxic byproducts of lipid and intrinsic alcohol metabolism . a general increase was found of the citric acid cycle enzymes , including citrate synthase , the isocitrate dehydrogenase complex , and the -ketoglutarate dehydrogenase complex , which was the main sites of regulation within this cycle ( figure 4 ) . this increase is accompanied with higher activity of fumarase and aconitase in vitro ( figure 4 ) . transcript profiles of daf-2 mutants did not show any significant change in mrna levels for citric acid cycle enzymes . expression of these genes therefore may be regulated post - transcriptionally or be the result of lowered turnover of these enzymes or the mitochondria in which they reside . here , daf-2 energy metabolism appears to differ from that of the dauer larva , which is characterized by both reduced gene expression and enzymatic activity of citric acid cycle genes . however , aco-1 is thought to be a cytosolic aconitase involved in the regulation of cellular iron concentrations and is therefore not part of the citric acid cycle . similarly , idh-1 is a predicted cytosolic nadp - dependent isocitrate dehydrogenase ( idh ) and shows no upregulation in daf-2 . we note here that cytosolic nadp - dependent idh activity is strongly increased in the daf-2 mutant ( data not shown ) . ( left ) heat map ( base-2 logarithmic scale ) and ( right ) schematic overview of enzymes in mitochondrial intermediary metabolism . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . inset bar graphs represent relative enzymatic activities of aconitase , fumarase , and isocitrate lyase . ( 1 ) pyruvate dehydrogenase complex , ( 2 ) citrate synthase , ( 3 ) aconitase , ( 4 ) isocitrate dehydrogenase complex , ( 5 ) -ketoglutarate dehydrogenase complex ( e1 , oxoglutarate dehydrogenase ; e2 , dihydrolipoyl succinyl transferase ; and e3 , dihydrolipoyl dehydrogenase ) , ( 6 ) succinate thiokinase , ( 7 ) succinate dehydrogenase , ( 8) fumarase , ( 9 ) malate dehydrogenase , ( 10 ) pyruvate carboxylase , ( 11 ) isocitrate lyase , ( 12 ) malate synthase , ( 13 ) mitochondrial tri / dicarboxylate carrier protein , ( 14 ) gdh-1 , glutamate dehydrogenase ; w07e11.1 , glutamate synthase , ( 15 ) malate--ketoglutarate antiporter ( malate shuttle ) , and ( 16 ) aspartate aminotransferase . succ , succinate ; succ - coa , succinyl - coa ; aco , aconitase ; and fum , fumarase . the glyoxylate shunt allows the synthesis of carbohydrates via gluconeogenesis from acetyl - coa obtained from fatty acid -oxidation and is generally known in plant and microorganism biochemistry . nematodes also have been shown to contain an active glyoxylate shunt , which is required for the full longevity phenotype of daf-2 and several etc - defective mutants ( mit mutants ) as well as the mitochondrial mutant clk-1(qm30 ) . in c. elegans , the key glyoxylate cycle enzymes , isocitrate lyase and malate synthase , we observed a very strong increase in icl-1 abundance , consistent with previous transcriptional evidence . consistently , in vitro activity of both isocitrate lyase and malate synthase is increased in daf-2 ( figure 4 ) and age-1(98 ) mutants , respectively . one - carbon metabolism involves the storage and utilization of one - carbon moieties in anabolic and transmethylation - based regulatory pathways . the carrier of activated one - carbon fragments is tetrahydrofolate ( thf ) , an essential water - soluble derivative of vitamin b9 involved in the formation of nucleotides for dna synthesis , the metabolism of certain amino acids , and the source of methyl groups for s - adenosyl methionine ( sam ) . a particularly strong increase was found in the protein levels of the putative c. elegans aicar formyltransferase ( c55f2.1 ) and c1-thf synthase ( k07e3.4 ) in daf-2 , both of which are involved in the interconversion of one - carbon derivates of thf ( figure 5 ) . serine is the major donor of one - carbon in the form of n , n - ch2-thf through its catabolism via glycine . we found increased levels in the daf-2 mutant of phosphoglycerate dehydrogenase ( c31c9.2 ) , which catalyzes the first step in glucose - derived serine biosynthesis , and f25b4.1 , the c. elegans orthologue of the glycine cleavage system t - protein , an aminomethyltransferase that catalyzes the actual transfer of methylene carbon from the decarboxylated glycine to tetrahydrofolate ( thf ) . taken together , our results suggest changes in thf - based anabolism in daf-2 mutants , possibly fueled via de novo synthesis of serine and subsequent oxidation over glycine to supply one - carbon units . ( right ) heat map ( base-2 logarithmic scale ) of enzymes part of c. elegans one - carbon metabolism . ( left ) shematic overview of one - carbon metabolism involving both the single - carbon carriers tetrahydrofolate and s - adenosylmethionine . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * p < 0.0005 . ( 1 ) phosphoglycerate dehydrogenase ( c31c9.2 ) , phosphoserine aminotransferase ( f26h9.6 ) , ( 2 ) serine hydroxymethyl transferase , ( 3 ) glycine cleavage system t - protein ( aminomethyltransferase ) , ( 4 ) aicar formyltransferase ( c55f2.1 ) , c1-thf - synthase ( k07e3.4 ) , ( 5 ) s - adenosyl methionine synthase , ( 6 ) methionine synthase , ( 7 ) s - adenosylhomocysteinase , ( 8 and 10 ) cystathionine -synthase / cysteine synthase , and ( 9 ) cystathionine -lyase . in contrast to thf - metabolism , the biosynthetic pathway of sam , the other main carrier for single - carbon units , appears repressed in daf-2 mutants ( figure 5 ) . s - adenosylmethionine synthase activity produces sam by catalyzing the condensation of methionine with atp . protein levels of this enzyme ( sams-1 ) are strongly reduced in the daf-2 mutant . demethylation of sam results in the production of s - adenosylhomocysteine ( sah ) , which is a strong inhibitor of all sam - dependent transmethylation reactions . s - adenosylhomocysteinase catalyzes the hydrolytic cleavage of sah into homocysteine plus adenosine , but expression of this enzyme ( ahcy-1 ) at the protein level also appears significantly reduced in daf-2 mutants . homocysteine lies at the cross - point of two competing pathways : it is either used to form methionine ( for sam synthesis ) via the remethylation pathway or to form cysteine via the transsulfuration pathway . transsulfuration of homocysteine to cysteine is catalyzed by two enzymes : cystathione -synthase ( cbs ) , which forms cystathionine from homocysteine and serine , and cystathionine -lyase , which converts cystathionine to cysteine , -ketobutyrate , and ammonia . the putative c. elegans orthologue of cbs ( k10h10.2 ) is strongly upregulated in daf-2 , suggesting increased funneling of homocysteine toward cysteine synthesis , away from remethylation of homocysteine into methionine . because cysteine is normally readily available from diet , the major role of the transsulfuration pathway in animals is thought to be the regulation of sam levels by controlling methionine / homocysteine degradation and not cysteine biosynthesis per se thus , high cbs activity in daf-2 mutants is an additional strong indication of repressed sam synthesis . in contrast , the putative c. elegans cystathionine -lyase orthologue cth-1 is downregulated , and its paralogue , cth-2 , shows no change in expression . because there is no apparent alternate route for the further processing of cystathionine , it seems that reduced cth-1 expression aims to temper the increased flux through cbs . taken together , our data strongly suggest a reduced flux through the methyl cycle in daf-2 mutants . in mammals , fatty acid synthesis is initiated by citrate transport across the mitochondrial membranes into the cytoplasm via tricarboxylate carriers ( tic ) in exchange with malate . once in the cytosplasm , citrate is cleaved by atp - citrate lyase ( acl ) into acetyl - coa and oxaloacetate . this enzyme is the main source for cytosolic acetyl - coa for fatty acid synthesis and represents a major link between the citric acid cycle and lipogenesis . in mammals , expression of this mitochondrial citrate carrier is activated by insulin signaling , and inhibition of acl was shown to reduce fatty acid synthesis significantly and to increase fatty acid -oxidation . we found strongly increased abundance of the c. elegans mitochondrial tic ( k11h3.3 ) and acl ( d1005.1 ) in daf-2 mutants compared to the reference strain , suggesting increased lipogenesis ( figure 6b ) . paradoxically , we found a strong suppression of the predicted c. elegans fatty acid synthase , fasn-1 , in daf-2 adults . this suggests that in adult daf-2 worms fatty acid synthesis is attenuated even in the presence of plenty of substrates . we note that the c. elegans genome contains at least three other putative fatty acid synthase genes ( c41a3.1 , f10g8.9 , and f32h2.6 ) , which could also be important determinants of fatty acid synthesis . fatty acid metabolism . ( right ) heat map and ( left ) schematic overview of enzymes implicated in fatty acid synthesis . ( 1 ) citrate synthase , ( 2 ) mitochondrial tricarboxylate / dicarboxylate carrier protein ( citrate transport protein ) , ( 3 ) atp - citrate lyase , and ( 4 ) fasn-1 , fatty acid synthase ; elo-1 , long - chain fatty acid elongase ; dhs-25 , mitochondrial beta - ketoacyl - acp reductase ; fat-6 and fat-2 encode fatty acyl desaturases . 3-hadh , 3-hydroxyacyl - coa dehydrogenase . ( right ) heat map and ( left ) schematic overview of detected fatty acid -oxidation enzymes . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . ( 1 ) acyl - coa synthetase , ( 2 ) carnitine o - acyltransferase , ( 3 ) acyl - coa dehydrogenase , ( 4 ) trans-2-enoyl - coa hydratase , ( 5 ) 3-hydroxyacyl - coa dehydrogenase , ( 6 ) thiolase , ( 7 ) acetyl - coa acetyltransferase ( thiolase ) , ( 8) 3-hydroxy-3-methylglutaryl - coa ( hmg - coa ) lyase , and ( 9 ) succinyl - coa - acetoacetate coa transferase . at the same time , we found increased expression in daf-2 adults of many fatty acid -oxidation proteins ( figure 6a ) as well as an enzyme involved in glycerol catabolism ( t24g3.4 ) and a choline / carnitine palmitoyl transferase ( b0395.3 ) . the latter is possibly responsible for the translocation of long - chain fatty acids into the mitochondrial matrix prior to oxidation . we also found increased protein levels of maoc-1 , a predicted peroxisomal fatty acid -oxidase enzyme . to assess whether fatty acid -oxidation is increased in the daf-2 mutant , we measured the in vitro activity of 3-hydroxyacyl coa dehydrogenase , which catalyzes the oxidation of l-3-hydroxyacyl coa , and found that its activity was unchanged ( figure 6 ) . nonetheless , the coordinated upregulation of most -oxidation enzymes clearly indicates that daf-2(e1370 ) worms tend to rely more on internal fat stores compared to wild type to fuel their energy needs . the formed acetyl - coa is either further oxidized to co2 and water in the citric acid cycle or , alternatively , acetyl - coa is converted into succinate and malate via the upregulated glyoxylate cycle for use in gluconeogenesis . in addition , glyceraldehyde-3-phosphate derived from the catabolism of glycerol can be fed into the glycolytic pathway for energy generation . we note that the relative increase in -oxidation proteins found in our study appears not to be reflected at the transcript level , as both sage- and microarray - based transcript profiling detected no change in the mrna expression of -oxidation enzymes in daf-2 mutants . on the basis of our results , we hypothesize that daf-2 mutants switch from fat synthesis and storage during development and early adulthood to controlled lipid breakdown for the remainder of life , mimicking the dauer larva . to test this hypothesis , we determined the age - dependent alterations in fat content of daf-2 mutants and daf16;daf-2 reference worms by staining with the fat - soluble dye oil red o ( figure 7 ) . we combined these results with data on the feeding behavior of the same iis mutants ( but lacking the glp-4 mutation ) collected over many aging series that were run independently in liquid cultures in our lab . as expected , our data show that daf-2 mutants accumulate significantly more fat than the daf-16;daf-2 reference at day 2 of young adulthood . although we noticed a sudden drop in oil red o intensity at day 6 of adulthood , daf-2 mutants were able to maintain relatively high fat content , whereas in reference worms , fat levels decreased steadily with age . this result is remarkable given that daf-2(e1370 ) mutants exhibit a clear eat phenotype ( reduced food uptake ) that manifests itself during early adulthood ( figure 7 ) . therefore , daf-2 fat metabolism is reminiscent to the nonfeeding dauer larva , which depends on the slow and controlled release of energy and anabolic intermediates via -oxidation of internal fat stores for its survival . nematodes were stained with oil red o to determine the relative fat content in individual worms . for each time point , the amount of e. coli in grams required each day to maintain a constant turbidity ( od550 = 1.8 ) in culture medium ( liquid cultures ) was used as a measure to assess the feeding behavior as a function of age . we note that zero values for oil red o intensity do not indicate that these animals are devoid of fat but merely indicate the detection limit of this dye for quantifying fat levels . the carbon atoms of propionate are recycled into the citric acid cycle component succinyl - coa via an evolutionary conserved multienzyme pathway that was shown to be fully functional in c. elegans ( figure 8) . we detected significantly increased abundance of the propionate catabolic enzymes methylmalonyl - coa racemase ( mce-1 ) , methylmalonyl - coa mutase ( mmcm-1 ) , and the alpha ( pcca-1 ) and beta ( pccb-1 ) subunits of propionyl - coa decarboxylase in daf-2 adult animals ( figure 8) . ( right ) heat map and ( left ) schematic overview of propionate metabolism enzymes . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . ( 2 ) propionyl - coa carboxylase , ( 3 ) methylmalonyl - coa racemase , and ( 4 ) methylmalonyl - coa isomerase . propionate is either activated to propionyl - coa by an appropriate acyl - coa synthetase or originates directly from the catabolism of the branched amino acids valine and isoleucine or -oxidation of odd - chain - length fatty acids . activation of this catabolic pathway would thus indicate increased catabolism of fatty acids or branched chain amino acids in the daf-2 mutant . recent metabolomic profiling studies of the daf-2 mutant have revealed an increase in the concentration of free amino acids , in particular of branched - chain amino acids ( bcaas ) , suggesting altered amino acid metabolism in this mutant . in accordance , we report an increase in the level of glutamate dehydrogenase ( gdh-1 ) , an enzyme essential in amino acid breakdown that catalyzes the deamination of glutamate into -ketoglutarate ( figure 4 ) . we also find a dramatic increase ( 16.6-fold ) in the putative glutamate synthase w07e11.1 , which catalyzes the formation of glutamate from glutamine . in mammals , glutamine serves as a universal transporter of nitrogen and is the most common free amino acid in human blood plasma . interestingly , martin et al . showed that the level of glutamine and glutamate were increased and decreased , respectively , in the daf-2 mutant . we speculate that the strong increase of glutamate synthase in combination with glutamate dehydrogenase is indicative of increased amino acid catabolism in the daf-2 mutant , fueling the tca cycle . also consistent with increased amino acid catabolism is the clear increase of most tyrosine catabolic enzymes , including hpd-1 ( figure 9a ) . our result is consistent with an earlier proteomics report where fumarylacetoacetate hydrolase ( k10c2.4 ) was found to be significantly upregulated in the daf-2 mutant . moreover , the transcript level of cytosolic tyrosine aminotransferase ( tat , f24d1.2 ) , which catalyzes the first step in tyrosine catabolism , was found to be highly enriched in dauers . ( right ) heat map and ( left ) schematic overview of tyrosine catabolism enzymes . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . ( 1 ) ( tyrosine / aspartate ) transaminase , ( 2 ) p - hydroxyphenylpyruvate hydroxylase , ( 3 ) homogentisate oxidase , ( 4 ) maleylacetoacetate isomerase , and ( 5 ) fumarylacetoacetate hydrolase . ( 1 ) branched - chain aminotransferase , ( 2 ) branched - chain -keto acid decarboxylase complex , and ( 3 ) ivd-1 , isovaryl - coa dehydrogenase ; b0250.5 , 3-hydroxyisobutyrate dehydrogenase . the level of cellular tyrosine is thought to be determined by insulin signaling through modulation of tyrosine catabolism . like in mammals , c. elegans tat and the gluconeogenic enzyme pepck both contain an insulin - responsive element ( ire ) in the promoter region , conferring insulin - induced inhibition of expression . thus , rather surprisingly and in contrast with our and others proteome data , reduced insulin signaling in c. elegans has been associated with the transcriptional repression of tyrosine catabolic enzyme hpd-1 . this discrepancy between transcript and protein levels indicates complex regulation of tyrosine catabolism enzymes . in contrast to tyrosine , we found reduced concentrations of bcaa catabolic enzymes , including the branched - chain aminotransferase enzyme ( bcat-1 ) , in the daf-2 mutant ( figure 9b ) , in line with transcriptional evidence and paralleled by increased levels of bcaas as reported previously by us and others . finally , we also found increased abundance of the aspartate aminotransferases t01c8.5 and c14f11.1 ( figure 4 ) , which catalyze the interconversion of aspartate and -ketoglutarate into oxaloacetate and glutamate . an increased protein level of several subunits of complexes i and ii and to a lesser extent complexes iii and v was found in daf-2 mutants ( figure 10 ) . remarkably , protein expression of cytochrome c oxidase ( complex iv ) was repressed in daf-2 . cytochrome c oxidase catalyzes the terminal reduction of one o2 molecule to two molecules of h2o with the additional translocation of four protons to the intermembrane space . if complex iv activity is reduced in daf-2 animals , then it can be expected that less oxygen is consumed compared to wild type . a slightly reduced mass - specific metabolic rate , as measured by carbon dioxide gas respirometry , has been reported in daf-2 mutants . however , no decrease in oxygen consumption between daf-2(e1370 ) and wild - type or daf-16(m26);daf-2(e1370 ) has been observed in locomotory active worms . carbon dioxide gas respirometry is expected to leave the worms practically undisturbed on a spot of e. coli bacteria , whereas direct oxygen consumption measurements in liquid require extensive stirring , stimulating worm movement and thus metabolic activity , which could explain the discrepancy in reported daf-2(e1370 ) metabolic rates . in addition , adult daf-2 mutants cultured on undisturbed agar plates appear to be rather inactive when compared to the reference strain , but they retain the ability to move freely in response to , for example , mechanical stimulation , similar to dauers.daf-2 mutants are also characterized by reduced food uptake ( as was discussed earlier ) and low heat output . therefore , we suggest that daf-2 standard metabolism , like dauers , is hypometabolic , but the metabolic rate is readily raised when necessary , and this is ensured by the availability of large amounts of fat and glycogen and the increased levels of enzymes of core intermediary metabolism . schematic representation of the respiratory chain ( complexes i iv ) together with heat maps of detected subunits . numbers next to each heat map row denote fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . green arrows indicate the possible alternative flux of electrons via rhodoquinone ( rq ) ( instead of ubiquinone , uq ) over complex ii using fumarate as the final electron acceptor . we also found elevated protein levels of the electron - transferring flavoproteins ( etf ) f27d4.1 and etf - dehydrogenase ( let-721 ) in daf-2 mutants , although the latter upregulation was not statistically significant . these proteins transfer the electron pair of fadh2 , resulting from the -oxidation of acyl - coa , to the flavo - iron sulfur protein etf - dehydrogenase , which in turn reduces coenzyme q in the electron transport chain in the mitochondrial inner membrane . this is in line with the enhanced fatty acid oxidation pathway in daf-2 mutants , as described above . interestingly , mai-2 , a member of the f1 atpase inhibitor ( if1 ) family , showed a modest but significant upregulation in daf-2 adults . if1 inhibitor proteins prevent the deleterious hydrolytic consumption of atp by complex v under anoxic conditions when the electrochemical gradient across the inner membrane collapses . this finding is consistent with an earlier report of elevated transcriptional expression of if1 inhibitors in daf-2 mutants and dauers . increased expression of mai-2 may , in part , explain the ability of daf-2 to withstand prolonged hypoxic insult better . we found increased levels of most glycolytic enzymes ( with the exception of pyruvate kinase ) and gluconeogenic enzymes , including pepck . together with an experimentally confirmed increased flux of carbon through the glyoxylate shunt and high levels of the storage sugars glycogen and trehalose , it was assumed gluconeogenic activity is high in the daf-2 mutant . however , despite high pepck enzyme levels , a significant decrease of in vitro pepck activity was detected for the young adult daf-2 , implying reduced gluconeogenesis . in contrast , pyruvate kinase activity was similar between daf-2 and the daf-16;daf-2 reference , suggesting glycolytic flux is not drastically altered in daf-2 . in the event pyruvate kinase activity would be a limiting factor , the conversion of pep into pyruvate is prevented , favoring glucose production via gluconeogenesis . alternatively , as was suggested by oriordan and burnell , pepck potentially acts in reverse , driving co2 fixation via pep and oxaloacetate with the subsequent formation of malate via malate dehydrogenase , as is seen in parasitic helminthes ( green arrows in figure 1 ) . although increased levels of malate have been found in the daf-2 mutant , malate synthesis is most likely also increased via the upregulated glyoxylate shunt . helminths such as fasciola hepatica and ascaris suum are able to survive under anaerobic conditions in their host environment by generating atp via the malate dismutation pathway . the malate dismutation pathway appears to be conserved in c. elegans and is thought to play an essential role in dauer energy metabolism and survival . furthermore , rea and johnson hypothesized that a shift from aerobic to mitochondrial anaerobic respiration underlies daf-2 longevity and possibly most , if not all , other life - span extending mutations , including mit mutants . in this pathway , glycolytic phosphoenolpyruvate is converted into oxaloacetate by pepck , which is then further reduced by cytosolic malate dehydrogenase to produce malate . once malate is transported into ( partially ) anaerobically functioning mitochondria , it is further catabolized to generate atp from the proton gradient generated across the inner mitochondrial membrane by electron transport , but fumarate is utilized as the terminal electron acceptor instead of o2 , producing succinate instead of h2o . we speculate the strong increased abundance of the putative mitochondrial tricarboxylate carrier k11h3.3 could be responsible for the increased import of cytosolic malate into the mitochondrial matrix in exchange for citrate ( figure 6b ) . as was noted earlier , strong activation of the glyoxylate shunt in daf-2 mutants could also constitute a considerable source of intramitochondrial malate to drive anaerobic respiration . although we were able to quantify several enzymes involved in malate dismutation , including malic enzyme ( y48b6a.12 ) , asc ( c05c10.3 ) , and stk subunits ( c50f7.4 , f47b10.1 , and f23h11.3 ) , none of them showed any significant alteration in protein expression ( data not shown ) . however , we detected a clear upregulation of all propionate catabolism enzymes in daf-2 . propionate is one of the principal waste products ( along with acetate and succinate ) of malate dismutation and was previously found to be present in higher levels in the daf-2 mutant . lastly , the conspicuous downregulation of complex iv components but increases in complexes i iii could indicate that mitochondrial respiration in daf-2 relies less on oxygen as the terminal electron acceptor to maintain the proton gradient . taken together , these results support activation of the malate dismutation pathway in daf-2 , a hypothesis that merits further study . protein expression of enzymes in thf anabolism and the sam biosynthetic pathway were increased and decreased in the daf-2 mutant , respectively . interestingly , previous proteomic and metabolomic analysis of the pept-1 mutant revealed a very similar decrease in the abundance of one - carbon metabolism enzymes , including s - adenosylmethionine synthase ( sams-1 ) and s - adenosylhomocysteine hydrolase ( ahcy-1 ) , and changes in methionine ( decreased ) and homocysteine ( increased).pept-1 encodes an intestinal peptide transporter required for the absorption of amino acids in form of di- and tripeptides from the environment , and its mutation further extends daf-2 lifespan by approximately 60% . in contrast with our findings , however , the same group was unable to find these changes for the daf-2 mutant . nevertheless , reduced sams-1 protein levels in daf-2 have been reported before , and daf-2 is characterized as a strong transcriptional repression of pept-1 . sams-1 was previously discovered to be a mediator of the dr response , possibly downstream of tor signaling , and is required for a normal protein synthesis rate . in addition , metformin - induced lifespan extension in c. elegans was recently shown to result from methionine restriction following disruption of folate metabolism in the e. coli food source . therefore , one attractive hypothesis is that alterations in the methionine cycle results in reduced protein synthesis in the daf-2 mutant , the latter of which has been experimentally confirmed by us and others . by extension , we postulate decreased protein synthesis in the pept-1 mutant and metformin - treated c. elegans , which could conceivably underlie their longevity phenotype . moreover , because rnai knockdown of pept-1 results in a significant decrease in the intracellular pool of free amino acids , a concomitant decrease in protein synthesis is to be expected . several metabolomic profiling studies have revealed changes in the metabolism of amino acids in the daf-2 mutant . in particular , changes in bcaa metabolism have been linked to daf-2 , but it remains unclear how these changes contribute to daf-2 longevity , if at all . in mammals , bcaas ( leucine in particular ) are well - known to stimulate protein synthesis through activation of tor ( target of rapamycin ) kinase . however , because we recently showed that protein synthesis is repressed in the daf-2 mutant , such action by bcaas would thus have to be restricted to certain tissues , such as body - wall muscles , which we have shown previouslty are preserved from degradation in daf-2 . more recently , increased levels of aromatic amino acids ( phe and tyr ) and especially bcaa degradation products , such as glu , ala , and c3 and c5 acylcarnitines , were found to be strongly associated with insulin resistance and type 2 diabetes in humans . it was suggested that accumulation of these incompletely oxidized intermediates causes mitochondrial stress , leading to impaired insulin action . it is therefore conceivable that the rationale for the inhibition of bcaa catabolism in the daf-2 mutant is a ( futile ) attempt to restore normal insulin / igf-1 signaling levels . in parallel , tyrosine was identified in c. elegans as a potent antagonist of insulin signaling , promoting dauer formation and inducing longevity ( a. ferguson , p. hu , d. kim , and a. fisher , personal communication ) , and enzymes of this pathway have been associated with c. elegans longevity before . similarly , the increased catabolism of tyrosine is conceivably part of a feedback mechanism for restoration of normal insulin / igf-1 signaling . finally , c3 and c5 acylcarnitines mainly arise from the incomplete catabolism of propionate ( following bcaa degradation , figure 8) . thus , increased expression of propionate catabolism enzymes should further prevent accumulation of these intermediates . interestingly , attenuation of intermediary metabolism ( glycolysis , the tca cycle , and gluconeogenesis ) and the electron transport chain has been reported to extend life - span of daf-2 mutants significantly further . this is unexpected because expression of all these enzymes is increased in response to reduced iis . it has been suggested that activation of metabolism is part of a compensatory mechanism triggered by reduced iis that antagonizes full life - span potential of daf-2 mutants . we additionally propose that further attenuation of an already reduced metabolic rate could have an additive effect on daf-2 lifespan , perhaps at the cost of reduced metabolic responsiveness to environmental triggers . note , however , that activation of the glyoxylate shunt ( isocitrate lyase / malate synthase , icl-1 ) has been shown to be required for full life - span extension in the daf-2 mutant as well as in ubiquinone - defective clk-1 , suggesting that increased activity of this pathway is indispensable for iis- or etc - induced longevity . in summary , our data suggests daf-2 mutant cells are equipped with highly bioenergetic competent mitochondria and a proficient , partly rerouted metabolic network that makes economical use of internal energy stores to maintain energy homeostasis during its long life . a major challenge for the field will be to unravel the multiplicity of tissue - specific metabolic changes and how these tissues in turn interact to achieve the extensive physiological transformation induced by mutation in daf-2 . ultimately , such detailed understanding of the altered metabolism of c. elegans iis mutants could also lead to important insights into various insulin - related disease pathologies in humans such as diabetes and obesity as well as aging .

the insulin / igf-1 receptor is a major known determinant of dauer formation , stress resistance , longevity , and metabolism in caenorhabditis elegans . in the past , whole - genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin / igf-1 signaling , including the expression levels of metabolism - associated genes . taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry ( lc ms)-based proteomics , we profiled the proteomic changes that occur in response to activation of the daf-16 transcription factor in the germline - less glp-4(bn2);daf-2(e1370 ) receptor mutant . strikingly , the daf-2 profile suggests extensive reorganization of intermediary metabolism , characterized by the upregulation of many core intermediary metabolic pathways . these include glycolysis / gluconeogenesis , glycogenesis , pentose phosphate cycle , citric acid cycle , glyoxylate shunt , fatty acid -oxidation , one - carbon metabolism , propionate and tyrosine catabolism , and complexes i , ii , iii , and v of the electron transport chain . interestingly , we found simultaneous activation of reciprocally regulated metabolic pathways , which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post - translational regulation of these enzymes . this restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers , allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy - generating pathways to sustain longevity .

Introduction Materials and Methods Results and Discussion Conclusions

in caenorhabditis elegans , attenuation of the insulin / igf-1 signaling ( iis ) pathway during adulthood leads to significant changes in worm physiology , including extended life - span , increased innate immunity and stress - resistance , and extensively altered metabolism . these phenotypic adaptations rely , for the most part , on the activation and nuclear translocation of the forkhead box o ( foxo ) transcription factor daf-16 , which triggers an extensive genetic program that is normally associated with an alternate developmental arrest state called the dauer diapause . although disruption of iis in nematodes does not lead to any clear pathological conditions , the conservation of this pathway in the regulation of fat and carbohydrate metabolism as well as in life - span determination from nematodes to mammals is at least striking . whole - genome transcript profiling of daf-2 mutants and/or dauers , using either microarray or sage analysis , has proven to be successful at identifying differentially expressed gene sets dependent on daf-16 , some of which are involved in energy metabolism . of daf-2(e1370 ) mutants has identified a limited set of enzymes in carbohydrate metabolism ( glycolysis / gluconeogenesis ) as well as the tca and glyoxylate cycles that are differentially expressed in daf-2 mutants and showed that these enzymes are important determinants of nematode life span . recently , several groups have published the first metabolite profiles of the daf-2 mutant , revealing significant shifts in carbohydrate , amino acid , and lipid metabolism , several of which were not apparent from previously published transcriptomics studies . using lc ms / ms - based proteomics analysis , we found that activation of daf-16 in the nucleus results in over - representation of many enzymes of major , often reciprocally regulated , metabolic pathways involved in the synthesis and utilization of internal carbohydrate ( glycolysis and gluconeogenesis ) , lipid stores ( lipogenesis and lipolysis ) , amino acid metabolism , and respiration ( aerobic and anaerobic ) . in the following sections , we integrate our proteomic data set together with a selection of enzymatic activity assays , available literature , and metabolomics data in an attempt to reconstruct and resolve the intricacies of altered daf-2 metabolism . however , we argue that our data reflect bona fide metabolic changes in response to the daf-2 mutation . in the absence of fudr identified a limited number of differentially expressed enzymes in carbohydrate , fatty acid , and amino acid metabolism whose expression pattern was confirmed in our study . for comparing the metabolic profile between long - lived glp-4(bn2);daf-2(e1370 ) and reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm strains , the accurate mass and time ( amt ) lc ms proteomics data set was used , which was published earlier by our group in depuydt et al . the ptm algorithm allows a data set to be searched for proteins of which the abundance profile matches a user - defined template profile , which is based on the pearson correlation between the template and the proteins in the data set.the kyoto encyclopedia of genes and genomes ( kegg ) pathway and uniprot knowledgebase were used in conjunction with the process of reconstructing the c. elegans metabolic network . functional annotation clustering of the proteomics data set showed a significant enrichment of proteins involved in intermediary metabolism among long - lived glp-4(bn2);daf-2(e1370 ) compared to reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm strains ( throughout the text , we will refer to both strains as daf-2 and daf-16;daf-2 , respectively ) . during culturing of the worms , because the use of fudr in c. elegans aging studies warrants caution , we discuss this issue in more detail in the materials and methods ( c. elegans culturing and sampling ) . table 1 shows the result of functional annotation clustering for entries in the kegg database category , which deals mainly with annotation of intermediary metabolism genes . by searching the uniprot protein knowledgebase and on the basis of the available literature , we were able to reconstruct many of the pathways in c. elegans intermediary metabolism from our data set , which is the focus of this article . the only exception occurs in the final ( irreversible ) step in glycolysis catalyzed by the pyruvate kinase enzymes pyk-1 and pyk-2 , which remain unaltered in daf-2 mutants . pepck constitutes a key activator of gluconeogenesis , and elevated levels of pepck in daf-2 mutants confirm earlier reports on pepck transcript and protein levels in insulin / igf-1 signaling - defective mutants . to investigate the relative activities of glycolysis / gluconeogenesis , we measured in vitro enzymatic activities of pepck ( gluconeogenesis ) and pyruvate kinase ( glycolysis ) ( figure 1 ) . in addition , in dauer larvae , high glycogen reserves likely serve to maintain their high degree of motility during the first few weeks after dauer formation , which is in agreement with the expression of glycogen phosphorylase in c. elegans muscles . we found significantly increased protein levels of tps-1 , one of two c. elegans trehalose-6-phosphate synthase genes responsible for trehalose biosynthesis , consistent with increased trehalose levels in iis - defective c. elegans mutants and dauers and daf-16-dependent transcription regulation . our data show a significant upregulation in the expression of three enzymes predicted to be part of the pentose phosphate pathway , including gspd-1 , the c. elegans orthologue of glucose-6-phosphate dehydrogenase ( g6pdh ) , which catalyzes the first step in the pentose phosphate pathway and converts nadp into nadph ( figure 3 ) . however , we found no significant difference in the activity of this pathway by measuring the enzymatic activity of 6-phosphogluconate dehydrogenase ( figure 3 ) . nonetheless , temporal activation of this pathway could be involved in the increased oxidative stress resistance of the daf-2 mutant because the rerouting of carbohydrate intermediates from glycolysis to the pentose phosphate shunt has been shown to be part of a coordinated response to oxidative stress by maintaining high cytoplasmatic nadph / nadp ratios in c. elegans , drosophila , and mammals . on the basis of these reports , the activation of alcohol - fermenting enzymes in daf-2 mutants may well be part of a coordinated stress response system that boosts the nematodes resistance to toxic byproducts of lipid and intrinsic alcohol metabolism . a general increase was found of the citric acid cycle enzymes , including citrate synthase , the isocitrate dehydrogenase complex , and the -ketoglutarate dehydrogenase complex , which was the main sites of regulation within this cycle ( figure 4 ) . transcript profiles of daf-2 mutants did not show any significant change in mrna levels for citric acid cycle enzymes . expression of these genes therefore may be regulated post - transcriptionally or be the result of lowered turnover of these enzymes or the mitochondria in which they reside . here , daf-2 energy metabolism appears to differ from that of the dauer larva , which is characterized by both reduced gene expression and enzymatic activity of citric acid cycle genes . however , aco-1 is thought to be a cytosolic aconitase involved in the regulation of cellular iron concentrations and is therefore not part of the citric acid cycle . the glyoxylate shunt allows the synthesis of carbohydrates via gluconeogenesis from acetyl - coa obtained from fatty acid -oxidation and is generally known in plant and microorganism biochemistry . nematodes also have been shown to contain an active glyoxylate shunt , which is required for the full longevity phenotype of daf-2 and several etc - defective mutants ( mit mutants ) as well as the mitochondrial mutant clk-1(qm30 ) . one - carbon metabolism involves the storage and utilization of one - carbon moieties in anabolic and transmethylation - based regulatory pathways . the carrier of activated one - carbon fragments is tetrahydrofolate ( thf ) , an essential water - soluble derivative of vitamin b9 involved in the formation of nucleotides for dna synthesis , the metabolism of certain amino acids , and the source of methyl groups for s - adenosyl methionine ( sam ) . a particularly strong increase was found in the protein levels of the putative c. elegans aicar formyltransferase ( c55f2.1 ) and c1-thf synthase ( k07e3.4 ) in daf-2 , both of which are involved in the interconversion of one - carbon derivates of thf ( figure 5 ) . we found increased levels in the daf-2 mutant of phosphoglycerate dehydrogenase ( c31c9.2 ) , which catalyzes the first step in glucose - derived serine biosynthesis , and f25b4.1 , the c. elegans orthologue of the glycine cleavage system t - protein , an aminomethyltransferase that catalyzes the actual transfer of methylene carbon from the decarboxylated glycine to tetrahydrofolate ( thf ) . ( left ) shematic overview of one - carbon metabolism involving both the single - carbon carriers tetrahydrofolate and s - adenosylmethionine . in contrast to thf - metabolism , the biosynthetic pathway of sam , the other main carrier for single - carbon units , appears repressed in daf-2 mutants ( figure 5 ) . protein levels of this enzyme ( sams-1 ) are strongly reduced in the daf-2 mutant . demethylation of sam results in the production of s - adenosylhomocysteine ( sah ) , which is a strong inhibitor of all sam - dependent transmethylation reactions . because cysteine is normally readily available from diet , the major role of the transsulfuration pathway in animals is thought to be the regulation of sam levels by controlling methionine / homocysteine degradation and not cysteine biosynthesis per se thus , high cbs activity in daf-2 mutants is an additional strong indication of repressed sam synthesis . this enzyme is the main source for cytosolic acetyl - coa for fatty acid synthesis and represents a major link between the citric acid cycle and lipogenesis . in mammals , expression of this mitochondrial citrate carrier is activated by insulin signaling , and inhibition of acl was shown to reduce fatty acid synthesis significantly and to increase fatty acid -oxidation . paradoxically , we found a strong suppression of the predicted c. elegans fatty acid synthase , fasn-1 , in daf-2 adults . ( 1 ) citrate synthase , ( 2 ) mitochondrial tricarboxylate / dicarboxylate carrier protein ( citrate transport protein ) , ( 3 ) atp - citrate lyase , and ( 4 ) fasn-1 , fatty acid synthase ; elo-1 , long - chain fatty acid elongase ; dhs-25 , mitochondrial beta - ketoacyl - acp reductase ; fat-6 and fat-2 encode fatty acyl desaturases . at the same time , we found increased expression in daf-2 adults of many fatty acid -oxidation proteins ( figure 6a ) as well as an enzyme involved in glycerol catabolism ( t24g3.4 ) and a choline / carnitine palmitoyl transferase ( b0395.3 ) . to assess whether fatty acid -oxidation is increased in the daf-2 mutant , we measured the in vitro activity of 3-hydroxyacyl coa dehydrogenase , which catalyzes the oxidation of l-3-hydroxyacyl coa , and found that its activity was unchanged ( figure 6 ) . therefore , daf-2 fat metabolism is reminiscent to the nonfeeding dauer larva , which depends on the slow and controlled release of energy and anabolic intermediates via -oxidation of internal fat stores for its survival . activation of this catabolic pathway would thus indicate increased catabolism of fatty acids or branched chain amino acids in the daf-2 mutant . recent metabolomic profiling studies of the daf-2 mutant have revealed an increase in the concentration of free amino acids , in particular of branched - chain amino acids ( bcaas ) , suggesting altered amino acid metabolism in this mutant . we speculate that the strong increase of glutamate synthase in combination with glutamate dehydrogenase is indicative of increased amino acid catabolism in the daf-2 mutant , fueling the tca cycle . moreover , the transcript level of cytosolic tyrosine aminotransferase ( tat , f24d1.2 ) , which catalyzes the first step in tyrosine catabolism , was found to be highly enriched in dauers . in contrast to tyrosine , we found reduced concentrations of bcaa catabolic enzymes , including the branched - chain aminotransferase enzyme ( bcat-1 ) , in the daf-2 mutant ( figure 9b ) , in line with transcriptional evidence and paralleled by increased levels of bcaas as reported previously by us and others . therefore , we suggest that daf-2 standard metabolism , like dauers , is hypometabolic , but the metabolic rate is readily raised when necessary , and this is ensured by the availability of large amounts of fat and glycogen and the increased levels of enzymes of core intermediary metabolism . we also found elevated protein levels of the electron - transferring flavoproteins ( etf ) f27d4.1 and etf - dehydrogenase ( let-721 ) in daf-2 mutants , although the latter upregulation was not statistically significant . these proteins transfer the electron pair of fadh2 , resulting from the -oxidation of acyl - coa , to the flavo - iron sulfur protein etf - dehydrogenase , which in turn reduces coenzyme q in the electron transport chain in the mitochondrial inner membrane . together with an experimentally confirmed increased flux of carbon through the glyoxylate shunt and high levels of the storage sugars glycogen and trehalose , it was assumed gluconeogenic activity is high in the daf-2 mutant . although increased levels of malate have been found in the daf-2 mutant , malate synthesis is most likely also increased via the upregulated glyoxylate shunt . interestingly , previous proteomic and metabolomic analysis of the pept-1 mutant revealed a very similar decrease in the abundance of one - carbon metabolism enzymes , including s - adenosylmethionine synthase ( sams-1 ) and s - adenosylhomocysteine hydrolase ( ahcy-1 ) , and changes in methionine ( decreased ) and homocysteine ( increased).pept-1 encodes an intestinal peptide transporter required for the absorption of amino acids in form of di- and tripeptides from the environment , and its mutation further extends daf-2 lifespan by approximately 60% . sams-1 was previously discovered to be a mediator of the dr response , possibly downstream of tor signaling , and is required for a normal protein synthesis rate . therefore , one attractive hypothesis is that alterations in the methionine cycle results in reduced protein synthesis in the daf-2 mutant , the latter of which has been experimentally confirmed by us and others . by extension , we postulate decreased protein synthesis in the pept-1 mutant and metformin - treated c. elegans , which could conceivably underlie their longevity phenotype . however , because we recently showed that protein synthesis is repressed in the daf-2 mutant , such action by bcaas would thus have to be restricted to certain tissues , such as body - wall muscles , which we have shown previouslty are preserved from degradation in daf-2 . it is therefore conceivable that the rationale for the inhibition of bcaa catabolism in the daf-2 mutant is a ( futile ) attempt to restore normal insulin / igf-1 signaling levels . similarly , the increased catabolism of tyrosine is conceivably part of a feedback mechanism for restoration of normal insulin / igf-1 signaling . interestingly , attenuation of intermediary metabolism ( glycolysis , the tca cycle , and gluconeogenesis ) and the electron transport chain has been reported to extend life - span of daf-2 mutants significantly further . this is unexpected because expression of all these enzymes is increased in response to reduced iis . it has been suggested that activation of metabolism is part of a compensatory mechanism triggered by reduced iis that antagonizes full life - span potential of daf-2 mutants . note , however , that activation of the glyoxylate shunt ( isocitrate lyase / malate synthase , icl-1 ) has been shown to be required for full life - span extension in the daf-2 mutant as well as in ubiquinone - defective clk-1 , suggesting that increased activity of this pathway is indispensable for iis- or etc - induced longevity .

although the results of clinical findings have shown that asthma control can be achieved in most patients , epidemiological evidence suggests that there is a significant gap between treatment goals and the actual level of control achieved with treatment in the general population . therefore , there remains the challenge of identifying the factors that are related to poor asthma control and of developing strategies to ensure that asthma control is achieved and maintained . incorrect handling of inhalers and inappropriate inhaler technique result in low bronchial deposition of the drug and can contribute to poor asthma control . understanding the frequency and type of inhaler technique errors , as well as their associations with the level of asthma control , may allow the development of educational strategies to help reduce the morbidity of the disease . the objective of the present article was to assess inhaler technique in outpatients with asthma and to determine associations between the correctness of that technique and the level of asthma control . the study protocol was approved by the research ethics committee of the hospital de clnicas de porto alegre ( hcpa , porto alegre hospital de clnicas ) , located in the city of porto alegre , brazil . written informed consent was obtained from all patients or their legal guardians , in the case of those under 18 years of age . the study population consisted of patients treated at the hcpa outpatient clinics specializing in asthma . individuals aged 18 years or older who had a previous diagnosis of asthma were sequentially recruited . a physician who was a member of the research team confirmed the diagnosis on the basis of the following criteria : symptoms consistent with asthma , accompanied by reversible airflow obstruction ( an increase in fev1 > 12% and > 200 ml after administration of an inhaled short - acting 2 agonist ) or by hyperresponsiveness to a bronchial challenge agent . patients should have made two prior visits to one of the outpatient clinics mentioned above , and the pharmacological treatment regimen should have already been adjusted to the level of asthma severity . patients should be receiving inhaled corticosteroids alone or in combination with long - acting 2 agonists . the exclusion criteria were declining to participate in the study , having another chronic lung disease ( emphysema , chronic bronchitis , or bronchiectasis ) , not using inhaled drugs , and failing to complete all of the evaluations required by the study protocol . the questionnaire used to interview patients included a checklist for assessing patient handling of the device used to inhale the corticosteroid . prior to the study outset , the principal investigator trained all members of the research team on the correct use of each device and on how to score each stage of the evaluation process . patients were asked to demonstrate their inhaler technique , using placebo . for metered dose inhalers , patients were assessed for their performance of the following steps : a ) shaking the inhaler before using it ; b ) exhaling normally before using the inhaler ; c ) holding the inhaler at an appropriate distance ( 3 - 5 cm ) from the lips if a spacer is not used or , if a spacer is used , placing the inhaler in the mouth and creating an adequate seal with the lips ; d ) inhaling slowly and deeply after squeezing the inhaler ; and e ) performing a breath - hold of at least 10 seconds ( after inhalation ) . for dry powder inhalers , patients were assessed for their performance of the following steps : a ) exhaling normally before using the inhaler ; b ) placing the inhaler in the mouth and creating an adequate seal with the lips ; c ) inhaling as forcefully and deeply as possible ; and d ) performing a breath - hold of at least 10 seconds ( after inhalation ) . asthma severity was categorized on the basis of the daily medication regimen in use , as proposed in the global initiative for asthma ( gina ) guidelines . the level of asthma control was assessed in accordance with the classification proposed in the 2011 gina guidelines ( chart 1 ) . chart 1criteria for assessing the level of asthma control.a ain accordance with the global initiative for asthma . pulmonary function was assessed with a computerized spirometer ( masterscreen v4.31 ; jaeger , wrzburg , germany ) . all parameters are expressed as a percentage of the predicted value for age , gender , and height . measurements of pef were performed with a portable peak flow monitor ( vitalograph ; boehringer ingelheim , ingelheim am rhein , germany ) . the results are expressed as a percentage of the predicted value for age , gender , and height . for the statistical analysis , we used the statistical package for the social sciences , version 18.0 ( spss inc . , data are expressed as number ( percentage ) of cases , mean sd , or median ( interquartile range ) . in the present sample , the association between the number of errors ( dichotomized into one or more errors and two or more errors ) and the level of asthma control ( controlled , partially controlled , and uncontrolled ) was analyzed by the chi - square test . we found that the cut - off point of one or more errors was not significantly associated with the level of asthma control ( p = 0.07 ) , whereas the cut - off point of two or more errors was significantly associated with the level of asthma control ( p = 0.002 ) . therefore , correct inhaler technique was defined as making less than two inhaler technique errors , whereas incorrect inhaler technique was defined as making two or more errors . patients with correct inhaler technique and patients with incorrect inhaler technique were compared for categorical variables using the chi - square test with adjusted standardized residuals and , when necessary , yates ' correction or fisher 's exact test . continuous variables were compared by the independent sample t - test or the mann - whitney u test . a binary logistic regression model ( enter method ) was used to identify characteristics predictive of incorrect inhaler technique . variables with a significance level less than 0.1 in univariate analysis , adjusted for gender and age , were included in a logistic regression model . thirty patients declined to participate , 27 patients were excluded because they had another chronic lung disease , 7 patients were excluded because they did not use the prescribed inhaled drug , and 2 patients were excluded because they failed to complete all of the evaluations required by the study protocol . of those , 187 patients showed correct inhaler technique and 81 ( 30.2% ) showed incorrect technique . one hundred and ninety - nine patients ( 74.3% ) were female , and 223 ( 83.2% ) were white . most patients ( 60.1% ) had had 8 years of schooling or less , and 186 ( 69.4% ) had a monthly family income of less than three times the national minimum wage . asthma severity was classified as mild persistent in 37 ( 13.8% ) of the patients , as moderate persistent in 89 ( 33.2% ) , and as severe persistent in 142 ( 53.0% ) . asthma was classified as controlled in 47 ( 17.5% ) of the patients , as partially controlled in 74 ( 27.6% ) , and as uncontrolled in 147 ( 54.9% ) . table 2 shows the comparison between groups formed on the basis of inhaler technique assessment . statistically significant differences were found for the following variables : marital status ( p = 0.002 ) , the proportion of widowed patients being higher among those with incorrect inhaler technique ; level of education ( p = 0.023 ) , the proportion of patients who had had 8 years of schooling or less being higher among those with incorrect inhaler technique ; monthly family income ( p = 0.016 ) , the proportion of patients with an income of less than three times the national minimum wage being higher among those with incorrect inhaler technique ; and level of asthma control ( p = 0.007 ) , the proportion of patients with uncontrolled asthma being higher among those with incorrect inhaler technique . table 3 shows that there was a statistically significant difference in type of inhaler used ( p < 0.001 ) between the two groups formed on the basis of inhaler technique assessment , the proportion of patients with correct technique being higher among those using dry powder inhalers than among those using metered dose inhalers . in addition , the proportion of patients with correct inhaler technique differed significantly among each specific type of inhaler used ( p < 0.001 ) , the proportion of patients with correct inhaler technique being higher among those using aerolizer or turbuhaler . in contrast , we found a higher proportion of patients with incorrect technique among those using metered dose inhalers without spacers . the following variables were independently associated with incorrect inhaler technique : being widowed ( or = 5.01 ; 95% ci , 1.74 - 14.41 ; p = 0.003 ) ; using metered dose inhalers ( or = 1.58 ; 95% ci , 1.35 - 1.85 ; p < 0.001 ) ; having a monthly family income of less than three times the national minimum wage ( or = 2.67 ; 95% ci , 1.35 - 1.85 ; p = 0.008 ) , and having two or more comorbidities ( or = 3.80 ; 95% ci , 1.03 - 14.02 ; p = 0.045 ) . the present study showed that the number of inhaler technique errors has a significant impact on the level of asthma control . the variables that were associated with incorrect inhaler technique were being widowed , using metered dose inhalers , having a monthly family income of less than three times the national minimum wage , and having two or more comorbidities . incorrect inhaler technique in asthma treatment can substantially reduce lung deposition of the drug , undermining the effectiveness of asthma treatment . in the present study , incorrect inhaler technique ( i.e. , making 2 or more errors ) was associated with poor asthma control . it is of note that a previous study , in which incorrect inhaler technique was defined as making one or more errors , found no association between the correctness of inhaler technique and the level of asthma control . studies have suggested that 32% to 96% of asthma patients make errors when using their inhalers , and that , in 28% to 68% of cases , those errors are important to the point of undermining the effects of treatment . in the present study , 30.2% of the patients made two or more inhaler technique errors , and this cut - off point was associated with asthma control . in contrast to these findings , in a study conducted in brazil , coelho et al . evaluated handling of inhaler devices by 467 patients with severe asthma who were followed at a center in the state of bahia and observed that most patients showed appropriate inhaler technique , a finding that was attributed to the intense educational intervention that those patients received at a referral center . performing the inhaler technique correctly depends on the type of inhaler . a systematic review has shown that patients using dry powder inhalers had lower rates of inhaler technique errors than did those using metered dose inhalers . the present study adds to the evidence that the proportion of patients with inappropriate inhaler technique is higher among patients using metered dose inhalers than among those using dry powder inhalers . this difference is even greater when patients using metered dose inhalers without spacers are considered . metered dose inhalers are more difficult to use , because they require greater motor coordination . the use of a spacer reduces the need for greater motor coordination , but , despite that , metered dose inhalers remain more difficult to use than dry powder inhalers , which leads to a higher proportion of inhaler technique errors . however , one factor to be considered in the present study is that the number of inhaler technique steps assessed was greater for metered dose inhaler use ( five steps ) than for dry powder inhaler use ( four steps ) . this may indicate a bias in the present study , with the requirement for classifying the technique as correct being more stringent for individuals using metered dose inhalers . however , this is more likely to represent the greater complexity of performing the inhaler technique with metered dose inhalers than with dry powder inhalers . in the present study , a higher proportion of patients with inappropriate technique were found among those with a monthly family income of less than three times the national minimum wage . a previous study has shown that , to ensure appropriate treatment and reduce asthma morbidity , it is necessary that socioeconomically disadvantaged patients receive a more intense educational approach . the level of support provided by family members or caregivers can also contribute to the appropriate performance of the inhaler technique . in our study widowhood can contribute to a varying degree of social isolation and loneliness that can negatively impact the treatment of chronic diseases . the physical or mental impairment induced by the presence of other diseases can negatively impact the use of inhalers . conditions such as tremors , vision impairment , hearing impairment , arthritis , mood disorders , and cognitive disorders can impair learning of the inhaler technique or its appropriate performance . in the present study , the presence of two or more comorbidities was associated with inappropriate inhaler technique . however , we did not specifically address which diseases were more prevalent in this association . since the present study found a large proportion of patients with uncontrolled asthma ( 69.1% ) , it is important to highlight the fact that a previous study conducted in brazil showed that the level of asthma control was associated with asthma severity , access to medication , and appropriate use of inhaled corticosteroids . therefore , since our study was carried out at a public tertiary care center , it is natural that cases that are more difficult to control will be referred there for treatment and that , in contrast , controlled asthma cases will be sent back for treatment at public primary care clinics . first , it was a cross - sectional study and , therefore , it does not allow the establishment of a temporal sequence between the quality of the patients ' performance of the inhaler technique and their level of asthma control . second , the study was carried out at a single center that provides care within the public health system . consequently , the study population consisted of individuals who had a low monthly family income and a low educational level , and this may limit the generalization of results . the clinical implications of this study lie primarily in the demonstration of the fact that two or more inhaler technique errors affect the level of asthma control , with 30.2% of the patients studied showing inappropriate inhaler technique on the basis of this definition . in addition , our findings indicate that target group patients such as widowed patients , patients using metered dose inhalers , patients with a monthly family income of less than three times the national minimum wage , and patients with two or more comorbidities require special attention in terms of inhaler technique education . therefore , it is important that educational strategies for asthma patients be developed to improve their performance of the inhaler technique and increase their level of asthma control . avaliar a tcnica inalatria em pacientes com asma atendidos ambulatorialmente , estabelecendo associaes dessa com o grau de controle da doena . estudo transversal envolvendo pacientes com idade > 14 anos e diagnstico mdico de asma , recrutados no ambulatrio de asma do hospital de clnicas de porto alegre , na cidade de porto alegre ( rs ) . os pacientes completaram dois questionrios ( um geral e um questionrio de controle da asma baseado nas diretrizes da global initiative for asthma de 2011 ) . a tcnica inalatria incorreta foi definida como a execuo incorreta de pelo menos duas etapas da avaliao . desses , 81 ( 30,2% ) apresentaram tcnica inalatria incorreta , que foi associada com falta de controle da asma ( p = 0,002 ) . a regresso logstica identificou os seguintes fatores associados com a tcnica inalatria incorreta : ser vivo ( or = 5,01 ; ic95% , 1,74 - 14,41 ; p = 0,003 ) ; utilizar inalador pressurizado ( or = 1,58 ; ic95% , 1,35 - 1,85 ; p < 0,001 ) ; ter renda familiar mensal < 3 salrios mnimos ( or = 2,67 ; ic95% , 1,35 - 1,85 ; p = 0,008 ) ; e ter > 2 comorbidades ( or = 3,80 ; ic95% , 1,03 - 14,02 ; p = 0,045 ) . na amostra estudada , a tcnica inalatria incorreta se associou com a falta de controle da asma . viuvez , uso de inalador pressurizado , baixo nvel socioeconmico e presena de > 2 comorbidades se associaram tcnica inalatria incorreta . ainda que resultados de ensaios clnicos tenham demonstrado que o controle da asma pode ser obtido na maioria dos pacientes , as evidncias epidemiolgicas sugerem que existe uma importante lacuna entre os objetivos do tratamento e o real grau de controle obtido com o mesmo para a populao geral . assim , persiste o desafio de identificar os fatores relacionados com a falta de controle da asma e desenvolver estratgias para garantir que esse controle seja alcanado e mantido . as medicaes inalatrias se constituem na terapia fundamental da asma . o manejo errneo dos dispositivos inalatrios e a tcnica inalatria inadequada acarretam baixa deposio brnquica da medicao e podem contribuir para o controle precrio da asma . a compreenso da frequncia e do tipo de erros na tcnica inalatria e de suas associaes com o grau de controle da asma poderia permitir o desenvolvimento de estratgias educativas que contribussem para reduzir a morbidade da doena . o objetivo do presente artigo foi avaliar a tcnica inalatria em pacientes com asma atendidos ambulatorialmente , estabelecendo associaes dessa com o grau de controle da doena . o delineamento constituiu - se em um estudo transversal . o protocolo foi aprovado pela comisso de tica e pesquisa do hospital de clnicas de porto alegre ( hcpa ) , em porto alegre ( rs ) . o termo de consentimento livre e esclarecido foi obtido de todos os pacientes ou de seus responsveis , no caso de menores de 18 anos . a populao do estudo constituiu - se de pacientes atendidos nos ambulatrios especializados em asma do hcpa . foram recrutados de maneira sequencial indivduos com idade igual ou superior a 14 anos com diagnstico prvio de asma . o diagnstico foi confirmado por um mdico , membro da equipe de pesquisa , de acordo com os seguintes critrios : sintomas compatveis com asma , associados a obstruo reversvel no fluxo areo ( aumento no vef1 > 12% e > 200 ml aps a administrao inalatria de um 2-agonista de curta durao ) ou a hiper - responsividade a um agente broncoprovocador . os pacientes deveriam ter pelo menos duas consultas prvias no referido ambulatrio , e a prescrio das medicaes j deveria estar ajustada de acordo com a classificao da gravidade da doena . os pacientes deveriam estar em uso de corticosteroide inalatrio em apresentao isolada ou combinada com 2-agonista de longa ao . os critrios de excluso foram recusa em participar do estudo , presena de outra doena pulmonar crnica ( enfisema , bronquite crnica ou bronquiectasias ) , no utilizao de medicao inalatria ou falha em completar todas as avaliaes exigidas pelo protocolo do estudo . o questionrio utilizado para entrevistar os pacientes inclua uma lista de controle para avaliar o adequado manejo do dispositivo utilizado pelo paciente para inalar o corticoide . todos os membros da equipe de pesquisa foram previamente treinados pelo investigador principal quanto utilizao correta de cada dispositivo e a como pontuar cada etapa do processo de avaliao . para o uso do inalador pressurizado , as seguintes etapas eram avaliadas : a ) agitar o aerossol antes do uso ; b ) realizar expirao normal antes do uso ; c ) manter distncia adequada de 3 - 5 cm do dispositivo at a boca , na ausncia de uso de espaador ou , se em uso de espaador , coloc - lo na boca e fechar os lbios adequadamente ; d ) realizar inspirao lenta e profunda aps disparar o aerossol ; e e ) fazer pausa ps - inspiratria de , no mnimo , 10 segundos . para a utilizao do inalador de p , as seguintes etapas eram avaliadas : a ) realizar expirao normal antes do uso ; b ) colocar o dispositivo na boca e fechar os lbios adequadamente ; c ) inspirar o mais vigorosa e profundamente possvel ; e d ) fazer pausa ps - inspiratria de , no mnimo , 10 segundos . para avaliar a gravidade da asma , foi utilizada a classificao de gravidade conforme o regime medicamentoso dirio usado , proposta pelas diretrizes da global initiative for asthma ( gina ) . para avaliar o grau de controle da asma , foi utilizada a classificao proposta pelas diretrizes da gina em 2011 ( quadro 1 ) . quadro 1critrios para avaliar o grau de controle da asma.a ade acordo com a global initiative for asthma.(4 ) a funo pulmonar foi avaliada utilizando um espirmetro computadorizado masterscreen v4.31 ( jaeger , wrzburg , alemanha ) . foram registrados cvf , vef1 e relao vef1/cvf . todos os parmetros foram expressos em percentagem do previsto para idade , sexo e altura . a medida do pfe foi realizada atravs de um aparelho porttil ( peak flow monitor vitalograph ; boehringer ingelheim , ingelheim am rhein , alemanha ) . o resultado foi expresso em percentagem do previsto para idade , sexo e altura . para a anlise estatstica foi empregado o programa statistical package for the social sciences verso 18.0 ( spss inc . , os dados foram expressos como nmero de casos ( proporo ) , mdia dp ou mediana ( amplitude interquartlica ) . o nmero de erros na realizao da tcnica inalatria foi registrado para cada paciente . na presente amostra , a associao entre o nmero de erros ( dicotomizado em um ou mais erros e dois ou mais erros ) e o grau de controle da asma ( controlada , parcialmente controlada e no controlada ) foi analisada com o teste do qui - quadrado . foi identificado que o ponto de corte de um ou mais erros no se associou significativamente com o grau de controle da asma ( p = 0,07 ) , enquanto o ponto de corte de dois ou mais erros se associou de forma significativa com o grau de controle da asma ( p = 0,002 ) . assim , a tcnica inalatria foi definida como correta frente identificao de menos de dois erros na avaliao da utilizao dos dispositivos , e definida como incorreta frente identificao de dois ou mais erros . a anlise comparativa entre o grupo com tcnica correta e o grupo com tcnica incorreta foi feita , para as variveis categricas , utilizando o teste do qui - quadrado com resduos ajustados padronizados , e , quando indicado , usando a correo de yates ou o teste exato de fisher . a comparao entre as variveis contnuas foi feita utilizando o teste t para amostras independentes ou o teste u de mann - whitney . o modelo de regresso logstica binria pelo mtodo enter foi utilizado para identificar caractersticas preditivas relacionadas com a tcnica inalatria incorreta . as variveis com significncia < 0,1 na anlise univariada , controladas por sexo e idade , foram includas no modelo de regresso logstica . trinta pacientes recusaram - se a participar , 27 foram excludos porque apresentavam outra doena pulmonar crnica , 7 pacientes foram excludos porque no utilizavam a medicao inalatria prescrita , e 2 foram excludos porque falharam em realizar todas as avaliaes preconizadas pelo estudo . desses , 187 pacientes apresentavam tcnica inalatria classificada como correta , e 81 ( 30,2% ) apresentavam tcnica incorreta . a tabela 1 mostra as caractersticas gerais dos pacientes estudados . cento e noventa e nove pacientes ( 74,3% ) eram do sexo feminino , e 223 ( 83,2% ) eram de raa branca . a mdia de idade foi de 50,9 16,5 anos . a grande maioria dos pacientes ( 60,1% ) tinha grau de instruo igual ou menor que 8 anos de estudo , e 186 ( 69,4% ) tinham renda familiar menor que trs salrios mnimos nacionais . a gravidade da asma foi classificada como persistente leve em 37 ( 13,8% ) dos pacientes , como persistente moderada em 89 ( 33,2% ) e como persistente grave em 142 ( 53,0% ) . a asma foi classificada como controlada em 47 ( 17,5% ) dos pacientes , parcialmente controlada em 74 ( 27,6% ) e no controlada em 147 ( 54,9% ) . a tabela 2 apresenta a comparao entre grupos de acordo com a avaliao da tcnica inalatria . foram observadas diferenas estatisticamente significativas para as seguintes variveis : estado civil ( p = 0,002 ) , sendo maior a proporo de pacientes vivos entre aqueles com tcnica inalatria incorreta ; grau de instruo ( p = 0,023 ) , sendo maior a proporo de pacientes com tempo de estudo < 8 anos entre aqueles com tcnica inalatria incorreta ; renda familiar ( p = 0,016 ) , sendo maior a proporo de pacientes com renda menor que trs salrios mnimos entre aqueles com tcnica inalatria incorreta ; e grau de controle da asma ( p = 0,007 ) , sendo maior a proporo de pacientes com asma no controlada entre aqueles com tcnica inalatria incorreta . tabela 2comparao entre grupos de acordo com a avaliao da tcnica inalatria . a tabela 3 mostra que houve diferena estatisticamente significativa entre os dois grupos de tcnica inalatria para o tipo geral de dispositivo utilizado ( p < 0,001 ) , observando - se maior proporo de pacientes com tcnica correta entre aqueles em uso de dispositivo de p do que aqueles em uso de inalador pressurizado . tambm a proporo de pacientes com tcnica inalatria correta diferiu significativamente entre o tipo especfico de dispositivo utilizado ( p < 0,001 ) , observando - se maior proporo de pacientes com tcnica inalatria correta entre aqueles em uso de aerolizer ou turbuhaler . por outro lado , evidenciou - se maior proporo de pacientes com tcnica incorreta entre aqueles em uso de inalador pressurizado sem espaador . a tabela 4 apresenta a regresso logstica para os fatores relacionados com a tcnica inalatria incorreta . as variveis que se associaram de forma independente com a tcnica inalatria incorreta foram : ser vivo ( or = 5,01 ; ic95% , 1,74 - 14,41 ; p = 0,003 ) ; usar inalador pressurizado ( or = 1,58 ; ic95% , 1,35 - 1,85 ; p < 0,001 ) ; ter renda familiar menor que trs salrios mnimos ( or = 2,67 ; ic95% , 1,35 - 1,85 ; p = 0,008 ) ; e apresentar duas ou mais comorbidades ( or = 3,80 ; ic95% , 1,03 - 14,02 ; p = 0,045 ) . tabela 4regresso logstica binria para fatores relacionados com a tcnica incorreta de uso dos dispositivos inalatrios . o presente estudo mostrou que o nmero de erros na tcnica inalatria tem um impacto significativo sobre o grau de controle da asma . as variveis que se associaram com a tcnica inalatria incorreta foram ser vivo , utilizar inalador pressurizado , ter renda familiar menor que trs salrios mnimos e apresentar duas ou mais comorbidades . a tcnica inalatria incorreta no tratamento da asma pode reduzir substancialmente a deposio pulmonar da medicao , prejudicando a efetividade do tratamento da asma . no presente estudo , a tcnica inalatria incorreta ( identificao de 2 ou mais erros ) se associou com a falta de controle da doena . vale ressaltar que um estudo prvio , no qual se considerou como tcnica inalatria incorreta a identificao de um ou mais erros , no indicou uma associao entre a tcnica inalatria e o grau de controle da doena . estudos tm sugerido que de 32% a 96% dos pacientes asmticos cometem erros quando utilizam seus dispositivos inalatrios , sendo que em 28% a 68% dos casos os erros so importantes a ponto de prejudicar a ao do tratamento . no presente estudo , 30,2% dos pacientes cometeram dois ou mais erros na tcnica inalatria , sendo esse ponto de corte associado com o controle da doena . em contraste a esses achados , em um estudo brasileiro , coelho et al . avaliaram o manuseio dos dispositivos por 467 asmticos graves acompanhados em um centro no estado da bahia , evidenciando que a maioria dos pacientes demonstrava tcnica inalatria adequada no uso dos dispositivos , fato esse atribudo intensa atividade educativa a que eles eram submetidos em um centro de referncia . a execuo correta da tcnica inalatria depende do tipo de inalador . uma reviso sistemtica demonstrou que pacientes em uso de dispositivos de p tinham taxas de erro na tcnica inalatria menores que pacientes em uso de inalador pressurizado . o presente estudo refora a evidncia de que a proporo de pacientes com tcnica inalatria inadequada maior entre os pacientes em uso de inalador pressurizado que entre os pacientes em uso de inaladores de p . essa diferena ainda maior quando considerados os pacientes em uso de inalador pressurizado sem espaador . o inalador pressurizado um dispositivo mais difcil de ser utilizado , pois requer maior coordenao motora no uso . a utilizao de espaador reduz a necessidade de maior coordenao , mas ainda assim a dificuldade de uso permanece maior do que para os dispositivos de p , levando a uma maior proporo de erros na tcnica inalatria . entretanto , uma considerao a ser feita no presente estudo que o nmero de etapas avaliadas na execuo da tcnica inalatria foi maior para o uso de inalador pressurizado ( cinco etapas ) do que para o uso dos dispositivos de p ( quatro etapas ) . isso poderia apontar para um vis na presente avaliao , com uma maior exigncia na classificao da tcnica correta para os indivduos em uso de inalador pressurizado . porm , o mais provvel que isso represente a maior complexidade de execuo da tcnica inalatria com o inalador pressurizado do que com os dispositivos de p . no presente estudo , foi observada uma maior proporo de pacientes com tcnica inalatria inadequada naqueles com renda familiar menor que trs salrios mnimos . um estudo prvio mostrou que pacientes em desvantagem socioeconmica necessitam um manejo educativo mais intenso para um tratamento adequado e reduo da morbidade da doena . o nvel de apoio fornecido por familiares ou cuidadores tambm pode contribuir para o adequado desempenho na tcnica inalatria . identificamos em nosso estudo que os pacientes vivos apresentaram tcnica inalatria inadequada mais frequentemente . o estado de viuvez pode contribuir para um grau variado de isolamento social e solido que pode interferir negativamente no tratamento de doenas crnicas . o prejuzo fsico ou mental provocado pela presena de outras doenas pode interferir negativamente no uso de dispositivos inalatrios . assim , condies como tremor , dificuldade visual , dificuldade de audio , artrite , alteraes do humor e distrbios cognitivos podem prejudicar o aprendizado da tcnica inalatria ou sua adequada execuo . no presente estudo , a presena de duas ou mais comorbidades se associou com tcnica inalatria inadequada . como o presente estudo mostrou uma proporo grande de pacientes com asma no controlada ( 69,1% ) , cabe salientar que um estudo prvio em nosso meio mostrou que o grau de controle da doena se associou com a gravidade da asma , com o acesso medicao e com o uso adequado do corticosteroide inalatrio . assim , como o presente estudo foi realizado em um centro tercirio do sistema pblico , natural que os casos de controle mais difcil sejam encaminhados para tratamento e , por outro lado , que os casos com doena controlada retornem para o atendimento na rede pblica . em primeiro lugar , um estudo transversal e , assim , no permite que se estabelea uma sequncia temporal entre a qualidade da tcnica inalatria e o grau de controle da asma . em segundo lugar , o estudo foi realizado em um centro nico , o qual fornece assistncia para o sistema pblico de sade . em consequncia , a populao foi constituda de indivduos com baixa renda familiar e baixo nvel educacional , o que poderia limitar a generalizao dos resultados . as implicaes clnicas do presente estudo envolvem primeiramente a demonstrao do fato de que dois ou mais erros na tcnica inalatria acarretam interferncias no grau de controle da asma , sendo que 30,2% dos pacientes estudados tiveram a tcnica inalatria inadequada por essa definio . alm disso , o trabalho sinaliza que pacientes de grupos alvo como vivos , pacientes em uso de inalador pressurizado , pacientes com renda familiar menor que trs salrios mnimos e aqueles com a presena de duas ou mais comorbidades necessitam ateno especial na educao da tcnica inalatria . dessa forma , importante que sejam desenvolvidas estratgias educativas para pacientes asmticos de forma a aprimorar a tcnica inalatria e melhorar o grau de controle da doena .

objective : to evaluate inhaler technique in outpatients with asthma and to determine associations between the correctness of that technique and the level of asthma control.methods:this was a cross - sectional study involving patients > 14 years of age with physician - diagnosed asthma . the patients were recruited from the asthma outpatient clinic of the hospital de clnicas de porto alegre , in the city of porto alegre , brazil . the patients completed two questionnaires ( a general questionnaire and an asthma control questionnaire based on the 2011 global initiative for asthma guidelines ) , demonstrated their inhaler technique , and performed pulmonary function tests . incorrect inhaler technique was defined as the incorrect execution of at least two of the predefined steps.results:we included 268 patients . of those , 81 ( 30.2% ) showed incorrect inhaler technique , which was associated with poor asthma control ( p = 0.002 ) . logistic regression analysis identified the following factors associated with incorrect inhaler technique : being widowed ( or = 5.01 ; 95% ci , 1.74 - 14.41 ; p = 0.003 ) ; using metered dose inhalers ( or = 1.58 ; 95% ci , 1.35 - 1.85 ; p < 0.001 ) ; having a monthly family income < 3 times the minimum wage ( or = 2.67 ; 95% ci , 1.35 - 1.85 ; p = 0.008 ) , and having > 2 comorbidities ( or = 3.80 ; 95% ci , 1.03 - 14.02 ; p = 0.045).conclusions : in the sample studied , incorrect inhaler technique was associated with poor asthma control . widowhood , use of metered dose inhalers , low socioeconomic level , and the presence of > 2 comorbidities were associated with incorrect inhaler technique .

Introduction Methods Results Discussion OBJETIVO: MTODOS: RESULTADOS: CONCLUSES: Introduo Mtodos Resultados Discusso

although the results of clinical findings have shown that asthma control can be achieved in most patients , epidemiological evidence suggests that there is a significant gap between treatment goals and the actual level of control achieved with treatment in the general population . therefore , there remains the challenge of identifying the factors that are related to poor asthma control and of developing strategies to ensure that asthma control is achieved and maintained . incorrect handling of inhalers and inappropriate inhaler technique result in low bronchial deposition of the drug and can contribute to poor asthma control . understanding the frequency and type of inhaler technique errors , as well as their associations with the level of asthma control , may allow the development of educational strategies to help reduce the morbidity of the disease . the objective of the present article was to assess inhaler technique in outpatients with asthma and to determine associations between the correctness of that technique and the level of asthma control . the study protocol was approved by the research ethics committee of the hospital de clnicas de porto alegre ( hcpa , porto alegre hospital de clnicas ) , located in the city of porto alegre , brazil . written informed consent was obtained from all patients or their legal guardians , in the case of those under 18 years of age . a physician who was a member of the research team confirmed the diagnosis on the basis of the following criteria : symptoms consistent with asthma , accompanied by reversible airflow obstruction ( an increase in fev1 > 12% and > 200 ml after administration of an inhaled short - acting 2 agonist ) or by hyperresponsiveness to a bronchial challenge agent . patients should have made two prior visits to one of the outpatient clinics mentioned above , and the pharmacological treatment regimen should have already been adjusted to the level of asthma severity . the exclusion criteria were declining to participate in the study , having another chronic lung disease ( emphysema , chronic bronchitis , or bronchiectasis ) , not using inhaled drugs , and failing to complete all of the evaluations required by the study protocol . prior to the study outset , the principal investigator trained all members of the research team on the correct use of each device and on how to score each stage of the evaluation process . patients were asked to demonstrate their inhaler technique , using placebo . for metered dose inhalers , patients were assessed for their performance of the following steps : a ) shaking the inhaler before using it ; b ) exhaling normally before using the inhaler ; c ) holding the inhaler at an appropriate distance ( 3 - 5 cm ) from the lips if a spacer is not used or , if a spacer is used , placing the inhaler in the mouth and creating an adequate seal with the lips ; d ) inhaling slowly and deeply after squeezing the inhaler ; and e ) performing a breath - hold of at least 10 seconds ( after inhalation ) . for dry powder inhalers , patients were assessed for their performance of the following steps : a ) exhaling normally before using the inhaler ; b ) placing the inhaler in the mouth and creating an adequate seal with the lips ; c ) inhaling as forcefully and deeply as possible ; and d ) performing a breath - hold of at least 10 seconds ( after inhalation ) . asthma severity was categorized on the basis of the daily medication regimen in use , as proposed in the global initiative for asthma ( gina ) guidelines . the level of asthma control was assessed in accordance with the classification proposed in the 2011 gina guidelines ( chart 1 ) . chart 1criteria for assessing the level of asthma control.a ain accordance with the global initiative for asthma . in the present sample , the association between the number of errors ( dichotomized into one or more errors and two or more errors ) and the level of asthma control ( controlled , partially controlled , and uncontrolled ) was analyzed by the chi - square test . we found that the cut - off point of one or more errors was not significantly associated with the level of asthma control ( p = 0.07 ) , whereas the cut - off point of two or more errors was significantly associated with the level of asthma control ( p = 0.002 ) . therefore , correct inhaler technique was defined as making less than two inhaler technique errors , whereas incorrect inhaler technique was defined as making two or more errors . patients with correct inhaler technique and patients with incorrect inhaler technique were compared for categorical variables using the chi - square test with adjusted standardized residuals and , when necessary , yates ' correction or fisher 's exact test . a binary logistic regression model ( enter method ) was used to identify characteristics predictive of incorrect inhaler technique . thirty patients declined to participate , 27 patients were excluded because they had another chronic lung disease , 7 patients were excluded because they did not use the prescribed inhaled drug , and 2 patients were excluded because they failed to complete all of the evaluations required by the study protocol . of those , 187 patients showed correct inhaler technique and 81 ( 30.2% ) showed incorrect technique . one hundred and ninety - nine patients ( 74.3% ) were female , and 223 ( 83.2% ) were white . most patients ( 60.1% ) had had 8 years of schooling or less , and 186 ( 69.4% ) had a monthly family income of less than three times the national minimum wage . asthma severity was classified as mild persistent in 37 ( 13.8% ) of the patients , as moderate persistent in 89 ( 33.2% ) , and as severe persistent in 142 ( 53.0% ) . asthma was classified as controlled in 47 ( 17.5% ) of the patients , as partially controlled in 74 ( 27.6% ) , and as uncontrolled in 147 ( 54.9% ) . statistically significant differences were found for the following variables : marital status ( p = 0.002 ) , the proportion of widowed patients being higher among those with incorrect inhaler technique ; level of education ( p = 0.023 ) , the proportion of patients who had had 8 years of schooling or less being higher among those with incorrect inhaler technique ; monthly family income ( p = 0.016 ) , the proportion of patients with an income of less than three times the national minimum wage being higher among those with incorrect inhaler technique ; and level of asthma control ( p = 0.007 ) , the proportion of patients with uncontrolled asthma being higher among those with incorrect inhaler technique . table 3 shows that there was a statistically significant difference in type of inhaler used ( p < 0.001 ) between the two groups formed on the basis of inhaler technique assessment , the proportion of patients with correct technique being higher among those using dry powder inhalers than among those using metered dose inhalers . in addition , the proportion of patients with correct inhaler technique differed significantly among each specific type of inhaler used ( p < 0.001 ) , the proportion of patients with correct inhaler technique being higher among those using aerolizer or turbuhaler . in contrast , we found a higher proportion of patients with incorrect technique among those using metered dose inhalers without spacers . the following variables were independently associated with incorrect inhaler technique : being widowed ( or = 5.01 ; 95% ci , 1.74 - 14.41 ; p = 0.003 ) ; using metered dose inhalers ( or = 1.58 ; 95% ci , 1.35 - 1.85 ; p < 0.001 ) ; having a monthly family income of less than three times the national minimum wage ( or = 2.67 ; 95% ci , 1.35 - 1.85 ; p = 0.008 ) , and having two or more comorbidities ( or = 3.80 ; 95% ci , 1.03 - 14.02 ; p = 0.045 ) . the present study showed that the number of inhaler technique errors has a significant impact on the level of asthma control . the variables that were associated with incorrect inhaler technique were being widowed , using metered dose inhalers , having a monthly family income of less than three times the national minimum wage , and having two or more comorbidities . incorrect inhaler technique in asthma treatment can substantially reduce lung deposition of the drug , undermining the effectiveness of asthma treatment . in the present study , incorrect inhaler technique ( i.e. , making 2 or more errors ) was associated with poor asthma control . it is of note that a previous study , in which incorrect inhaler technique was defined as making one or more errors , found no association between the correctness of inhaler technique and the level of asthma control . studies have suggested that 32% to 96% of asthma patients make errors when using their inhalers , and that , in 28% to 68% of cases , those errors are important to the point of undermining the effects of treatment . in the present study , 30.2% of the patients made two or more inhaler technique errors , and this cut - off point was associated with asthma control . evaluated handling of inhaler devices by 467 patients with severe asthma who were followed at a center in the state of bahia and observed that most patients showed appropriate inhaler technique , a finding that was attributed to the intense educational intervention that those patients received at a referral center . a systematic review has shown that patients using dry powder inhalers had lower rates of inhaler technique errors than did those using metered dose inhalers . the present study adds to the evidence that the proportion of patients with inappropriate inhaler technique is higher among patients using metered dose inhalers than among those using dry powder inhalers . this difference is even greater when patients using metered dose inhalers without spacers are considered . metered dose inhalers are more difficult to use , because they require greater motor coordination . the use of a spacer reduces the need for greater motor coordination , but , despite that , metered dose inhalers remain more difficult to use than dry powder inhalers , which leads to a higher proportion of inhaler technique errors . however , one factor to be considered in the present study is that the number of inhaler technique steps assessed was greater for metered dose inhaler use ( five steps ) than for dry powder inhaler use ( four steps ) . this may indicate a bias in the present study , with the requirement for classifying the technique as correct being more stringent for individuals using metered dose inhalers . however , this is more likely to represent the greater complexity of performing the inhaler technique with metered dose inhalers than with dry powder inhalers . in the present study , a higher proportion of patients with inappropriate technique were found among those with a monthly family income of less than three times the national minimum wage . the level of support provided by family members or caregivers can also contribute to the appropriate performance of the inhaler technique . the physical or mental impairment induced by the presence of other diseases can negatively impact the use of inhalers . conditions such as tremors , vision impairment , hearing impairment , arthritis , mood disorders , and cognitive disorders can impair learning of the inhaler technique or its appropriate performance . in the present study , the presence of two or more comorbidities was associated with inappropriate inhaler technique . since the present study found a large proportion of patients with uncontrolled asthma ( 69.1% ) , it is important to highlight the fact that a previous study conducted in brazil showed that the level of asthma control was associated with asthma severity , access to medication , and appropriate use of inhaled corticosteroids . first , it was a cross - sectional study and , therefore , it does not allow the establishment of a temporal sequence between the quality of the patients ' performance of the inhaler technique and their level of asthma control . consequently , the study population consisted of individuals who had a low monthly family income and a low educational level , and this may limit the generalization of results . the clinical implications of this study lie primarily in the demonstration of the fact that two or more inhaler technique errors affect the level of asthma control , with 30.2% of the patients studied showing inappropriate inhaler technique on the basis of this definition . in addition , our findings indicate that target group patients such as widowed patients , patients using metered dose inhalers , patients with a monthly family income of less than three times the national minimum wage , and patients with two or more comorbidities require special attention in terms of inhaler technique education . therefore , it is important that educational strategies for asthma patients be developed to improve their performance of the inhaler technique and increase their level of asthma control . estudo transversal envolvendo pacientes com idade > 14 anos e diagnstico mdico de asma , recrutados no ambulatrio de asma do hospital de clnicas de porto alegre , na cidade de porto alegre ( rs ) . os pacientes completaram dois questionrios ( um geral e um questionrio de controle da asma baseado nas diretrizes da global initiative for asthma de 2011 ) . desses , 81 ( 30,2% ) apresentaram tcnica inalatria incorreta , que foi associada com falta de controle da asma ( p = 0,002 ) . a regresso logstica identificou os seguintes fatores associados com a tcnica inalatria incorreta : ser vivo ( or = 5,01 ; ic95% , 1,74 - 14,41 ; p = 0,003 ) ; utilizar inalador pressurizado ( or = 1,58 ; ic95% , 1,35 - 1,85 ; p < 0,001 ) ; ter renda familiar mensal < 3 salrios mnimos ( or = 2,67 ; ic95% , 1,35 - 1,85 ; p = 0,008 ) ; e ter > 2 comorbidades ( or = 3,80 ; ic95% , 1,03 - 14,02 ; p = 0,045 ) . o protocolo foi aprovado pela comisso de tica e pesquisa do hospital de clnicas de porto alegre ( hcpa ) , em porto alegre ( rs ) . para avaliar a gravidade da asma , foi utilizada a classificao de gravidade conforme o regime medicamentoso dirio usado , proposta pelas diretrizes da global initiative for asthma ( gina ) . quadro 1critrios para avaliar o grau de controle da asma.a ade acordo com a global initiative for asthma. foi identificado que o ponto de corte de um ou mais erros no se associou significativamente com o grau de controle da asma ( p = 0,07 ) , enquanto o ponto de corte de dois ou mais erros se associou de forma significativa com o grau de controle da asma ( p = 0,002 ) . foram observadas diferenas estatisticamente significativas para as seguintes variveis : estado civil ( p = 0,002 ) , sendo maior a proporo de pacientes vivos entre aqueles com tcnica inalatria incorreta ; grau de instruo ( p = 0,023 ) , sendo maior a proporo de pacientes com tempo de estudo < 8 anos entre aqueles com tcnica inalatria incorreta ; renda familiar ( p = 0,016 ) , sendo maior a proporo de pacientes com renda menor que trs salrios mnimos entre aqueles com tcnica inalatria incorreta ; e grau de controle da asma ( p = 0,007 ) , sendo maior a proporo de pacientes com asma no controlada entre aqueles com tcnica inalatria incorreta . a tabela 3 mostra que houve diferena estatisticamente significativa entre os dois grupos de tcnica inalatria para o tipo geral de dispositivo utilizado ( p < 0,001 ) , observando - se maior proporo de pacientes com tcnica correta entre aqueles em uso de dispositivo de p do que aqueles em uso de inalador pressurizado . tambm a proporo de pacientes com tcnica inalatria correta diferiu significativamente entre o tipo especfico de dispositivo utilizado ( p < 0,001 ) , observando - se maior proporo de pacientes com tcnica inalatria correta entre aqueles em uso de aerolizer ou turbuhaler . as variveis que se associaram de forma independente com a tcnica inalatria incorreta foram : ser vivo ( or = 5,01 ; ic95% , 1,74 - 14,41 ; p = 0,003 ) ; usar inalador pressurizado ( or = 1,58 ; ic95% , 1,35 - 1,85 ; p < 0,001 ) ; ter renda familiar menor que trs salrios mnimos ( or = 2,67 ; ic95% , 1,35 - 1,85 ; p = 0,008 ) ; e apresentar duas ou mais comorbidades ( or = 3,80 ; ic95% , 1,03 - 14,02 ; p = 0,045 ) .

overall mortality rate decreased , expectancy of life increased , a lot of new life saving drugs discovered which helps us to fight against several infectious and other diseases , and new advancement in the field of technology has boosted the capacity of modern science . in spite of such incredible advancement whether such benefit of modern science / medicine has reach to the every door of the world ? world health organization ( who ) in an international conference on primary health care in 1978 commonly known as declaration of alma - ata express the need to achieve the goal health for all step by step manner through tackling the poverty , illiteracy and poor sanitation . in 1998 , who incorporates a new global health policy health for all in the 21st century and set the goal to achieve health security , health equity , increased healthy life expectancy and to ensure access to essential quality healthcare for all.1 , 2 modern medical science , despite so many achievements and progress , is finding itself difficult to reach to every people and deal with the ever - increasing diseases and disorders . still , majority of world population mainly in developing and underdeveloped countries does not have access to modern medicine and depends on the time - tested traditional / alternative or complementary systems of medicine , many of these systems is much older compared to the allopathic medical wisdom.3 , 4 therefore , the major questions still exist ( 1 ) whether the goal has achieved ? ( 2 ) whether health for all can be possible without scientific integration of traditional herbal medicine in clinical practice ? in the 21st century , pollution , unhealthy lifestyle , environmental toxins increases the risk of diseases . the side effects , overuse / misuses of allopathic drugs are also a major concern . in 2013 , who developed and lunched who traditional medicine strategy 20142023 and emphasised to integrate traditional and complementary medicine to promote universal healthcare and to ensure the quality , safety and effectiveness of such medicine . therefore , the world is looking for cost effective , easily available , better physiological compatible traditional systems of medicine and holistic approach to avert such problem and provide the basic healthcare to all . knowledge regarding the therapeutic , toxicological effect of plants , minerals and other substances go back to the prehistoric times when people have migrated to into the indian subcontinent . several evidences indicated that in indian subcontinent medical intervention like dentistry and trepanation were exercised as early as 7000 bce . current archaeo - botanical excavations pointed towards the evidence regarding the use of medicinal plants in the middle gangetic region since the 2nd millennium bce which are still found in ayurvedic folk medicine . india is a land of different group of people who have their own religion , beliefs , culture , language and dialects . a number of medicinal systems also introduced here from outside and enriched in india . since ancient time , indian society depends on traditional medicinal systems practiced here . introduction of allopathic drug during british era and neglecting indian traditional medicine by british ruler are responsible for significant erosion of indian traditional medicine . high scientific progress in allopathic medicine and modern facilities also resists the growth of traditional medicine . still , about 70% rural populations of india are believed in traditional medicine for primary healthcare.7 , 8 ayurveda is a comprehensive scientific medicinal system indigenous to india . knowledge of life , which comprises two sanskrit words , ayu ( life ) and veda ( knowledge or science ) . four vedas , considered as the oldest indian literature ( 50001000 bc ) contain information about natural remedies . ayurveda was established as a fully grown medicinal system.9 , 10 charaka samhita ( focussing on internal medicine ) and susruta samhita ( focussing on surgery ) were written systematically and considered as classical text of ayurveda . vital details of charaka samhita and susruta samhita were complied together and updated additionally in astanga sangraha and astanga hrdaya . some other ancient classics which include minor work of ayurveda includes madhava nidana ( focussing on diagnosis of disease ) , bhava prakasa ( focussing on additional information related to plant and diet ) , sarngadhara samhita ( focussing on formulation and dosage form).9 , kayacikitsa ( internal medicine ) , salya tantra ( surgery ) , salakya ( diseases of supra - clavicular origin ) , kaumarabhrtya ( paediatrics , obstetrics and gynaecology ) , bhutavidya ( psychiatry ) , agada tantra ( toxicology ) , rasayana tantra ( rejuvenation and geriatrics ) , vajikarana ( aphrodisiology and eugenics).9 , 10 , 11 siddha system of medicine is believed as a brilliant achievement and symbol of tamil culture which originated in southern parts of india . siddha medicine invented from dravidian culture and is grown in the time of indus valley civilization . chinese alchemy , taoism , and taoist patrology are considered as a main source of inspiration for siddha alchemy . it is believed that in ancient time , the system was developed by eighteen siddhar ( a class of tamil sages ) . though siddha system of medicine resembles with ayurveda in many aspects it has own philosophy and concept , holistic approach , and lifestyle oriented measures.12 , 13 , 14 , 15 unani system of medicine is the fusion of contemporary traditional medicinal system in egypt , syria , iran , iraq , china , india and several other east countries . arab and persian settlers in 11th century introduced unani medicine in india , the system gets recognition and enriched during mughul rule.16 , 17 , 18 amchi or sowa - rigpa is another ancient well documented traditional medicinal system , which was popular in tibet , mongolia , nepal , bhutan , himalayan region of india , some parts of china and former soviet union . though conflicts exist on the origin place of amchi medicine as some believed it originated in india , some say tibetan region and other considered it as chinese origin . amchi has close similarity with ayurveda , though influence of chinese traditional medicine and tibetan folklore also observed in this system . other than codified traditional medicinal system the uncodified folk medicine also plays a vital role in maintenance of health and cure of diseases for large number of people belongs to rural / indigenous / ethnic communities . nearly 8000 plants species are utilized in folk medicine and approximately 25,000 effective plant - based formulations used by the rural and ethnic communities in india . plants are always the key source of drug or treatment strategy in different traditional medicinal systems . in recent years , many people are choosing to plant based medicines or products to improve their health conditions or as curative substance either alone or in combination with others . according to the who , herbs or herbal products are used by the large number of populations for basic healthcare needs . herbal medicine includes herbs , herbal materials ( like plant parts ) or preparations , processed and finished herbal products , active ingredients.20 , 21 in recent years , a huge resurgence of the use of herbal product due to the side effects of modern drugs , failure of modern therapies for against chronic diseases , and microbial resistance . it is estimated that nearly 75% of the plant based therapeutic entities used worldwide were included from traditional / folk medicine . in india , approximately 70% of modern drug are discovered from natural resources and number of other synthetic analogues have been prepared from prototype compounds isolated from plants.20 , 22 , 23 it was reported that more than 60% of cancer drug available in market or in testing are based on natural products . currently , about 80% of antimicrobial , immunosuppressive , cardiovascular , and anticancer drugs are derived from plant sources . more than 70% entities among 177 anticancer drugs approved are based on natural products or mimetic . about 25% prescription drug found globally are derived from plant sources , and nearly 121 such drugs entity are in use . thirteen drugs of natural origin are approved in united states between 2005 and 2007 , and clinical trials are going on more than 100 natural product - based drugs . it was also estimated that 11% of the total 252 drugs found in essential medicine list of who are exclusively of plant origin.24 , 25 in indian traditional medicine a large number of plants are used . it was estimated that ayurveda uses 12001800 plants , siddha medicine includes 500900 plants , unani utilize 400700 medicinal plants and amchi medicine uses nearly 300 plants while folk healers of india use more than 7500 medicinal plants in different medicine . three classical ayurvedic literature charaka samhita , sushruta samhita and astanga hridaya mentioned about 526,573 and 902 number of plants.17 , 26 , 27 in spite of global reorganization and very sound history of traditional uses , promotion of herbal medicine faces number of challenges around the glove mainly in developed nations . following problems need to be overcome before the promotion of traditional herbal knowledge around the world.28 , 29 , 30 , 31 , 32 , 33 , 34quality issues : adulteration , misidentification of plant , faulty collection and preparation , incorrect formulation process are the main problems that reduces the effectiveness of herbal preparation and can be considered as key factors affecting quality and purity of herbal medicines.processing and harvesting issues : indiscriminate harvesting , poor agriculture and propagation method , poor pre and post harvest practices , lack of processing techniques leads to the substandard quality of herbal drugs.quality control related issues : standardization , poor quality control procedure and lack of good manufacturing practices ( gmp ) are the main hurdle to maintain the quality of herbal drugs . lack of awareness regarding the guideline among growers and manufacturers , lack of implementation and regulation of the guideline are also frequent in small and medium scale industries.administrative issues : lack of regulation and controlling authority in herbal sector , lack of proper monitoring and controlling are absolute need for the quality of drugs.infrastructure related issue : lack of processing technique , trained personal , sophisticated instrument , utilization of modern techniques , facility to fabricate instrument locally are the major problems.pharmacogivilane : proper pharmacogivilane in herbal sector is the need of time to find the toxicological data and adverse drug reaction of herbal drugs . adverse reactions , contraindications , interactions with other drug , food and existing orthodox pharmaceuticals need to be monitor properly.clinical trial : since the safety continues to be a foremost issue with the use of herbal remedies therefore , clinical trials are necessary to understand the safety and efficacy of these drugs before introduced them in global market.ipr and biopiracy : biopiracy is the major difficulty in promotion of herbal traditional medicine . documentation of folk knowledge thus important for our future.irrational use : it is generally believed that herbal products do n't have any side effects , interaction , but unfortunately is not true . thus , irrational practice of these drugs can lead to various problems which can hinder the promotion of such drugs.r&d : research and development on dosage , processing , techniques are the key need for any drug , but in herbal sector it is quite less compare to allopathic medicine . research to understand the mode of action and pharmacokinetics phenomenon , improvement / creation of monographs and reference standards for marker - based analysis are necessary of time . decisive gap in current ethnopharmacological and modern medicinal plant research is another problem for sustainable , socio - culturally equitable and safe supply of herbal medicines.other issues : unethical practice of herbal medicine , lack of qualified physician , exposure of unreliable and misleading information , lack of sufficient fund , absence of focused marketing and branding , lack of knowledge sharing also hold back the global promotion of herbal medicine . lack of protection of biodiversity and protecting the traditional medicinal plants are also a big challenge . quality issues : adulteration , misidentification of plant , faulty collection and preparation , incorrect formulation process are the main problems that reduces the effectiveness of herbal preparation and can be considered as key factors affecting quality and purity of herbal medicines . processing and harvesting issues : indiscriminate harvesting , poor agriculture and propagation method , poor pre and post harvest practices , lack of processing techniques leads to the substandard quality of herbal drugs . quality control related issues : standardization , poor quality control procedure and lack of good manufacturing practices ( gmp ) are the main hurdle to maintain the quality of herbal drugs . lack of awareness regarding the guideline among growers and manufacturers , lack of implementation and regulation of the guideline are also frequent in small and medium scale industries . administrative issues : lack of regulation and controlling authority in herbal sector , lack of proper monitoring and controlling are absolute need for the quality of drugs . infrastructure related issue : lack of processing technique , trained personal , sophisticated instrument , utilization of modern techniques , facility to fabricate instrument locally are the major problems . pharmacogivilane : proper pharmacogivilane in herbal sector is the need of time to find the toxicological data and adverse drug reaction of herbal drugs . adverse reactions , contraindications , interactions with other drug , food and existing orthodox pharmaceuticals need to be monitor properly . clinical trial : since the safety continues to be a foremost issue with the use of herbal remedies therefore , clinical trials are necessary to understand the safety and efficacy of these drugs before introduced them in global market . ipr and biopiracy : biopiracy is the major difficulty in promotion of herbal traditional medicine . irrational use : it is generally believed that herbal products do n't have any side effects , interaction , but unfortunately is not true . thus , irrational practice of these drugs can lead to various problems which can hinder the promotion of such drugs . r&d : research and development on dosage , processing , techniques are the key need for any drug , but in herbal sector it is quite less compare to allopathic medicine . research to understand the mode of action and pharmacokinetics phenomenon , improvement / creation of monographs and reference standards for marker - based analysis are necessary of time . decisive gap in current ethnopharmacological and modern medicinal plant research is another problem for sustainable , socio - culturally equitable and safe supply of herbal medicines . other issues : unethical practice of herbal medicine , lack of qualified physician , exposure of unreliable and misleading information , lack of sufficient fund , absence of focused marketing and branding , lack of knowledge sharing also hold back the global promotion of herbal medicine . lack of protection of biodiversity and protecting the traditional medicinal plants are also a big challenge . in spite of number of hurdles , the traditional medicine of india in acknowledged widely around the world and the demand is increasing continuously . combined effort of public and government sector is essential for the promotion of herbal medicine . here we are discussing about the situation and possibilities of promotion of indian traditional herbal medicine in india . in india , the national policy on traditional and alternative medicine was introduced in 1940 in the form of drug and cosmetic act 1940 and drug and cosmetic rule , which was updated in several instate . in 1959 , govt of india recognized traditional indian system of medicine ( ism ) and updated drug and cosmetic act . several expert committees for different ism were established time to time and the earliest was established in 1962 . in the year 1969 , separate chapter related to ayurveda , siddha and unani drugs was inserted by act 13 of 1964 in the act , which partly similar as those for conventional pharmaceuticals . later the act was modified again with some substitutions in the year 1983 , 1987 , 1994 and 2002 . in 2006 and 2008 guideline for evaluation and analysis of drugs under ism was given under drug and cosmetic rule 1945 . the central council of indian medicine ( ccim ) is constituted in the year 1970 , which involved in the framing and implementing different regulations including the curricula and syllabii in ism ( i.e. ayurveda , siddha and unani ) . in 2012 , department of indian medicine and homeopathy ( ism & h ) was formed with the objective to develop the ism . in 2003 , this department was renamed as department of ayurveda , yoga and naturopathy , unani , siddha and homoeopathy ( ayush ) , and in 2014 separate ministry on ayush was formed.35 , 36 , 37 , 38 , 39 department of ayush concentrates on the overall governance , education , regulation , development and growth of ism in the india and abroad . the department has few subordinate offices , several autonomous bodies in the form of research councils , professional council , pharmacopoeia laboratories , national institutes , academy and hospitals . in the year 2002 major objectives of this policy are,37 , 40utilize the ayush to endorse good health and spread out the outreach of healthcare to our people ( mainly who can not afford or reach to the modern healthcare facilities ) through preventive , promotive , mitigative and curative approaches.to provide affordable ayush services & drugs which are safe and efficacies;to ensure the availability and genuine of raw drugs as required by pharmacopoeial standards to help improve quality of ayush drugs , for domestic and/or export purpose.incorporate ayush in healthcare delivery system and national programmes and to ensure the best possible utilization of huge infrastructure of hospitals , dispensaries and physicians.to offer full opportunity for the expansion and development of ism and utilization of the potentiality , strength and revival of their glory . utilize the ayush to endorse good health and spread out the outreach of healthcare to our people ( mainly who can not afford or reach to the modern healthcare facilities ) through preventive , promotive , mitigative and curative approaches . to provide affordable ayush services & drugs which are safe and efficacies ; to ensure the availability and genuine of raw drugs as required by pharmacopoeial standards to help improve quality of ayush drugs , for domestic and/or export purpose . incorporate ayush in healthcare delivery system and national programmes and to ensure the best possible utilization of huge infrastructure of hospitals , dispensaries and physicians . to offer full opportunity for the expansion and development of ism and utilization of the potentiality , strength and revival of their glory . in india , several legislative and administrative measures are in place to control the manufacturing and sale of ayurveda , siddha and unani ( asu ) medicine . chapter iva in the drugs & cosmetics act , 1940 describe the provisions for regulation of manufacturing , packaging , labelling and sale of asu drugs . periodic revision of such act is also a part of betterment of asu drugs , as the latest amendments in this chapter were done on march 2013 . a separate ayurveda , siddha and unani technical advisory board ( asudtab ) was also formed which deals and advice the authorities on the technical matters involved in the regulation of asu drugs . another ayurveda , siddha and unani drugs consultative committee ( asudcc ) also constituted which advice to attain the uniformity in the administration of drugs & cosmetics act , 1940 ( related to asu drugs ) throughout india . t of the indian drugs & cosmetics act , 1940 and rules , 1945 was notified in 2000 . in view of world concern related to the presence of heavy metals in asu drugs guideline was issued to test the asu drug to find the presence of heavy metal and check the limit of heavy metal if any . guidelines have also been issued to curb growth of irrational asu combinations.40 , 41 several rules / guidelines like inclusion of proper botanical name in label , include the caution regarding the consumption of drug under medical supervision if ingredient contain any hazardous / other substance specified in schedule e(1 ) of the drugs and cosmetics rules 1945 , introduction of rule regarding the maintenance of records of raw materials , guideline of permitted excipients along with their standards , law to test heavy metals , stipulation of expiry date for ayurvedic medicines . in 1970 , pharmacopoeial laboratory of indian medicine was formed to ensure standardization and testing of asu drugs . several others government recognized laboratories are also involved to lay down pharmacopoeial standards , preparation of monographs and standard operating procedures ( sops ) for asu drugs . pharmacopoeial committees for asu systems involve in the lay down of standards for quality , purity and strength of drugs and approve drug formularies . pharmacopoeial laboratories , central council for research in ayurveda and siddha ( ccras ) laboratories , laboratories of central council for research in unani medicine ( ccrum ) , council for scientific & industrial research ( csir ) laboratories and several other laboratories of private sector are involved in the mammoth job of controlling and maintaining of quality , formulation of standard for ensuring safety and quality of polyherbal / herbomineral preparations.17 , 40 , 41 both traditional and modern parameters are used to test the quality and to standardize the raw and finished products . several methods like organoleptic standardization of drugs , chemical investigation , and bioassay are used in this regard . ayurvedic pharmacopoeia of india ( part i contain eight volumes and part ii contain three volume of compound formulation ) , unani pharmacopoeia of india ( part i on signal drug contain six volume , part ii on formulation contain two volume ) , siddha pharmacopoeia of india ( volume i and ii ) , ayurvedic formulary of india ( part i & ii ) , unani formulary of india ( part i vi ) , siddha formulary of india ( part i & ii ) are playing a significant role in this area . pharmacognostical , chemical and standards of the plant drugs used in ism are mentioned in such publications . these pharmacopoeias and formularies contain information about the biological source , synonyms , description , tlc , important formulation , therapeutic indication , and details related to identity , purity , strength . identification and estimation of active therapeutic ingredients and marker compounds with reference to which drugs of ayurveda , siddha and unani can be standardized are still developing and all these parameters are being added to pharmacopoeias . inclusion of monograph on herbal drug in indian pharmacopeia and formulation of herbal pharmacopeia are also a major step to achieve the goal . along with pharmacopoeias and formularies other publications like production of ism drugs with current good manufacturing practices , quality standards of indian medicinal plants could also be useful to maintain the standard and quality of ism.37 , 42 , 43 , 44 several research works are going on the asu drugs . basic research to preclinical or clinical study , investigation on standardization and formulation on ism are a hot area of research in current time . central council for research in ayurvedic sciences , central council for research in unani medicine , central council for research in siddha , central council for research in yoga & naturopathy , csir , central drug research institute ( cdri ) , several private research centre , institution and universities are actively engaged in research , development and promotion of traditional herbal medicine . nearly , 9000 manufacturing units of indian traditional medicine are present in the india as on april 2013 . though the majority of them ( 7744 ) are involved in manufacturing of ayurveda drugs , whereas , 485 , 344 and 323 manufacturing units were engaging in manufacturing of unani , siddha and homoeopathy drugs respectively . statistics also suggest that only 0.1% per annum growth was observed realized in total ayush drug manufacturing units during last two decade . a recent statistics showed that among the 10,000 ayurvedic , siddha and unani medicine manufacturing unit about 54% is gmp complying units.37 , 45 ccim was established under the indian medicine central council act , 1970 and involve in the regulation of education and practice of ism . a significant increase in ayush colleges more than 500 ayush under graduate colleges with admission capacities more than 25,000 was recorded in india . ayurveda have 261 under graduate colleges , whereas homoeopathy , siddha , unani and naturopathy have 188 , 9 , 41 and 17 under graduate colleges respectively . more than 125 post graduate colleges ( ayurveda 76 , homoeopathy 40 , siddha 3 , unani 8) with more than 2700 admission capacities were in existence in the india in 2013 . national institute of ayurveda ( jaipur ) , national institute of naturopathy ( pune ) , institute of post graduate teaching and research in ayurveda ( jamnagar ) , national institute of unani medicine ( bengaluru ) , national institute of siddha ( chennai ) are some of the apex educational institute of traditional medicine in india . several courses like bachelor degree medicine and surgery in different indian system of medicine , doctor of medicine ( md ) , doctor of surgery ( ms ) , pg diploma courses , diploma citification courses , phd courses in several branches of ism are being offered by the several institutions certificate course , currently pharmacy degree i.e. d. pharm , b. pharm , m. pharm in indian traditional medicine also started.40 , 45 , 46 it was counted more than 3100 ayush hospitals with 57,056 beds strength runs in india as on april 2013 . maximum number of hospitals are on ayurveda ( 2408 ) , whereas , 255 , 267 , 29 , 201 and 7 hospitals pertain to unani , siddha , naturopathy , homoeopathy and yoga systems respectively . more than 26,000 ayush dispensaries also deliver the primary healthcare facility around the country . number of dispensaries in ayurveda , unani , siddha , yoga , naturopathy , homoeopathy and sowa - rigpa recorded as 15,927 , 1483 , 830 , 140 , 120 , 7585 and 22 , respectively . in 2013 , 686,319 ayush registered practitioners ( maximum 387,976 practitioners have been registered under ayurveda system ) were available which is increasing continuously . the number of institutionally qualified ( iq ) registered practitioners has also been increased in last few years , in 2013 the total number of iq registered practitioners was 461,032.40 , 45 tkdl is an exclusive digital database regarding the medical knowledge mentioned in several ism that are available in the public domain . the database consists the information about the medicinal plant , formulation of ayurveda , siddha , unani , yoga etc . tkdl is a collaborative project of csir and ayush , and the information 's are also available in different national and international language . the access to 2.5 lakh medicinal formulations different to patent offices under tkdl access agreement is considered as unique process to protect traditional knowledge from biopiracy . based on the third party notes submitted by the tkdl , so far a large number of patent applications in european patent offices , canadian intellectual property office , united states patent and trademark office , united kingdom patent and trademark office , controller general of patents , designs and trademarks , india has been either set aside , or withdrawn / cancelled , or declared dead . there has been a steady policy support to promote the traditional medicine in india . the government also supporting different plans related to research - development related to medicinal plant research . the budget allocation for the dept of ayush has been increasing gradually over the years . in the 12th five year plan of india ( 20122017 ) , total allocation for ayush was inr . 10,044 crore , which was 235% more than the actual expenditure of 11th plan . in the view of integrate the ayush system for healthcare system several policies are formulated,40 , 48 , 49 like:utilization of ayush doctors in national reproductive & child health and population stabilization programmesinclusion of several traditional drugs ( i.e. ayush ghutti , bal rasayana , soubhagya shunthi , ark ajwain , ark pudina , punarnavadi mandoor and ksheerbala tel . ) in the national reproductive & child health ( rch ) programme for use by mothers & children.a pilot project to monitor the effect of ayurveda treatment in the ante - natal and post - natal careutilization of available facilities of ism in rural health care mission ( nrhm ) . like , appointing ayurveda doctors and paramedics in the primary healthcare delivery system and in national health programs.inclusion of ayush drug ( i.e. punarnavadi mandoor for management of anaemia during pregnancy ) kit of asha ( asha or accredited social health activist act as an interface between the community and the public health system in rural india ) in addition to generic drugs for common ailments at sub - centre / primary health centre / community health centre.ensure the availability of ayurvedic , siddha and unani essential drug to primary health centre.to find the way of inclusion of ayush medicine in schemes such as janani suraksha yojana ( jsy - ayush ) , icds - ayush , reproductive child health ( rch ) , early breastfeeding , growth monitoring of children , ante and post natal care , etc . and find their effectiveness . utilization of ayush doctors in national reproductive & child health and population stabilization programmes inclusion of several traditional drugs ( i.e. ayush ghutti , bal rasayana , soubhagya shunthi , ark ajwain , ark pudina , punarnavadi mandoor and ksheerbala tel . ) in the national reproductive & child health ( rch ) programme for use by mothers & children . a pilot project to monitor the effect of ayurveda treatment in the ante - natal and post - natal care utilization of available facilities of ism in rural health care mission ( nrhm ) . like , appointing ayurveda doctors and paramedics in the primary healthcare delivery system and in national health programs . inclusion of ayush drug ( i.e. punarnavadi mandoor for management of anaemia during pregnancy ) kit of asha ( asha or accredited social health activist act as an interface between the community and the public health system in rural india ) in addition to generic drugs for common ailments at sub - centre / primary health centre / community health centre . ensure the availability of ayurvedic , siddha and unani essential drug to primary health centre . to find the way of inclusion of ayush medicine in schemes such as janani suraksha yojana ( jsy - ayush ) , icds - ayush , reproductive child health ( rch ) , early breastfeeding , growth monitoring of children , ante and post natal care , etc . and find their effectiveness . recently , there are some suggestion to allow the ayush doctor to prescribe modern medicine if rural areas in view of shortage of allopathic doctors . but there is strong oppose as it may violate the rule present in india . collaboration , establishment of research institute in foreign , conduct of seminar - conferences are the key way to promote the ism globally . ayush opened an indo - us joint center for research on indian systems of medicine ( crism ) at the university of mississippi usa in 2008 . ayurveda education & research , gujarat ayurveda university , jamnagar singed a mou with institutions in japan , australia , netherlands , argentina , italy , and usa . several other national institutions also have mou with foreign institutions in the field of research and education in ism . institutions of india offering various courses for the students from japan , russia , south africa , netherlands , france , hungary , canada , usa , poland , germany , brazil , switzerland , ukraine , sri lanka etc . india and russia had concluded a mou on the cooperation in the field of ayurveda teaching , treatment and research . in 2004 , russian - indian centre on ayurvedic research was established in moscow.40 , 51 protection of biodiversity and conservation of medicinal plants is essential for the livelihood security and ensure the availability of medicinal plant in future . ayush drug manufacturing units use almost 90% of the raw materials of medicinal plants from natural forests , but during such process we overlook the environmental and social issues . therefore , sustainable use of medicinal plants and good field collection practice is important . in situ ( conservation of plants in their natural habitat outside the native habitat ) and ex situ conservation ( like , seed storage , dna storage , pollen storage , in vitro conservation , field gene banks and botanical garden etc ) are the key approaches to protect and conserve medicinal plant species.52 , 53 in recent years there is a huge upserge in the use of traditional and complementary medicine around the glove . in africa nearly 80% of population uses such medicine for their primary healthcare . in china , it was estimated that traditional herbal medicine account for 3050% of the total medicinal consumption . majority of the people ( around 60% ) uses traditional herbal drugs as a first line medicine for treatment high fever resulting from malaria in countries like ghana , mali , nigeria and zambia . in australia about 48% , in canada 70% , in germany 80% , in usa 42% , in belgium 39% and in france 76% of population uses traditional / complementary medicine at least once . around 75% of the hiv positive / aids patients living in san francisco , london and south africa use traditional and complementary medicine . in malaysia importance of herbal medicines in terms of healthcare provider and economy are growing steadily.53 , 54 , 55 therefore , india has a great opportunity to promote ism globally . ayurveda is well recognized in asian countries like nepal , sri lanka , bangladesh and other asian countries . about 75% of 100 million subcontinental people are enjoying the benefits of ayurveda . in japan , osaka medical school has formed the society of ayurveda in 1969 , and since last 40 years study , research and spread of ayurveda is being carried out passionately . ism mainly ayurveda is increasing in argentina , brazil , venezuela , chile , nicaragua , costa rica , guatemala , germany , austria , switzerland , france , czech republic , greece , israel . countries like south africa , uae , russia , sweden , indonesia , netherlands , italy , spain , australia , new zealand , hungary have acknowledged ayurveda . several other countries are on the verge of doing the same.56 , 57 approximately 25,000 effective plant - based formulations are available in ism which is commonly used by rural and ethnic people n india and the popularity of such medicine is also increasing among the common people . it was also estimated that > 2000 tons of medicinal plant raw material is required annually . it was also estimated that nearly 960 species of medicinal plants are in trade , among them 178 species have annual consumption levels more than 100 metric tones . 8090 billion , and export value of medicinal plants and related products from india is approximately l10 billion . in 20122013 , 24,741.2 crores , though in next financial year ( 20132014 ) it was reduced slightly . the percentage share of ayush products in the total trade of india in 20132014 was 0.36% . the global market for herbal drugs is increasing in steady manner and the global herbal trade will reach usd 7 trillion by 2050.8 , 17 , 58 ayurveda and other isms are involved in the curative and preventive measures to promote the health . rasayana ( rejuvenation therapy ) , branch of ayurveda involve in the preservation and promotion of health by promoting longevity and also prevents or delays the ageing process , which another speciality of ayurveda namely panchakarma ( purification therapy ) removes the toxins and waste materials from the body and thus purify the biological system to disease completely . ayurvedic formulations are highly effective against various common diseases like common cold , fever , hyperacidity , ulcer , cough , gastro - intestinal problems , diarrhoea , amoebic dysentery , liver diseases , uterine bleeding , urinary tract infection , arthritic condition , gout , bronchial asthma , eye diseases etc . at the primary healthcare level . formulation of ayurveda also shown prominent effect in the treatment of several chronic diseases like cardiovascular disease ( hypertension , angina , cardiomyopathies , myocardial infarction , congenital heart disease ) , cancer , dengue , anti - inflammatory disease , kidney diseases etc.59 , 60 , 61 , 62 , 63 some studies have suggested that ayurvedic medicine is also useful to manage some emergency conditions like severe diarrhoea and vomiting , patient suffering from typhoid suffered from semi consciousness and also muttering delirium , burns and seals , poison , threatened abortion , abortion & miscarriage etc . medicine from siddha system is used to cure diverse diseases like skin problems ( psoriasis ) , sexual transmitted diseases , urinary tract infections , liver and gastro - intestinal diseases , diabetes , general debility , postpartum anaemia , diarrhoea , rheumatic diseases , prostate enlargement , bleeding piles , peptic ulcer , venereal diseases , fever , allergic disorders and general fevers other than emergency cases.65 , 66 unani drugs are used to treat hepatitis , gastroenteritis & uteritis , fever , cardiovascular problems , palpitation , nausea , vomiting , diarrhoea , gastro intestinal trouble , fever , insomnia , schizophrenia , epilepsy , gonorrhoea , urinary tract infection , kidney stone , headache , dizziness , common cold , migraine , colic pain , arthritis , syphilis , paralysis , diabetes insipidus , bad wetting , anxiety , typhoid fever , measles , small pox , premature ejaculation etc . medicinal plants found in india and utilized by the different folk and codified medicine are utilized to cure diverse diseases . effectiveness of ism particularly ayurveda has been evident in sub - continent as people enjoys the benefit of such medicine since the pre - biblical era . core strength in these systems is the holistic approach to health and disease using natural substances derived from medicinal plants , minerals and animal sources . but in current situation proper clinical trial , quality assurance and pharmacogivilance are the important area to promote such drug worldwide . in recent year 's national and international level a number of collaboration in this sector has been increasingly attracting several entrepreneurs and organizations . ayush research portal ( http://ayushportal.nic.in/ ) included information regarding 4175 clinical trial , 8514 pre - clinical research , 6002 drug research and 2926 fundamental research information related to ism or plants used in these systems as on january 2016 . safety evolution and to find the mode of administration or use also a critical criteria of these study . for example , a study showed that polyherbal drug , ab - fn-02 having significant anti - osteoarthritic effect and non - toxic when administered in traditional method with milk , but it may produce toxic effect when administered as a drug otherwise . information about 500 medicinal plants / formulations used by folk medicine practitioners ( non - codified system ) was included and currently scientific validation ( safety , efficacy , mode of action and clinical trials ) of few selected leads are going on different parts of india . although it is true that proper high value clinical trial of such medicine is comparatively les due to small size of population , problems with research designs , lack of appropriate control groups etc . drug discovery from medicinal plants used in different indian medicinal systems is a hot spot of research . a number of drugs were obtained from the plant sources and several others have discovered by using natural substance as lead . investigations in india and abroad became played a key role in such research . in 1931 , sen and bose reported two alkaloids from rauwolfia serpentina , siddiqui and siddiqui in same year isolated five alkaloids which named as ajmaline , ajmalinine , ajmalicine , scrpentine , and serpentinine . chopra and his colleagues in 1933 isolate an alkaloid from the plant and observed the hypotensive and cns depressant activity . several others investigations had also been made by the indian researchers in subsequent years . in 1949 , a historical paper by dr vakil in british heart journal reported the antihypertensive activity of rauwolfia in patients . during that time nearly 90% of doctors in india used it as a routine hypotensive drug and about 50 million tablets had been sold by a manufacturing agency alone.72 , 73 peruvoside , a cardiac glycoside was isolated from thevetia peruviana at the indian laboratory and developed in germany . taxol , potent anticancer drug discovered from taxus brevifolia , the plant has been utilized by western indian cultures as a medicine since long time . a national / international research discovered a number of drugs from plant which has been used indian traditional medicine since ancient time , like , vasicine and vasicinone from adhatoda vasica , bacosoids from bacopa monnieri , tylophorine from tylophora indica , homoharringtonine from cephalotaxus , camptothecin from camptotheca acuminate , conessine from holarrhena antidysentrica , morphine and codeine from papaver som - niferum , sarsasapogenin , asparanin a and asparanin b from asparagus adscendens , shatavarin from asparagus racemosus , atropine from atropa belladonna , glycyrrhizin from glycyrrhiza glabra , aloin from aloe vera , protodioscin from tribulus terrestris , sophoradin from sophora subprostrata , quinine from cinchona spp . , trigonelline from trigonella foenum - graecum , catechin from acacia catechu , withanolides from withania somnifera , tinosporic acid from tinospora cordifolia , cocaine from erythroxy - lum coca , aegelin and marmelosin from aegle marmelos , pris - timerin from celastrus paniculata , asiaticoside from centella asiatica , emetine from cephaelis ipecacuanha , psoralen from psoralea corylifolia , glycyrrhizin from g. glabra , boeravinones from boerrhavia diffusa , berberine from berberis aristata , plumbagin from plumbago indica , curcumin from curcuma longa , podophyllin from podophyllum emodi , jatamansone from nardostachys jatamansi , quassinoids from ailanthus spp . , arjunolic acid from terminalia arjuna , gingerols from zingiber officinale , digoxin and digitoxin from digitalis lantana , paclitaxel from taxus baccata and taxus brevifolia , allicin from allium sativum , nimbidin from azadirachta indica , forskolin from coleus forskohlii , pilocarpine from pilocarpus jaborandi , dysobinin from dysoxylum binectariferum , diosgenin from t. foenum - graecum and plants of dioscorea spp . , vinblastine and vincristine from catharanthus roseus and many more.17 , 75 , 76 novel semisynthetic derivatives of rohitukine ( from the plant amoora rohituka & d. binectariferum ) named as flavopiridol and p-276 - 00 are in the advance clinical trial as anticancer drug . guggulu , an oleo - gum resin obtained from the bark of commiphora wightii has been used in ayurveda for the treatment of inflammation , gout , rheumatism , obesity , and disorders of lipids metabolism . several compounds namely z - guggulsterone , e - guggulsterone , guggulsterol - i , guggulsterol - ii etc . have been isolated from guggulu . csir and its constituents laboratories are involved in the development of new herbal drug or formulation . some of the key developments in central drug research institute ( cdri ) in this area are , ( i ) standardized fraction of gugulipid was developed by cdri and marketed ( guglip , cipla ltd ) as a drug for hyperlipidaemia and atherosclerosis , ( ii ) arteether ( a semisynthetic derivative of artemisinin , the active constituent of artemisia annua ) as antimalarial drug which is marketed by themis chemicals ltd . , mumbai under the trade name e - mal , ( iii ) consap ( a local spermicidal cream ) contain saponins from sapindus mukorossi , ( iv ) picroliv , an iridoid glycoside mixture containing 60% picroside i and kutoside obtained from picrorhiza kurroa developed as hepatoprotective agent , ( v ) a standardize herbal preparation derived from the plant b. monnieri as memory enhancer . rrl jammu has commercialized boswellia serrata gum resin as nsaid ( non - steroidal anti - inflammatory drug ) ( sallaki gufic ) . a number of herbal pain reliever , antifungal cream , anti - dandruff shampoo has been developed by different csir labs across the country . very recently , an antidiabetic drug ( bgr-34 ) has developed jointly developed by scientist of csir - nbri & csir - cimap . under the golden triangle partnership project between ayush , indian council of medical research ( icmr ) , csir there is an attempt to find few formulations and developing new drugs . in recent years research on these areas is increasing and lot more drug / formulations investigated by public or private sector in india are in queue or in under clinical trial . india has great pool of diverse medicinal plant sources , a long and well characterized traditional medicinal system which makes india a unique place of new drug discovery . global pharmaceutical companies and researchers equipped with modern scientific knowledge , technology , idea and started to rediscover medicinal plants as a source of new drug candidates based on traditional knowledge.17 , 20 modern technology and techniques have revolutionized the progression of drug discovery from medicinal plants . new approaches / concepts / technologies became a key tool in the development of traditional medicine further . research utilizing modern equipments or methods helped us to isolate and develop phytoconstituents as new drug present in traditional herbal formulation or medicinal plants . modern technology and techniques became an essential tool to monitor and maintain the quality of traditional formulation , use phytochemical as lead to discover new drugs , to find pharmacokinetic profile and toxicity , to find out mechanism , to find the new use of existing drug or formulation , acceleration of drug discovery process , synthetic and semisynthetic process to manufacture a natural constituent etc . drug discovery based on traditional information is a key path towards the discovery of new drug . reverse pharmacology is an approach where discovery of leads / formulations is based on the documented clinical experiences and scientific observations through series of studies . reverse pharmacology based on traditional knowledge concentrate on the reversing routine laboratory - to - clinic development to clinics - to - laboratories. safety is considered as most significant point remains and the effectiveness becomes a matter of validation . in 21st century , tremendous advances in healthcare sector coexist with inequities in accessibility , availability and affordability of the healthcare facilities in many parts of the world . since last few decades , there is a growing interest in traditional medicine in all over the globe . variety , flexibility , easy availability , religious / social acceptance , relative low side effect and cost became the key factor for the growth of traditional medicine . of course , these also provide us the opportunity to integrate such medicine in primary healthcare to facilitate the people health . a number of approaches have been formulated toward the mainstreaming of traditional medicine in healthcare system . romantic approach suggested that traditional medicine is good as such and should be remained as it is , trans - cultural and transdisciplinary synergy approach support the fact that sciences recognize that they stand for one type of knowledge among others and that information is always culturally entrenched and forming part of historic advance , syncretic approach considered to be merging two system to form a new system , in complementarity approach one system provides as supportive role to another . but evidence based approach to utilize both conventional as well as traditional medicine in conjunction could be the best option to provide healthcare facility to all . a number of case studies or surveys had shown the importance of traditional medicine in primary healthcare service . a study in rural area of west bengal shows that folk medicine play a key role to prevent common diseases likes small injuries , skin disease , fever , dehydration , diabetes , high bp , liver disease etc in better way . in rural areas of meghalaya , indigenous medicine play significant role in primary healthcare for prevention / management of common ailments . another study reported the efficacy of ayurvedic multimodal management in osteoarthritis , the study also proposed that the ayurvedic drugs can be an alternative of nsaids in such treatment . beneficial effect of integration of ayurveda with modern system of medicine in tertiary care hospital for management of osteoarthritis ( knee ) also investigated . ayurvedic treatment proved effective with respect to reducing the symptoms , improving quality of life and reducing the side effect of allopathic pain killers . it was also suggested that ayurveda offers outstanding drugs and treatments which can be easily included along with the mainstream cancer medicines . ayurvedic treatment is effective to reduce the side effects of chemotherapy and to improve general well - being of the patient . herbal remedies are found most useful to manage simple healthcare problems like fever , diarrhoea , dysentery , upper respiratory tract infections , worm infestations , certain liver diseases , hepatitis , anaemia , arthritis , few gynaecological problem along with different communicable diseases such as malaria , hiv . in india , the ratio of the doctor - patient is 1:1700 if we consider only allopathic doctors , the ratio will come to 1:800 if the ayush practitioners are added . , an acute shortage of allopathic doctors exists in the india and in rural areas this problem is higher and with very less presence in remote areas . traditional medicine particularly herbal medicine playing important role in maintain of health in rural and remote areas . indian traditional medicine like ayurveda and others have sound scientific background of effectiveness and also acknowledged by the recent researches . although efforts are needed to overcome barriers like irrational use , quality control and standardization issues , high pharmacovogilance etc . stick implementation of rules , monitoring and periodic revision of regulations are absolute necessary to promote indian traditional medicine . overall , adequate knowledge about the system , high quality clinical trial , proper information about such drugs and their effectiveness among common people required towards the promotion of such medicine . integration of ayurvedic and others indian traditional medicine in clinical practice will helpful to promote the health of the people who are unable to access the modern medicine properly . utilization of such medicine along with conventional drug surly put more values to promote health or cure diseases in the better way . therefore , mainstreaming of ism along with allopathic drugs and healthy lifestyle will be helpful to provide healthcare service in best possible way to all people not only in india but around the globe .

in spite of incredible advances in modern science , technology and allopathic medicine a large we are unable to provide quality healthcare to all . traditional medicine particularly herbal medicine considered as a major healthcare provider around the globe particularly in rural and remote areas . a large section of people depends on such medicine for their primary healthcare mainly in underdeveloped or developing countries . indian traditional medicinal system like ayurveda , siddha and unani has a very rich history of their effectiveness ; modern research also acknowledged the importance of such medicine . indian traditional medicine or medicinal plants are also considered as a vital source of new drug . mainstreaming of such medicine is important for the people . several steps have been taken in india to promote such medicine and to integrate them into clinical practice . evidence based incorporation of indian traditional medicine in clinical practice will help to provide quality healthcare to all .

Introduction Indian society and traditional medicine Herbal medicine and its importance Promotion of herbal medicine problems need to be addressed Modernization & integration of herbal medicine in clinical practice experience from India Indian traditional medicine revival story and globalization Towards the achievement of universal healthcare Conclusion

overall mortality rate decreased , expectancy of life increased , a lot of new life saving drugs discovered which helps us to fight against several infectious and other diseases , and new advancement in the field of technology has boosted the capacity of modern science . in spite of such incredible advancement whether such benefit of modern science / medicine has reach to the every door of the world ? in 1998 , who incorporates a new global health policy health for all in the 21st century and set the goal to achieve health security , health equity , increased healthy life expectancy and to ensure access to essential quality healthcare for all.1 , 2 modern medical science , despite so many achievements and progress , is finding itself difficult to reach to every people and deal with the ever - increasing diseases and disorders . still , majority of world population mainly in developing and underdeveloped countries does not have access to modern medicine and depends on the time - tested traditional / alternative or complementary systems of medicine , many of these systems is much older compared to the allopathic medical wisdom.3 , 4 therefore , the major questions still exist ( 1 ) whether the goal has achieved ? ( 2 ) whether health for all can be possible without scientific integration of traditional herbal medicine in clinical practice ? the side effects , overuse / misuses of allopathic drugs are also a major concern . in 2013 , who developed and lunched who traditional medicine strategy 20142023 and emphasised to integrate traditional and complementary medicine to promote universal healthcare and to ensure the quality , safety and effectiveness of such medicine . therefore , the world is looking for cost effective , easily available , better physiological compatible traditional systems of medicine and holistic approach to avert such problem and provide the basic healthcare to all . since ancient time , indian society depends on traditional medicinal systems practiced here . introduction of allopathic drug during british era and neglecting indian traditional medicine by british ruler are responsible for significant erosion of indian traditional medicine . high scientific progress in allopathic medicine and modern facilities also resists the growth of traditional medicine . still , about 70% rural populations of india are believed in traditional medicine for primary healthcare.7 , 8 ayurveda is a comprehensive scientific medicinal system indigenous to india . ayurveda was established as a fully grown medicinal system.9 , 10 charaka samhita ( focussing on internal medicine ) and susruta samhita ( focussing on surgery ) were written systematically and considered as classical text of ayurveda . some other ancient classics which include minor work of ayurveda includes madhava nidana ( focussing on diagnosis of disease ) , bhava prakasa ( focussing on additional information related to plant and diet ) , sarngadhara samhita ( focussing on formulation and dosage form).9 , kayacikitsa ( internal medicine ) , salya tantra ( surgery ) , salakya ( diseases of supra - clavicular origin ) , kaumarabhrtya ( paediatrics , obstetrics and gynaecology ) , bhutavidya ( psychiatry ) , agada tantra ( toxicology ) , rasayana tantra ( rejuvenation and geriatrics ) , vajikarana ( aphrodisiology and eugenics).9 , 10 , 11 siddha system of medicine is believed as a brilliant achievement and symbol of tamil culture which originated in southern parts of india . chinese alchemy , taoism , and taoist patrology are considered as a main source of inspiration for siddha alchemy . though siddha system of medicine resembles with ayurveda in many aspects it has own philosophy and concept , holistic approach , and lifestyle oriented measures.12 , 13 , 14 , 15 unani system of medicine is the fusion of contemporary traditional medicinal system in egypt , syria , iran , iraq , china , india and several other east countries . arab and persian settlers in 11th century introduced unani medicine in india , the system gets recognition and enriched during mughul rule.16 , 17 , 18 amchi or sowa - rigpa is another ancient well documented traditional medicinal system , which was popular in tibet , mongolia , nepal , bhutan , himalayan region of india , some parts of china and former soviet union . amchi has close similarity with ayurveda , though influence of chinese traditional medicine and tibetan folklore also observed in this system . other than codified traditional medicinal system the uncodified folk medicine also plays a vital role in maintenance of health and cure of diseases for large number of people belongs to rural / indigenous / ethnic communities . nearly 8000 plants species are utilized in folk medicine and approximately 25,000 effective plant - based formulations used by the rural and ethnic communities in india . plants are always the key source of drug or treatment strategy in different traditional medicinal systems . in india , approximately 70% of modern drug are discovered from natural resources and number of other synthetic analogues have been prepared from prototype compounds isolated from plants.20 , 22 , 23 it was reported that more than 60% of cancer drug available in market or in testing are based on natural products . it was also estimated that 11% of the total 252 drugs found in essential medicine list of who are exclusively of plant origin.24 , 25 in indian traditional medicine a large number of plants are used . it was estimated that ayurveda uses 12001800 plants , siddha medicine includes 500900 plants , unani utilize 400700 medicinal plants and amchi medicine uses nearly 300 plants while folk healers of india use more than 7500 medicinal plants in different medicine . three classical ayurvedic literature charaka samhita , sushruta samhita and astanga hridaya mentioned about 526,573 and 902 number of plants.17 , 26 , 27 in spite of global reorganization and very sound history of traditional uses , promotion of herbal medicine faces number of challenges around the glove mainly in developed nations . following problems need to be overcome before the promotion of traditional herbal knowledge around the world.28 , 29 , 30 , 31 , 32 , 33 , 34quality issues : adulteration , misidentification of plant , faulty collection and preparation , incorrect formulation process are the main problems that reduces the effectiveness of herbal preparation and can be considered as key factors affecting quality and purity of herbal medicines.processing and harvesting issues : indiscriminate harvesting , poor agriculture and propagation method , poor pre and post harvest practices , lack of processing techniques leads to the substandard quality of herbal drugs.quality control related issues : standardization , poor quality control procedure and lack of good manufacturing practices ( gmp ) are the main hurdle to maintain the quality of herbal drugs . lack of awareness regarding the guideline among growers and manufacturers , lack of implementation and regulation of the guideline are also frequent in small and medium scale industries.administrative issues : lack of regulation and controlling authority in herbal sector , lack of proper monitoring and controlling are absolute need for the quality of drugs.infrastructure related issue : lack of processing technique , trained personal , sophisticated instrument , utilization of modern techniques , facility to fabricate instrument locally are the major problems.pharmacogivilane : proper pharmacogivilane in herbal sector is the need of time to find the toxicological data and adverse drug reaction of herbal drugs . thus , irrational practice of these drugs can lead to various problems which can hinder the promotion of such drugs.r&d : research and development on dosage , processing , techniques are the key need for any drug , but in herbal sector it is quite less compare to allopathic medicine . lack of protection of biodiversity and protecting the traditional medicinal plants are also a big challenge . lack of protection of biodiversity and protecting the traditional medicinal plants are also a big challenge . in spite of number of hurdles , the traditional medicine of india in acknowledged widely around the world and the demand is increasing continuously . here we are discussing about the situation and possibilities of promotion of indian traditional herbal medicine in india . in the year 1969 , separate chapter related to ayurveda , siddha and unani drugs was inserted by act 13 of 1964 in the act , which partly similar as those for conventional pharmaceuticals . ayurveda , siddha and unani ) . in 2012 , department of indian medicine and homeopathy ( ism & h ) was formed with the objective to develop the ism . in 2003 , this department was renamed as department of ayurveda , yoga and naturopathy , unani , siddha and homoeopathy ( ayush ) , and in 2014 separate ministry on ayush was formed.35 , 36 , 37 , 38 , 39 department of ayush concentrates on the overall governance , education , regulation , development and growth of ism in the india and abroad . in the year 2002 major objectives of this policy are,37 , 40utilize the ayush to endorse good health and spread out the outreach of healthcare to our people ( mainly who can not afford or reach to the modern healthcare facilities ) through preventive , promotive , mitigative and curative approaches.to provide affordable ayush services & drugs which are safe and efficacies;to ensure the availability and genuine of raw drugs as required by pharmacopoeial standards to help improve quality of ayush drugs , for domestic and/or export purpose.incorporate ayush in healthcare delivery system and national programmes and to ensure the best possible utilization of huge infrastructure of hospitals , dispensaries and physicians.to offer full opportunity for the expansion and development of ism and utilization of the potentiality , strength and revival of their glory . to offer full opportunity for the expansion and development of ism and utilization of the potentiality , strength and revival of their glory . in india , several legislative and administrative measures are in place to control the manufacturing and sale of ayurveda , siddha and unani ( asu ) medicine . a separate ayurveda , siddha and unani technical advisory board ( asudtab ) was also formed which deals and advice the authorities on the technical matters involved in the regulation of asu drugs . another ayurveda , siddha and unani drugs consultative committee ( asudcc ) also constituted which advice to attain the uniformity in the administration of drugs & cosmetics act , 1940 ( related to asu drugs ) throughout india . identification and estimation of active therapeutic ingredients and marker compounds with reference to which drugs of ayurveda , siddha and unani can be standardized are still developing and all these parameters are being added to pharmacopoeias . inclusion of monograph on herbal drug in indian pharmacopeia and formulation of herbal pharmacopeia are also a major step to achieve the goal . along with pharmacopoeias and formularies other publications like production of ism drugs with current good manufacturing practices , quality standards of indian medicinal plants could also be useful to maintain the standard and quality of ism.37 , 42 , 43 , 44 several research works are going on the asu drugs . nearly , 9000 manufacturing units of indian traditional medicine are present in the india as on april 2013 . though the majority of them ( 7744 ) are involved in manufacturing of ayurveda drugs , whereas , 485 , 344 and 323 manufacturing units were engaging in manufacturing of unani , siddha and homoeopathy drugs respectively . a recent statistics showed that among the 10,000 ayurvedic , siddha and unani medicine manufacturing unit about 54% is gmp complying units.37 , 45 ccim was established under the indian medicine central council act , 1970 and involve in the regulation of education and practice of ism . national institute of ayurveda ( jaipur ) , national institute of naturopathy ( pune ) , institute of post graduate teaching and research in ayurveda ( jamnagar ) , national institute of unani medicine ( bengaluru ) , national institute of siddha ( chennai ) are some of the apex educational institute of traditional medicine in india . d. pharm , b. pharm , m. pharm in indian traditional medicine also started.40 , 45 , 46 it was counted more than 3100 ayush hospitals with 57,056 beds strength runs in india as on april 2013 . more than 26,000 ayush dispensaries also deliver the primary healthcare facility around the country . number of dispensaries in ayurveda , unani , siddha , yoga , naturopathy , homoeopathy and sowa - rigpa recorded as 15,927 , 1483 , 830 , 140 , 120 , 7585 and 22 , respectively . the database consists the information about the medicinal plant , formulation of ayurveda , siddha , unani , yoga etc . there has been a steady policy support to promote the traditional medicine in india . punarnavadi mandoor for management of anaemia during pregnancy ) kit of asha ( asha or accredited social health activist act as an interface between the community and the public health system in rural india ) in addition to generic drugs for common ailments at sub - centre / primary health centre / community health centre.ensure the availability of ayurvedic , siddha and unani essential drug to primary health centre.to find the way of inclusion of ayush medicine in schemes such as janani suraksha yojana ( jsy - ayush ) , icds - ayush , reproductive child health ( rch ) , early breastfeeding , growth monitoring of children , ante and post natal care , etc . ensure the availability of ayurvedic , siddha and unani essential drug to primary health centre . in 2004 , russian - indian centre on ayurvedic research was established in moscow.40 , 51 protection of biodiversity and conservation of medicinal plants is essential for the livelihood security and ensure the availability of medicinal plant in future . therefore , sustainable use of medicinal plants and good field collection practice is important . in africa nearly 80% of population uses such medicine for their primary healthcare . majority of the people ( around 60% ) uses traditional herbal drugs as a first line medicine for treatment high fever resulting from malaria in countries like ghana , mali , nigeria and zambia . in malaysia importance of herbal medicines in terms of healthcare provider and economy are growing steadily.53 , 54 , 55 therefore , india has a great opportunity to promote ism globally . several other countries are on the verge of doing the same.56 , 57 approximately 25,000 effective plant - based formulations are available in ism which is commonly used by rural and ethnic people n india and the popularity of such medicine is also increasing among the common people . it was also estimated that nearly 960 species of medicinal plants are in trade , among them 178 species have annual consumption levels more than 100 metric tones . medicinal plants found in india and utilized by the different folk and codified medicine are utilized to cure diverse diseases . effectiveness of ism particularly ayurveda has been evident in sub - continent as people enjoys the benefit of such medicine since the pre - biblical era . but in current situation proper clinical trial , quality assurance and pharmacogivilance are the important area to promote such drug worldwide . although it is true that proper high value clinical trial of such medicine is comparatively les due to small size of population , problems with research designs , lack of appropriate control groups etc . during that time nearly 90% of doctors in india used it as a routine hypotensive drug and about 50 million tablets had been sold by a manufacturing agency alone.72 , 73 peruvoside , a cardiac glycoside was isolated from thevetia peruviana at the indian laboratory and developed in germany . a national / international research discovered a number of drugs from plant which has been used indian traditional medicine since ancient time , like , vasicine and vasicinone from adhatoda vasica , bacosoids from bacopa monnieri , tylophorine from tylophora indica , homoharringtonine from cephalotaxus , camptothecin from camptotheca acuminate , conessine from holarrhena antidysentrica , morphine and codeine from papaver som - niferum , sarsasapogenin , asparanin a and asparanin b from asparagus adscendens , shatavarin from asparagus racemosus , atropine from atropa belladonna , glycyrrhizin from glycyrrhiza glabra , aloin from aloe vera , protodioscin from tribulus terrestris , sophoradin from sophora subprostrata , quinine from cinchona spp . india has great pool of diverse medicinal plant sources , a long and well characterized traditional medicinal system which makes india a unique place of new drug discovery . global pharmaceutical companies and researchers equipped with modern scientific knowledge , technology , idea and started to rediscover medicinal plants as a source of new drug candidates based on traditional knowledge.17 , 20 modern technology and techniques have revolutionized the progression of drug discovery from medicinal plants . research utilizing modern equipments or methods helped us to isolate and develop phytoconstituents as new drug present in traditional herbal formulation or medicinal plants . drug discovery based on traditional information is a key path towards the discovery of new drug . since last few decades , there is a growing interest in traditional medicine in all over the globe . variety , flexibility , easy availability , religious / social acceptance , relative low side effect and cost became the key factor for the growth of traditional medicine . of course , these also provide us the opportunity to integrate such medicine in primary healthcare to facilitate the people health . a number of approaches have been formulated toward the mainstreaming of traditional medicine in healthcare system . romantic approach suggested that traditional medicine is good as such and should be remained as it is , trans - cultural and transdisciplinary synergy approach support the fact that sciences recognize that they stand for one type of knowledge among others and that information is always culturally entrenched and forming part of historic advance , syncretic approach considered to be merging two system to form a new system , in complementarity approach one system provides as supportive role to another . but evidence based approach to utilize both conventional as well as traditional medicine in conjunction could be the best option to provide healthcare facility to all . a number of case studies or surveys had shown the importance of traditional medicine in primary healthcare service . in rural areas of meghalaya , indigenous medicine play significant role in primary healthcare for prevention / management of common ailments . , an acute shortage of allopathic doctors exists in the india and in rural areas this problem is higher and with very less presence in remote areas . traditional medicine particularly herbal medicine playing important role in maintain of health in rural and remote areas . indian traditional medicine like ayurveda and others have sound scientific background of effectiveness and also acknowledged by the recent researches . stick implementation of rules , monitoring and periodic revision of regulations are absolute necessary to promote indian traditional medicine . overall , adequate knowledge about the system , high quality clinical trial , proper information about such drugs and their effectiveness among common people required towards the promotion of such medicine . integration of ayurvedic and others indian traditional medicine in clinical practice will helpful to promote the health of the people who are unable to access the modern medicine properly . utilization of such medicine along with conventional drug surly put more values to promote health or cure diseases in the better way . therefore , mainstreaming of ism along with allopathic drugs and healthy lifestyle will be helpful to provide healthcare service in best possible way to all people not only in india but around the globe .

asthma is a chronic inflammatory disease that is highly prevalent worldwide and it is characterized by the recruitment of leukocytes , mainly eosinophils , airway hyperreactivity , ige production , and mucus hypersecretion . the physiopathology of allergic asthma is coordinated mainly by th2-type immune responses which are characterized by release of cytokines such as interleukin- ( il- ) 4 , il-5 , and il-13 and chemokines such as rantes and ccl11 [ 13 ] . most patients with asthma have symptoms that are readily controllable by standard asthma therapies , including 2-adrenergic agonists , low doses of inhaled corticosteroids , or leukotriene modifiers . although these drugs have potent activity , they also have various and severe adverse effects [ 5 , 6 ] . therefore , agents of natural origin with very few side effects are required as substitutes for chemical therapeutics . natural products have long been used in folk medicine as alternative treatment for various diseases , including inflammatory processes of diverse origin . many medicinal plants provide relief of symptoms comparable to that obtained with allopathic medicines [ 5 , 7 , 8 ] . in the course of an ongoing search for bioactive plant - derived natural products , several groups , including our own , have successfully employed experimental methods to screen plant extracts and plant secondary metabolites for pharmacological activity [ 9 , 10 ] . ellagic acid , a polyphenol , is widely found in fruits ( e.g. , pomegranates , persimmon , raspberries , black raspberries , strawberries , peach , and plumes ) , nuts ( e.g. , walnuts and almonds ) , vegetables , and wine [ 11 , 12 ] . ellagic acid is widely known by its antioxidant effects ; however , it also demonstrates other biological effects such as anti - inflammatory proprieties [ 1113 ] . studies from our groups and others have demonstrated the anti - inflammatory activity of ellagic acid and extracts which contain it in the airways [ 7 , 10 , 14 ] . here , we determined the impact of ellagic acid on the resolution of allergic airways responses . ellagic acid ( 95% of purity hplc ; sigma - aldrich , mo , usa ) ; dexamethasone ( decadron teuto - brasileiro , go , bra ) ; ovalbumin ( sigma - aldrich , mo , usa ) ; aluminum hydroxide ( sigma chemical : missouri , usa ) ; edta ( sigma chemical : missouri , usa ) ; isoflurane ( anesthetic forane , abbott : abbott park , usa ) . all animal care and procedures used in this study were in compliance with the guidelines on the use of animals of the uftm ethics committee ( protocol number 162 ) , which follow the nih principles of laboratory animal care publication no . 8523 . the experiments were conducted using female balb / c mice ( 57 weeks old and weighing 2025 g ) that were kept in controlled temperature ( 22 2c ) and humidity ( 4555% ) under a 12 : 12 h light - dark cycle ( lights on 07 : 00 h ) . mice were sensitized on days 0 and 7 , by subcutaneous injection of 10 g of ovalbumin ( grade iii ) plus 1 mg of aluminum hydroxide at 0,2 ml of saline . after , sensitization protocol was followed by intranasal challenge ( days 14 , 15 , 16 , and 17 ) with 10 g of ovalbumin ( ova ) in saline and 50 l of this solution was delivered into the nostrils under isoflurane anesthesia with the aid of a micropipette . once ellagic acid demonstrates poor solubility in water the treatment in each animal was carried out with a suspension of ellagic acid in water at dose 10 mg / kg . the suspension was homogenized with the syringe used in the oral administration before each animal treatment . to study the preventive anti - inflammatory effects , mice received ellagic acid ( 10 mg / kg ) or vehicle ( water ) by gavage 30 minutes prior to intranasal ovalbumin challenge on days 14 , 15 , 16 , and 17 after sensitization . in a second cohort ( therapeutic treatment ) , the mice were treated with ellagic acid ( 10 mg / kg ) or vehicle ( water ) by oral gavage on resolution phase on days 18 , 19 , and 20 of the protocol . as a positive control , the mice were treated with dexamethasone as described in the previous schemes ( 1 mg / kg ; subcutaneous route ) . on the days 18 , 21 , or 25 following the inflammation , the mice were euthanised by sodium pentobarbital overdose ( 70 mg / kg , intraperitoneal ) , and the balf and lung were collected . balf was performed with 1 ml of phosphate - buffered saline ( pbs ) plus 0.6 mm methylenediaminetetraacetic acid ( edta ) and placed on ice . the total cell and differential leukocyte counts were made according to alves et al . . following centrifugation ( 400 g , 5 min , 4c ) , the supernatants of the balf were collected and stored at 80c for subsequent cytokine determination . the resolution was quantified by calculating the rate of resolution ( time interval required for the number of cell eosinophils fall half the maximum value ) using the balf [ 15 , 17 ] . in selected animals , after balf had been collected , lungs were collected , fixed with 10% phosphate - buffered formalin ( v / v ) , and embedded in paraffin . the histological slides were silanized and the tissues were cut into 5-m sections , which were then stained with haematoxylin and eosin ( h&e ) or periodic acid - schiff ( pas ) reagent ( sigma - aldrich ) for light microscopy examination . scores for peribronchiolar inflammatory cell infiltrates in sections from each lung were graded ; a score of 0 indicated the absence of inflammatory cell infiltrates ; a score of 1 , less than five layers of inflammatory cells in < 50% of the bronchiolar submucosa ; 2 , more than five layers of inflammatory cells in < 50% of the bronchiolar submucosa ; 3 , less than five layers of inflammatory cells in > 50% of the bronchiolar submucosa ; and 4 , more than five layers of inflammatory cells in > 50% of the bronchiolar submucosa . the number of pas staining ( pas+ ) cells in individual bronchioles was counted as described previously . for histological analyses the lungs were removed , postfixed for 24 h in the same solution , placed in ethanol ( 70% v / v ) , and then submerged in paraffin . slides were deparaffinized through a series of xylene baths and rehydrated through graded alcohol solutions . high temperature antigen retrieval was performed by immersion of the slides in a water bath at 9598c in 10 mmol / l trisodium citrate buffer ph 6.0 , for 40 min . after overnight incubation with primary antibody ( goat anti - p - selectin ( 1 : 1000 , santa cruz biotechnology , california , usa ) ) , at 4c , the slides were washed with pbs and incubated with appropriate biotin - coupled secondary antibody ( 1 : 250 ; dakocytomation , carpinteria , ca ) for 1 h at room temperature . the sections were then washed in pbs and incubated with streptavidin - peroxidase ( 1 : 250 ; invitrogen ) for 1 h. the visualization was completed by the use of 3,3-diaminobenzidine ( dab ) ( dakocytomation ) in chromogen solution and light counterstaining with harris 's haematoxylin solution ( merck , darmstadt , germany ) . images were obtained with a microscope ( eclipse 50i ; nikon , melville , ny ) and digital sight camera ( ds - fi1 ; nikon ) . control and experimental tissues were placed on the same slide and processed under the same conditions . the images were transferred to a computer , and the average pixel color intensity of p - selectin staining was calculated as described in a previous study . the lungs were removed and homogenized , and the assays were performed as described by rogerio et al . . alveolar macrophages from control or ovalbumin - sensitized and -challenged animals were obtained at therapeutic protocol at day 21 from balf as described previously by rogerio et al . and gilberti et al . . cells were placed on coverslips in 96-well plates ( 1.5 10 cells / well ) in media ( rpmi 1640 plus 10% fcs containing l - glutamine and antibiotics ) and incubated overnight at 37c . macrophages ( from balf ) were treated with ellagic acid ( 1 , 10 , or 100 m ) , dexamethasone ( 0.1 m ) , or vehicle ( dmso ) and incubated in the dark ( 20 min , 37c ) . ellagic acid was dissolved in dimethylsulfoxide ( dmso , final concentration is 0.1% ) and was diluted in 1x pbs to prepare required concentrations . in each treatment , the cells were treated with vehicle or with ellagic acid ( 1 , 10 , and 100 m ) or dexamethasone ( 1 m ) in the presence of alveolar macrophages . rabbit anti - ovalbumin igg - ab - coated polybead microsphere beads were prepared ( according to the manufacturer 's instructions ( polysciences ) ) and added to the cells at a ratio of 13 beads / cell . immediately ( time 0 ) or after 15 min , cells were washed with pbs and paraformaldehyde ( 4% ) was added . after 30 min , cells were washed again with pbs , and fitc - conjugated goat anti - rabbit ab ( 1 : 150 ) was added ( 35 min , room temperature , in the dark ) . supernatants were removed , and , after washing in pbs , the cells on the coverslips were mounted for fluorescent microscopy . beads were counted in both light and fluorescence images that were acquired for 50 cells in each incubation . because abs are not membrane permeable , only adherent noninternalized beads are fluorescent . this allows for distinction between internalized and cell - adherent beads . to quantify particle internalization , the number of surface - bound beads was counted from the fluorescence images and the total number of beads from the nonfluorescent images . the phagocytosis index was determined by subtracting the number of fluorescent beads from the total number of beads ( nonfluorescent images ) to derive the number of internalized beads . for each cell counted , the number of internalized beads was divided by the total number of beads to derive its phagocytosis index . the means from the different treatments in each individual experiment were compared by anova . when significant differences were identified , the individual comparisons were subsequently made with the tukey 's test . we first evaluated the preventive effect of ellagic acid ( 10 mg / kg ) on the resolution of allergic airways inflammation . so , ellagic acid was given to sensitized animals 30 min prior to each daily allergen intranasal challenge for a period of 4 d ( see section 2 ) ( figure 1(a ) ) . in all time points analyzed ( days 18 ( peak of inflammation ; data not shown ) , 21 ( figure 1(b ) ) , and 25 ( figure 1(c ) ) ) , the numbers of balf total cells , eosinophils , and/or macrophages , but not lymphocytes , from the vehicle - treated group were significantly increased compared to the control group . in agreement with previous studies we confirmed the anti - inflammatory effect of ellagic acid in the peak of inflammation ( day 18 ) ( data not shown ) . we extend this result and we demonstrate that ellagic acid accelerated the resolution of allergic airways inflammation ( at day 21 ) . ellagic acid or dexamethasone significantly reduced the total leukocytes number ~50% from 1.03 0.04 ( vehicle ) ( mean 10/ml sem ) to 0.53 0.14 ( ellagic acid ) or 0.37 0.06 ( dexamethasone ) ( figure 1(b ) ) . the balf eosinophil numbers of mice treated with ellagic acid were also reduced by approximately ~60% from 0.86 0.07 ( vehicle ) to 0.34 0.08 ( ellagic acid ) , while the dexamethasone treatment reduced by ~70% ( 0.23 0.05 ) ( figure 1(b ) ) . in addition , balf neutrophils were also reduced from 0.09 0.07 ( vehicle ) to 0.01 0.00 ( ellagic acid ) while no neutrophils were counted in dexamethasone treatment . moreover , the balf macrophages were significantly reduced in ellagic acid treatment from 0.22 0.11 ( vehicle ) to 0.05 0.01 ( ellagic acid ) ( figure 1(b ) ) . the results from day 25 ( figure 1(c ) ) were similar to day 21 . ellagic acid and dexamethasone reduced the number of total leukocytes of 0.08 1.76 ( vehicle ) to 1.12 0.23 ( ellagic acid ) ( mean 10/ml sem ) and 0.48 0.05 ( dexamethasone ) . in addition , the eosinophils numbers in balf were reduced also from 1.37 0.08 ( vehicle ) to 0.33 0.12 ( ellagic acid ) and 0.16 0.09 ( dexamethasone ) ( figure 1(c ) ) . no difference of lymphocytes , neutrophils , and macrophages numbers among the groups was observed . in the view of ellagic acid 's protective actions in the airways , we next evaluated the influence of ellagic acid on the resolution of established airway inflammation . after animals were ovalbumin - sensitized and intranasal - challenged , ellagic acid , dexamethasone , or vehicle was administered for 3 consecutive days ( protocol days 1820 ) , and no further allergen challenges were performed ( figure 2(a ) ) . balf leukocytes were enumerated on days 21 and 25 . in both days , the numbers of balf total cells , eosinophils , macrophages , and lymphocytes from the vehicle - treated group were significantly increased compared to the control group ( figures 2(b ) and 2(c ) ) . ellagic acid or dexamethasone decreased the balf eosinophil numbers at day 21 about 70% from 1.31 0.11 ( vehicle ) to 0.39 0.07 ( ellagic acid ) or 0.82 0.07 ( ~37% ) ( dexamethasone ) ( mean 10/ml sem ) . no significant difference was observed in the neutrophils , lymphocytes , and macrophages number of animals treated with ellagic acid or dexamethasone compared to vehicle - treated group ( figure 2(b ) ) . at day 25 , ellagic acid and dexamethasone treatment decreased eosinophil numbers in the balf ( figure 2(c ) ) . in this point , balf eosinophils were decreased by 80% from 1.10 0.12 ( vehicle ) to 0.23 0.05 ( ellagic acid ) or 0.38 0.05 ( ~65% ) ( dexamethasone ) . ellagic acid , different from dexamethasone , increased the number of lymphocytes from 0.10 0.02 ( vehicle ) to 0.32 0.03 ( ellagic acid ) ( figure 2(c ) ) . no significant difference was observed in the neutrophils and macrophages number of animals treated with ellagic acid or dexamethasone compared to vehicle - treated group . we next determined the influence of ellagic acid on the resolution of established eosinophilic airway inflammation . resolution interval ( ri ) is defined as the time required for the cell numbers to decrease to 50% of the maximum at peak inflammation ( the interval between peak of inflammation , at 18th day ) . in vehicle - exposed mice , the endogenous resolution interval for balf eosinophils was 5 days ( figure 2(d ) ) . ellagic acid , similar to dexamethasone , displayed an important decrease on resolution interval for balf eosinophils ( 2 days to 50% of the resolution interval ) compared to vehicle ( figure 2(d ) ) , indicative of more rapid resolution of allergic airway inflammation . we also determined the effect of ellagic acid on eosinophil peroxidase ( epo ) activity in the lung at day 21 . in the time point analyzed , the epo activity from the vehicle - treated group was significantly increased compared to the control group ( figure 2(e ) ) . ellagic acid and dexamethasone reduced epo activity from 2.42 0.16 ( vehicle ) to 1.54 0.11 ( ellagic acid ) or 1.72 0.15 ( dexamethasone ) ( figure 2(e ) ) . the proresolving actions of ellagic acid were also evident for lung inflammation and airways mucus metaplasia . the lungs of the vehicle - treated mice demonstrated increased edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the control group ( figures 3(a ) and 3(b ) ) . in addition , vehicle - treated mice demonstrated increase of mucus production ( figure 3(a ) ) . ellagic acid and dexamethasone - treatment reduced all of the aforementioned inflammatory parameters compared to the vehicle - treated mice , especially leukocyte infiltration . ellagic acid and dexamethasone treatment reduced the airways inflammation by ~30% or ~60% , respectively ( figures 3(a ) and 3(b ) ) . in addition , ellagic acid and dexamethasone also decreased the amount of mucus secretion by 80% and 90% , respectively , as indicated in arrows ( figure 3(a ) ) . in order to assess the effects of ellagic acid on the resolution phase ( at day 21 ) we evaluated the p - selectin , nf-b , and ap-1 expression in the lung . constitutive p - selectin expression was observed in control mice . in the vehicle - treated and ovalbumin - immunized and -challenged group , we observed an upregulation of p - selectin expression along the bronchial epithelium compared to control group . of note , ellagic acid and dexamethasone treatment significantly reduced the expression of p - selectin in the lung ( by 35% and 33% , resp . ) compared to the vehicle - treated animals ( figures 4(a ) and 4(b ) ) . no significant alterations were observed in p65 nf-b or ap-1 among the groups ( data not shown ) . we next evaluated the therapeutic effect of ellagic acid on the balf cytokine levels at 21 days . the concentrations of il-5 were increased in the vehicle - treated mice compared to control mice ( figure 4(c ) ) . ellagic acid and dexamethasone reduced the il-5 concentration 88% from 220 60 g / ml ( vehicle ) to 58 11 g / ml ( ellagic acid ) and 119 27 g / ml ( ~55% ) ( dexamethasone ) ( mean sem ) , respectively ( figure 4(c ) ) . no significant differences or detections were observed on il-10 , il-17 , and ifn- concentration among the groups or when compared to the vehicle - treated group control ( data not shown ) . we used macrophages from balf mice that were ovalbumin - sensitized and intranasal - challenged ( ex vivo ) of protocol at day 21 to evaluate the phagocytosis . ellagic acid ( 1 m ) , similar to dexamethasone , increased the macrophage phagocytosis within 15 min ( 37c ) for allergen - coated beads ( figure 5 ) . in the few decades , the number of the new drugs discovered that originated from nature has increased considerably [ 9 , 10 ] . the current therapy for the treatment of airway inflammation has not changed to the same degree . inhaled corticosteroids and 2-adrenoceptor agonists remain as the mainstay of asthma treatment . thus , the identification of new molecules that are able to prevent or treat inflammatory airway diseases is highly desirable . like other polyphenols , ellagic acid demonstrates a wide range of biological activities , which suggest that they could have beneficial effects on human health . several studies have demonstrated that ellagic acid possesses anti - inflammatory properties in the airways [ 7 , 10 , 23 ] . in the present work we extend previous results and highlight for the first time the effect of ellagic acid on the resolution of airway inflammation in an airways allergic inflammation experimental model . ellagic acid dampens the inflammation , mainly the eosinophilic inflammation , as in the balf and in the lung and reduced the mucus production . these effects could be associated to reduction of il-5 concentration in the balf and p - selectin expression in the lung . phytochemical and pharmacological studies have identified many potential anti - inflammatory substances , especially those derived from plants used in folk medicine . lafoensia pacari ( lythraceae ) has been used in traditional medicine to treat gastric ulcers and inflammation in the state of mato grosso ( brazil ) . in a bioassay - guided fractionation of the lafoensia pacari identified the ellagic acid as the compound responsible for the reduction of eosinophils recruitment into the peritoneal cavity of mice induced to injury by histoplasma capsulatum - derived -glucan . the reduction in the recruitment of eosinophils to the balf by ellagic acid has also been observed in an ovalbumin - induced experimental allergic airway inflammation . in addition , lafoensia pacari extract and ellagic acid demonstrated also antiedematous activity in a mouse paw edema model in mice . ellagitannins present in the pomegranate ( punica granatum l. ) fruit , which has been used for centuries for medical purposes , are hydrolysed to ellagic acid in the gut and are then metabolised by the colonic microflora to form urolithins ( a and b ) [ 24 , 25 ] . interestingly , these ellagic acid metabolites , urolithins , have also demonstrated anti - inflammatory effects , which could potentially promote synergic effect with ellagic acid [ 26 , 27 ] . studies with pomegranate extract have demonstrated its anti - inflammatory effects in murine models of collagen - induced arthritis and murine model experimental colitis [ 29 , 30 ] . in an experimental model of acute lung injury ( ali ) ( lps initiated ) , the pomegranate extract also reduced the myeloperoxidase ( a heme enzyme present in the primary granules of polymorphonuclear leukocytes neutrophils ) in the lungs of mice , suggesting that ellagic acid might be involved in this process . in fact , our group demonstrated the anti - inflammatory effects of ellagic acid in hcl acid - initiated ali . in asthma , a complex network of cytokines and chemokines il-5 is essential for eosinophil migration from the bone marrow to the blood and specifically supports terminal differentiation and proliferation of eosinophil precursors as well as activating mature eosinophils . in the resolution phase , ellagic acid accelerated the resolution of eosinophilic inflammation by reducing the eosinophils number in the balf and in the lung as well as eosinoperoxidase ( epo ) activity in the lung . there is a great amount of evidence indicating that adhesion molecules are critically involved in leukocyte control ; we next evaluated the expression of p - selectin in the lungs , which is also considered to be an important target for modulating eosinophilic influx to the inflammatory tissue [ 34 , 35 ] . our findings revealed that the ellagic acid consistently decreased the expression of p - selectin in the bronchial epithelium of ovalbumin - immunized and -challenged mice . therefore , the reported inhibition of p - selectin expression is expected to contribute to anti - inflammatory actions in synergism of inhibition of il-5 . in the setting of allergen - driven inflammation , inhaled allergen needs to be cleared to facilitate resolution of inflammation [ 17 , 36 ] . ellagic acid increased alveolar macrophage phagocytosis of allergen - coated beads in vitro without a concentration - dependent manner . these results demonstrate protective anti - inflammatory and proresolving actions for ellagic acid in airway inflammation . ellagic acid decreased eosinophil recruitment and airway mucus metaplasia ; moreover it promoted the resolution of allergic airway enhancing allergen clearance . together , these results point ellagic acid as a therapeutic candidate to treat airways diseases such asthma .

asthma is a disease of airway inflammation characterized by airway hyperresponsiveness , eosinophilic inflammation , and hypersecretion of mucus . ellagic acid , a compound derived from medicinal plants and fruits , has shown anti - inflammatory activity in several experimental disease models . we used the classical experimental model , in balb / c mice , of sensibilization with ovalbumin to determine the effect of ellagic acid ( 10 mg / kg ; oral route ) in the resolution of allergic airways response . dexamethasone ( 1 mg / kg ; subcutaneous route ) was used as a positive control . the control group consisted of nonimmunized mice that received challenge with ovalbumin . ellagic acid and dexamethasone or vehicle ( water ) were administered before or after intranasal allergen challenge . ellagic acid accelerated the resolution of airways inflammation by decreasing total leukocytes and eosinophils numbers in the bronchoalveolar lavage fluid ( balf ) , the mucus production and lung inflammation in part by reducing il-5 concentration , eosinophil peroxidase ( epo ) activity , and p - selectin expression , but not activator protein 1 ( ap-1 ) and nuclear factor kappa b ( nf-b ) pathways . in addition , ellagic acid enhanced alveolar macrophage phagocytosis of igg - ova - coated beads ex vivo , a new proresolving mechanism for the clearance of allergen from the airways . together , these findings identify ellagic acid as a potential therapeutic agent for accelerating the resolution of allergic airways inflammation .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

asthma is a chronic inflammatory disease that is highly prevalent worldwide and it is characterized by the recruitment of leukocytes , mainly eosinophils , airway hyperreactivity , ige production , and mucus hypersecretion . the physiopathology of allergic asthma is coordinated mainly by th2-type immune responses which are characterized by release of cytokines such as interleukin- ( il- ) 4 , il-5 , and il-13 and chemokines such as rantes and ccl11 [ 13 ] . ellagic acid , a polyphenol , is widely found in fruits ( e.g. , pomegranates , persimmon , raspberries , black raspberries , strawberries , peach , and plumes ) , nuts ( e.g. , walnuts and almonds ) , vegetables , and wine [ 11 , 12 ] . ellagic acid is widely known by its antioxidant effects ; however , it also demonstrates other biological effects such as anti - inflammatory proprieties [ 1113 ] . studies from our groups and others have demonstrated the anti - inflammatory activity of ellagic acid and extracts which contain it in the airways [ 7 , 10 , 14 ] . here , we determined the impact of ellagic acid on the resolution of allergic airways responses . ellagic acid ( 95% of purity hplc ; sigma - aldrich , mo , usa ) ; dexamethasone ( decadron teuto - brasileiro , go , bra ) ; ovalbumin ( sigma - aldrich , mo , usa ) ; aluminum hydroxide ( sigma chemical : missouri , usa ) ; edta ( sigma chemical : missouri , usa ) ; isoflurane ( anesthetic forane , abbott : abbott park , usa ) . the experiments were conducted using female balb / c mice ( 57 weeks old and weighing 2025 g ) that were kept in controlled temperature ( 22 2c ) and humidity ( 4555% ) under a 12 : 12 h light - dark cycle ( lights on 07 : 00 h ) . after , sensitization protocol was followed by intranasal challenge ( days 14 , 15 , 16 , and 17 ) with 10 g of ovalbumin ( ova ) in saline and 50 l of this solution was delivered into the nostrils under isoflurane anesthesia with the aid of a micropipette . once ellagic acid demonstrates poor solubility in water the treatment in each animal was carried out with a suspension of ellagic acid in water at dose 10 mg / kg . to study the preventive anti - inflammatory effects , mice received ellagic acid ( 10 mg / kg ) or vehicle ( water ) by gavage 30 minutes prior to intranasal ovalbumin challenge on days 14 , 15 , 16 , and 17 after sensitization . in a second cohort ( therapeutic treatment ) , the mice were treated with ellagic acid ( 10 mg / kg ) or vehicle ( water ) by oral gavage on resolution phase on days 18 , 19 , and 20 of the protocol . as a positive control , the mice were treated with dexamethasone as described in the previous schemes ( 1 mg / kg ; subcutaneous route ) . on the days 18 , 21 , or 25 following the inflammation , the mice were euthanised by sodium pentobarbital overdose ( 70 mg / kg , intraperitoneal ) , and the balf and lung were collected . balf was performed with 1 ml of phosphate - buffered saline ( pbs ) plus 0.6 mm methylenediaminetetraacetic acid ( edta ) and placed on ice . following centrifugation ( 400 g , 5 min , 4c ) , the supernatants of the balf were collected and stored at 80c for subsequent cytokine determination . the resolution was quantified by calculating the rate of resolution ( time interval required for the number of cell eosinophils fall half the maximum value ) using the balf [ 15 , 17 ] . in selected animals , after balf had been collected , lungs were collected , fixed with 10% phosphate - buffered formalin ( v / v ) , and embedded in paraffin . for histological analyses the lungs were removed , postfixed for 24 h in the same solution , placed in ethanol ( 70% v / v ) , and then submerged in paraffin . after overnight incubation with primary antibody ( goat anti - p - selectin ( 1 : 1000 , santa cruz biotechnology , california , usa ) ) , at 4c , the slides were washed with pbs and incubated with appropriate biotin - coupled secondary antibody ( 1 : 250 ; dakocytomation , carpinteria , ca ) for 1 h at room temperature . the sections were then washed in pbs and incubated with streptavidin - peroxidase ( 1 : 250 ; invitrogen ) for 1 h. the visualization was completed by the use of 3,3-diaminobenzidine ( dab ) ( dakocytomation ) in chromogen solution and light counterstaining with harris 's haematoxylin solution ( merck , darmstadt , germany ) . the images were transferred to a computer , and the average pixel color intensity of p - selectin staining was calculated as described in a previous study . cells were placed on coverslips in 96-well plates ( 1.5 10 cells / well ) in media ( rpmi 1640 plus 10% fcs containing l - glutamine and antibiotics ) and incubated overnight at 37c . macrophages ( from balf ) were treated with ellagic acid ( 1 , 10 , or 100 m ) , dexamethasone ( 0.1 m ) , or vehicle ( dmso ) and incubated in the dark ( 20 min , 37c ) . ellagic acid was dissolved in dimethylsulfoxide ( dmso , final concentration is 0.1% ) and was diluted in 1x pbs to prepare required concentrations . in each treatment , the cells were treated with vehicle or with ellagic acid ( 1 , 10 , and 100 m ) or dexamethasone ( 1 m ) in the presence of alveolar macrophages . rabbit anti - ovalbumin igg - ab - coated polybead microsphere beads were prepared ( according to the manufacturer 's instructions ( polysciences ) ) and added to the cells at a ratio of 13 beads / cell . after 30 min , cells were washed again with pbs , and fitc - conjugated goat anti - rabbit ab ( 1 : 150 ) was added ( 35 min , room temperature , in the dark ) . to quantify particle internalization , the number of surface - bound beads was counted from the fluorescence images and the total number of beads from the nonfluorescent images . we first evaluated the preventive effect of ellagic acid ( 10 mg / kg ) on the resolution of allergic airways inflammation . so , ellagic acid was given to sensitized animals 30 min prior to each daily allergen intranasal challenge for a period of 4 d ( see section 2 ) ( figure 1(a ) ) . in all time points analyzed ( days 18 ( peak of inflammation ; data not shown ) , 21 ( figure 1(b ) ) , and 25 ( figure 1(c ) ) ) , the numbers of balf total cells , eosinophils , and/or macrophages , but not lymphocytes , from the vehicle - treated group were significantly increased compared to the control group . in agreement with previous studies we confirmed the anti - inflammatory effect of ellagic acid in the peak of inflammation ( day 18 ) ( data not shown ) . we extend this result and we demonstrate that ellagic acid accelerated the resolution of allergic airways inflammation ( at day 21 ) . the balf eosinophil numbers of mice treated with ellagic acid were also reduced by approximately ~60% from 0.86 0.07 ( vehicle ) to 0.34 0.08 ( ellagic acid ) , while the dexamethasone treatment reduced by ~70% ( 0.23 0.05 ) ( figure 1(b ) ) . in addition , balf neutrophils were also reduced from 0.09 0.07 ( vehicle ) to 0.01 0.00 ( ellagic acid ) while no neutrophils were counted in dexamethasone treatment . moreover , the balf macrophages were significantly reduced in ellagic acid treatment from 0.22 0.11 ( vehicle ) to 0.05 0.01 ( ellagic acid ) ( figure 1(b ) ) . ellagic acid and dexamethasone reduced the number of total leukocytes of 0.08 1.76 ( vehicle ) to 1.12 0.23 ( ellagic acid ) ( mean 10/ml sem ) and 0.48 0.05 ( dexamethasone ) . in addition , the eosinophils numbers in balf were reduced also from 1.37 0.08 ( vehicle ) to 0.33 0.12 ( ellagic acid ) and 0.16 0.09 ( dexamethasone ) ( figure 1(c ) ) . in the view of ellagic acid 's protective actions in the airways , we next evaluated the influence of ellagic acid on the resolution of established airway inflammation . after animals were ovalbumin - sensitized and intranasal - challenged , ellagic acid , dexamethasone , or vehicle was administered for 3 consecutive days ( protocol days 1820 ) , and no further allergen challenges were performed ( figure 2(a ) ) . in both days , the numbers of balf total cells , eosinophils , macrophages , and lymphocytes from the vehicle - treated group were significantly increased compared to the control group ( figures 2(b ) and 2(c ) ) . no significant difference was observed in the neutrophils , lymphocytes , and macrophages number of animals treated with ellagic acid or dexamethasone compared to vehicle - treated group ( figure 2(b ) ) . at day 25 , ellagic acid and dexamethasone treatment decreased eosinophil numbers in the balf ( figure 2(c ) ) . ellagic acid , different from dexamethasone , increased the number of lymphocytes from 0.10 0.02 ( vehicle ) to 0.32 0.03 ( ellagic acid ) ( figure 2(c ) ) . no significant difference was observed in the neutrophils and macrophages number of animals treated with ellagic acid or dexamethasone compared to vehicle - treated group . we next determined the influence of ellagic acid on the resolution of established eosinophilic airway inflammation . resolution interval ( ri ) is defined as the time required for the cell numbers to decrease to 50% of the maximum at peak inflammation ( the interval between peak of inflammation , at 18th day ) . in vehicle - exposed mice , the endogenous resolution interval for balf eosinophils was 5 days ( figure 2(d ) ) . ellagic acid , similar to dexamethasone , displayed an important decrease on resolution interval for balf eosinophils ( 2 days to 50% of the resolution interval ) compared to vehicle ( figure 2(d ) ) , indicative of more rapid resolution of allergic airway inflammation . we also determined the effect of ellagic acid on eosinophil peroxidase ( epo ) activity in the lung at day 21 . in the time point analyzed , the epo activity from the vehicle - treated group was significantly increased compared to the control group ( figure 2(e ) ) . ellagic acid and dexamethasone reduced epo activity from 2.42 0.16 ( vehicle ) to 1.54 0.11 ( ellagic acid ) or 1.72 0.15 ( dexamethasone ) ( figure 2(e ) ) . the proresolving actions of ellagic acid were also evident for lung inflammation and airways mucus metaplasia . the lungs of the vehicle - treated mice demonstrated increased edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the control group ( figures 3(a ) and 3(b ) ) . in addition , vehicle - treated mice demonstrated increase of mucus production ( figure 3(a ) ) . ellagic acid and dexamethasone - treatment reduced all of the aforementioned inflammatory parameters compared to the vehicle - treated mice , especially leukocyte infiltration . ellagic acid and dexamethasone treatment reduced the airways inflammation by ~30% or ~60% , respectively ( figures 3(a ) and 3(b ) ) . in addition , ellagic acid and dexamethasone also decreased the amount of mucus secretion by 80% and 90% , respectively , as indicated in arrows ( figure 3(a ) ) . in order to assess the effects of ellagic acid on the resolution phase ( at day 21 ) we evaluated the p - selectin , nf-b , and ap-1 expression in the lung . constitutive p - selectin expression was observed in control mice . in the vehicle - treated and ovalbumin - immunized and -challenged group , we observed an upregulation of p - selectin expression along the bronchial epithelium compared to control group . of note , ellagic acid and dexamethasone treatment significantly reduced the expression of p - selectin in the lung ( by 35% and 33% , resp . ) we next evaluated the therapeutic effect of ellagic acid on the balf cytokine levels at 21 days . ellagic acid and dexamethasone reduced the il-5 concentration 88% from 220 60 g / ml ( vehicle ) to 58 11 g / ml ( ellagic acid ) and 119 27 g / ml ( ~55% ) ( dexamethasone ) ( mean sem ) , respectively ( figure 4(c ) ) . we used macrophages from balf mice that were ovalbumin - sensitized and intranasal - challenged ( ex vivo ) of protocol at day 21 to evaluate the phagocytosis . ellagic acid ( 1 m ) , similar to dexamethasone , increased the macrophage phagocytosis within 15 min ( 37c ) for allergen - coated beads ( figure 5 ) . the current therapy for the treatment of airway inflammation has not changed to the same degree . like other polyphenols , ellagic acid demonstrates a wide range of biological activities , which suggest that they could have beneficial effects on human health . several studies have demonstrated that ellagic acid possesses anti - inflammatory properties in the airways [ 7 , 10 , 23 ] . in the present work we extend previous results and highlight for the first time the effect of ellagic acid on the resolution of airway inflammation in an airways allergic inflammation experimental model . ellagic acid dampens the inflammation , mainly the eosinophilic inflammation , as in the balf and in the lung and reduced the mucus production . these effects could be associated to reduction of il-5 concentration in the balf and p - selectin expression in the lung . phytochemical and pharmacological studies have identified many potential anti - inflammatory substances , especially those derived from plants used in folk medicine . lafoensia pacari ( lythraceae ) has been used in traditional medicine to treat gastric ulcers and inflammation in the state of mato grosso ( brazil ) . in a bioassay - guided fractionation of the lafoensia pacari identified the ellagic acid as the compound responsible for the reduction of eosinophils recruitment into the peritoneal cavity of mice induced to injury by histoplasma capsulatum - derived -glucan . the reduction in the recruitment of eosinophils to the balf by ellagic acid has also been observed in an ovalbumin - induced experimental allergic airway inflammation . in addition , lafoensia pacari extract and ellagic acid demonstrated also antiedematous activity in a mouse paw edema model in mice . ellagitannins present in the pomegranate ( punica granatum l. ) fruit , which has been used for centuries for medical purposes , are hydrolysed to ellagic acid in the gut and are then metabolised by the colonic microflora to form urolithins ( a and b ) [ 24 , 25 ] . interestingly , these ellagic acid metabolites , urolithins , have also demonstrated anti - inflammatory effects , which could potentially promote synergic effect with ellagic acid [ 26 , 27 ] . studies with pomegranate extract have demonstrated its anti - inflammatory effects in murine models of collagen - induced arthritis and murine model experimental colitis [ 29 , 30 ] . in an experimental model of acute lung injury ( ali ) ( lps initiated ) , the pomegranate extract also reduced the myeloperoxidase ( a heme enzyme present in the primary granules of polymorphonuclear leukocytes neutrophils ) in the lungs of mice , suggesting that ellagic acid might be involved in this process . in fact , our group demonstrated the anti - inflammatory effects of ellagic acid in hcl acid - initiated ali . in asthma , a complex network of cytokines and chemokines il-5 is essential for eosinophil migration from the bone marrow to the blood and specifically supports terminal differentiation and proliferation of eosinophil precursors as well as activating mature eosinophils . in the resolution phase , ellagic acid accelerated the resolution of eosinophilic inflammation by reducing the eosinophils number in the balf and in the lung as well as eosinoperoxidase ( epo ) activity in the lung . there is a great amount of evidence indicating that adhesion molecules are critically involved in leukocyte control ; we next evaluated the expression of p - selectin in the lungs , which is also considered to be an important target for modulating eosinophilic influx to the inflammatory tissue [ 34 , 35 ] . our findings revealed that the ellagic acid consistently decreased the expression of p - selectin in the bronchial epithelium of ovalbumin - immunized and -challenged mice . therefore , the reported inhibition of p - selectin expression is expected to contribute to anti - inflammatory actions in synergism of inhibition of il-5 . in the setting of allergen - driven inflammation , inhaled allergen needs to be cleared to facilitate resolution of inflammation [ 17 , 36 ] . ellagic acid increased alveolar macrophage phagocytosis of allergen - coated beads in vitro without a concentration - dependent manner . these results demonstrate protective anti - inflammatory and proresolving actions for ellagic acid in airway inflammation . ellagic acid decreased eosinophil recruitment and airway mucus metaplasia ; moreover it promoted the resolution of allergic airway enhancing allergen clearance . together , these results point ellagic acid as a therapeutic candidate to treat airways diseases such asthma .

since the herbicide glyphosate ( n-(phosphonomethyl)glycine ) was commercialized in 1974 , it has become the most widely used herbicide in the world , due largely to the wide scale adoption of transgenic , glyphosate - resistant ( gr ) crops after their introduction in 1996 ( figure 1 ) . in gr crops , this relatively high use rate herbicide ( commonly 0.5 to 2.0 kg / ha / application ) is often used multiple times in a growing season . use of other herbicides declined steadily , while glyphosate use increased in the three major gr crops ( figure 2 ) . the increasing incidence of evolved , gr weeds , as well as weed shifts to naturally glyphosate - tolerant weed species , has resulted in increased use rates and numbers of applications of glyphosate , as well as other herbicides , per growing season in gr crops . since its introduction , glyphosate has been considered a toxicologically and environmentally safe pesticide , due to its low mammalian toxicity , relatively short environmental half - life , and extremely low activity in soil due to its binding to soil minerals ( reviewed by duke et al . ) . furthermore , only green plants , some fungi , and a limited number of microorganisms have the target site , 5-enolpyruvylshikimic acid-3-phosphate synthase ( epsps ) , of the herbicide . epsps is an enzyme required for synthesis of the essential aromatic amino acids phenylalanine , tyrosine , and tryptophan . adoption of the three most widely grown , herbicide - resistant ( hr ) crops in the united states . . data for each crop category include varieties with both hr as a single and stacked trait with insect resistance . data for 20002012 are available in the usda , economic research service data product , adoption of genetically engineered crops in the u.s . note that adoption data for 19961999 include hr corn and soybeans obtained using traditional breeding methods ( not transgenic ) . the more recent data ( 20002011 ) closed circles = all herbicides minus glyphosate , open circles = glyphosate only . department of agriculture , national agricultural statistics service data and statistics site http://www.nass.usda.gov/data_and_statistics/quick_stats/ ( accessed september 12 , 2012 ) . glyphosate is a nonselective herbicide , that is , it can kill all plant species , although there is variation between species with regard to levels of natural tolerance . glyphosate has little or no herbicidal activity in soil and , thus , is used only with foliar spray applications . due to crop sensitivity , its use was limited in crop production prior to the introduction of gr crops , after which its use greatly expanded with the widespread adoption of these crops worldwide . during its several decades of use over vast areas , no significant adverse secondary effects of the herbicide have been established , other than the intense selection pressure that has resulted in the evolution of gr weeds . in fact , its use in gr crops has been associated with several environmental benefits . the topic of evolution of gr weeds has been dealt with in detail in many research papers and reviews . several papers have been published recently that conclude that glyphosate adversely affects mineral nutrition in gr crops , leading to several adverse effects , including increased plant disease . others have indicated that gr crops are more susceptible to plant diseases due to other mechanisms . this review addresses these concerns in the context of the available literature on glyphosate ( ca . 8000 peer - reviewed papers according to scifinder ) . in order to understand possible effects of glyphosate on mineral nutrition of plants , it is necessary to understand the processes that affect glyphosate in soil . it is also necessary to understand how glyphosate interacts with minerals in soil and with soil microorganisms . once glyphosate interacts with soil , whether applied directly to the soil surface , exuded from a plant root , or released from decomposing plant tissue , it is subject to various processes that control its environmental behavior and fate , including retention ( sorption desorption ) , transport , and degradation . of these processes , sorption is arguably the most important as it controls the availability for degradation , plant uptake , and offsite transport . sorption of glyphosate to soil has been extensively reviewed . because glyphosate is a small polyprotic molecule ( pka1 = 2.27 , pka2 = 5.58 , pka3 = 10.25 ) with three polar functional groups and can be sorbed on minerals and organic matter , its sorption on soil as a whole is generally much greater compared to other pesticides , which are larger molecules with fewer functional groups and are primarily sorbed onto organic matter . glyphosate is primarily sorbed on variable - charge surfaces such as iron and aluminum oxides , aluminum silicates ( allophone and imogolite ) , and goethite ( -feooh ) , and to a lesser extent on the fe - oxide coatings of permanent charge minerals ( illite , smectite , and vermiculite ) and organic matter . the primary mechanisms responsible for sorption are ligand exchange and complex formation with mineral oxide surfaces . the magnitude of sorption increases with increased surface area of the minerals and decreased ph . the sorption onto the sorbent surface is fast initially for most of the glyphosate added to soil , which is then followed by slower sorption . further details of these processes can be found in the above cited reviews and the references cited therein . the magnitude of sorption has traditionally been characterized as the ratio of glyphosate bound to soil to that in solution for a single concentration ( kd ) or at multiple concentrations ( kf and 1/n values from the freundlich sorption isotherm ) . sorption coefficients are often expressed on a soil organic carbon basis ( koc , kfoc ) to normalize values between different soils . , regardless of soil properties in a cultivated prairie , glyphosate mean kd was 108 to133 l kg ( koc = 10 90014 900 l kg ) , depending on landscape position , and these values were 100 greater than those for the commonly used herbicide 2,4-d . in a study of 20 different soils , kd ranged from 41 to 303 l kg with a median value of 97 l kg . in column leaching experiments , coarse textured soils retained most all ( 8595% ) of the glyphosate applied despite the fact that higher than agronomic rates , 7.414.8 mg kg , of glyphosate were used . depending on soil type , glyphosate is weakly desorbed , that is , 524% of initially sorbed glyphosate . the strong adsorption of glyphosate to most soils , and its low desorbability , leaves little glyphosate in soil solution available for microbial degradation , interaction with trace metal cations , plant uptake , or offsite transport . sorbed glyphosate has been postulated to be released into soil solution upon addition of phosphate ( po4 ) , which can compete with glyphosate for sorption sites on soil . however , it appears there is only limited competition for sorption sites between glyphosate and po4 , even when much higher than agronomic rates of glyphosate are applied . for soils , competitive sorption studies between glyphosate and po4 showed that displacement of glyphosate by po4 was related to the amount of clays , cec , and ph , but glyphosate was not easily displaced by po4 from the clays . even when sorption competition has been shown to occur , glyphosate still remains strongly sorbed . for example , increasing extractable p by a factor of 10 in soil , only decreased the sorption coefficient kf from 215 to 106 l kg in loamy sand and 154 to 84 l kg in coarse sand soils . as a result , solution concentration of glyphosate does not appreciably increase upon the addition of po4 at environmentally relevant p concentrations . also , the competition between glyphosate and po4 ( if it occurs ) appears to be temporary ; the same amounts of glyphosate and po4 were sorbed after 7 days whether the compounds are present alone or together . therefore , it seems likely that glyphosate and po4 are specifically sorbed on to common as well as specific sites on various soil components . glyphosate can form chelates or complexes with micronutrient metal ions in solution . at physiologically relevant ph levels , and ph levels of most soils , cu and zn ions in solution can be relatively strongly complexed with glyphosate , whereas fe , ca , mg , and mn are complexed to lesser degrees . because of the ability of glyphosate to complex metal ions , glyphosate has been postulated to affect plant uptake of trace nutrients such as mn or zn . for plants grown in hydroponic solutions , mixed results for glyphosate effects on plants have been shown . in contrast , andrade and rosolem reported that glyphosate did not affect mn absorption and transport in gr soybean plants in the field . this topic is discussed in more detail in the section below on the effects of glyphosate on mineral nutrition of plants . it is difficult to extrapolate hydroponic studies to field situations where there are numerous cations at varying concentrations that can form complexes with glyphosate , and where soluble metal - glyphosate complexes are subject to sorption processes on soil , as are glyphosate and metal ions . for example , the presence of zn increased glyphosate sorption on two soils as a result of decreased solution ph resulting from zn exchanging with h on the soil surface . in bioassay experiments using tomato plants and white spruce seedlings , soils containing saturated solutions of glyphosate - metal complexes had little or no effect on the plants . in a recent study of micronutrient accumulation in soybean grown using standard agronomic practices in indiana , results showed that while there were differences in accumulation of micronutrients between cultivars , there was no consistent effect due to glyphosate treatment . for example , in five woodland sites , the mean soil solution concentrations of total mn and zn were 69 and 1.8 mol l , respectively , while in grassland sites , total mn concentrations varied by a factor of 6 in a clay soil and a factor of 2 in a sandy soil . in a soil toposequence under natural vegetation , total mn concentration in soil solution varied by a factor of 900 , depending on topographic soil position . in addition , the metal cations in soil solution are not necessarily free ions ; they can form complexes with dissolved soil organic matter and other ligands . for example , in a study of 15 agricultural soils , total soil solution concentrations of cu ranged from 0.023 to 1.03 mol l , with free cu comprising 773% of total dissolved cu . in the same soils , total zn solution concentration ranged from 0.4 to 43 mol l , with free zn comprising 4799% of total dissolved zn . glyphosate can only compete with other soil ligands and sorbent surfaces for free metal ion activity , with most glyphosate being adsorbed by the soil rather than remaining in the soil solution where it can complex with metal ions . in spite of the wide range of micronutrient cation concentrations in soil solutions , their concentrations are much greater than would be found for glyphosate in soil solutions . using a glyphosate application rate of 1 kg ha ( soil concentration = 0.75 g g , assuming incorporation to a depth of 10 cm and a soil bulk density of 1.33 ) , and an average soil kd = 100 , the amount of glyphosate in soil solution would be 0.044 mol l , which is much smaller than typical mn , zn , cu , and fe concentrations found in soil solutions from agricultural soils . under agricultural production , concentrations of , mn , zn , cu , and fe in soil solutions of holtville ( typic torrifluvent ) and altamont ( typic chromoxerert ) soils would be on average 480 , 220 , 80 , and 310 greater than the glyphosate in solution , respectively . therefore , free cation concentrations , such as mn , would not be reduced appreciably by glyphosate addition to soil , even at the highest recommended application rates and assuming all the glyphosate in solution formed a chelate with mn . furthermore , glyphosate degrades rapidly ( see degradation section below ) , whereas although micronutrient concentrations in soil can fluctuate temporally during the year , micronutrients do not degrade . the primary degradation pathway is the cleavage of glyphosate by glyphosate oxidoreductase to glyoxylate and ampa ( aminomethylphosphonic acid ) ( figure 3 ) , with the latter subsequently degraded to methylamine and inorganic phosphate by c p lyase enzymes . both glyoxylate and methylamine can support the growth of microorganisms . alternatively , the transformation of glyphosate to ampa and glyoxylate can also be performed by glycine oxidase . a second degradation pathway is the cleavage of inorganic phosphate from glyphosate by c sarcosine is further degraded into formaldehyde and glycine , which are utilized by a wide variety of soil microorganisms . soil microorganisms utilizing the sarcosine pathway have been isolated and characterized , including members of the soybean root symbionts , the rhizobiaceae . soil fungi also degrade glyphosate , and ampa was reported as a metabolite . since some microorganisms are sensitive to glyphosate ( see this review ) , the degradation of glyphosate in situ represents the activity of the glyphosate - degrading microbial community modulated by relative resistance or sensitivity to the herbicide . several studies have utilized c - labeled glyphosate to examine the fate of glyphosate in soil . these methods are useful because they provide an integrated assessment of glyphosate degradation by measurement of co2 production ( mineralization ) and the incorporation of glyphosate and glyphosate degradation products into soil organic matter and biota ( bound c residue ) . the co2 produced from microbial degradation after glyphosate addition to soil is variable , ranging from 10% in 332 d , to < 1040% in 96 d , to 5070% in 35 d , to > 70% in 140 d , and depends on soil type and c - label location ( phosphomethyl - c versus aminomethyl - c ) . the production of co2 begins after addition of glyphosate to soil without a lag period , showing that microorganisms with the capacity to degrade glyphosate are present in most all soils . the studies conducted with c label in the phosphonomethyl carbon show degradation of the ampa metabolite . the variability in the amount of co2 produced from glyphosate degradation in soil is likely due to the variability in the population of glyphosate - degrading microorganisms present in soil ( the glyphosate - degrading microbial community in soil has not been fully characterized ) and their biological activity and to competing sorption and binding processes . in addition to the direct application of glyphosate to soil , any glyphosate remaining in gr crop residues will be released to the soil as those crop residues degrade . at 35 days after treatment , glyphosate residues within corn leaves mineralized more slowly ( 61% ) than glyphosate applied directly to soil ( 77% ) . the use of gr crops allows for multiple applications ( commonly two or three ) of glyphosate within a field each growing season . c - labeled glyphosate has also been used to determine whether repeated applications of glyphosate can affect glyphosate degradation in soil . repeated applications reduced the rate of glyphosate mineralization by 28% from the initial application to the fifth application in a 10-wk period ( figure 4 ) . however , there was no difference in the rate of c - glyphosate mineralization between one , two , three , or four glyphosate applications . while the rate of mineralization was decreased after the fifth application relative to the first , it was not reduced relative to the second , third or fourth application . in a similar experiment , andra et al . found that the initial , immediately mineralizable glyphosate decreased after sequential applications as compared to the initial application . however , after the initial mineralization , the rate of mineralization was the same . for instance , the differences in the total amount of co2 detected 8 wks after treatment and the immediately mineralizable glyphosate were 16 and 19% after 1 and 4 sequential applications , respectively . weaver et al . reported greater mineralization ( % of applied ) from glyphosate added at 1 the field rate ( 47 mg glyphosate / kg soil , assuming 0.84 kg / ha application distributed in the surface 2 mm of soil ) than at the 3 rate of application . however , on a mass basis more glyphosate was degraded in the soil receiving the 3 application . these studies show that repeated glyphosate applications are unlikely to severely reduce the ability of the soil microbial community to metabolize glyphosate . broad spectrum measures of microbial activity ( respiration and enzyme activities ) and community structure show inconsistent or no response to glyphosate use ( see effects on rhizosphere populations and community structure section ) . distribution of c after the ( a ) first or ( b ) fifth glyphosate application to silt - loam soil the sequential glyphosate applications were made at 2 week intervals . the extractable fraction includes glyphosate and its transformation products extracted with 0.1 m naoh .. in addition to c - glyphosate being mineralized , a portion of the c - labeled glyphosate or its metabolites is converted to microbial biomass and some remains unextractable from soil . the amount of bound c - residue formed depends upon the molecular location of the c - label , the soil interaction , and the extraction methods used . mamy et al . compared the bound residue formed from several pesticides and found that glyphosate - bound residues generally accounted for less than 20% of the initial c added , equivalent to trifluralin , but less than those formed after application of the c - labeled herbicides metazachlor , metamitron , and sulcotrione . weaver et al . applied glyphosate equivalent to 1 or 3 recommended field rates , and less than 10% of the c was present in bound residues at 42 days after application . after four or five sequential applications at 14-day intervals , less than 30% of the c from applied glyphosate was present as bound residue in the soil , and there was no difference in bound residue formation after the first or the fifth application . however , greater accumulation of bound residues would occur from multiple applications than a single application . simonsen et al . assessed the bioavailability of bound residues formed after a single glyphosate application to plants by incubating c - glyphosate in soil for 6.5 months , followed by planting canola or barley . glyphosate was not detectable in soil at planting , and after 41 days of plant growth only 0.006% of the applied c was detected in the plants . field dissipation rates of glyphosate are affected by soil properties , method of application , and environmental conditions such as moisture and temperature , and therefore are extremely variable . field studies often result in longer estimated times to 50% dissipation ( dt50 ) as compared to laboratory studies , which are generally conducted under optimum conditions for degradation . in one field study , under the application and weather conditions that prevailed ( which resulted in water - saturated soils ) , no glyphosate was detected 24 h after treatment , with a trace amount detected in one replicate soil sample after 1 year . warm temperatures at the time of and in the season before the application are thought to explain the fast degradation rate in the water - saturated soil samples . in a comparison among glyphosate treatments between a forest floor and mineral soil , the dt50 times for glyphosate were 12 and 10 days for the forest floor matrix and mineral soil , respectively . simonsen et al . measured a 9-day dt50 for glyphosate and 32-day dt50 for ampa in soil . in an agronomic field study , glyphosate dissipation in the surface soil was rapid ( dt50 = 25 days ) and only 10% of applied chemical was present at 34 days after application . in another field study , glyphosate had an estimated dt50 of 4560 days , with total soil residues of glyphosate accounting for 618% of initial chemical at 360 days after application . reported a 110 to 151 day dt50 for glyphosate in a clay soil and attributed the long persistence to adsorption ( kf > 118 ) . as a result of strong sorption and slow desorption , some glyphosate residues tend to stay in the surface soils through the growing season . for example , of the initial amount of glyphosate added to a clay soil , 59% ( glyphosate + ampa residues ) remained primarily in the surface soil 748 days after application , despite large amounts of precipitation after application . also , only 0.009 and 0.019% of the initial amount of glyphosate added leached from the sand and clay soils , respectively , during the study period . no leaching of ampa occurred in the sand , whereas 0.03 g ha leached in the clay soil . the dt50 time of glyphosate was generally < 5 months in swedish railway embankments . in northern climates with seasonally frozen soils , field studies have shown clear overwinter persistence for glyphosate . after glyphosate applications in june and july , about 1020% of applied glyphosate was detected in the subsequent june in two field sites , demonstrating that the time for 90% ( dt90 ) dissipation of glyphosate was about 11 months . similar overwinter persistence was observed in agricultural fields in southeast finland . under warmer climates , glyphosate did not persist past the growing season , even after 15 consecutive annual applications . after either pre - emergence or postemergence applications of glyphosate , the distribution of residues is nonuniform in soils and is more concentrated near the surface of the soil . almost two years after application to a tilled soil in an outdoor lysimeter , glyphosate accounted for 1% and ampa for 19% of the applied glyphosate in the 010 cm depth increment . in the 1020 cm depth increment , glyphosate was not detectable , and ampa accounted for 5% of the applied herbicide . deep ( > 1 m ) leaching of glyphosate has been observed , but concentrations in leachate were < 0.07 g l. this was attributed to movement in macropore flow , rather than leaching through the bulk soil . deep movement of glyphosate might be also expected via translocation of the herbicide sprayed on to foliage of crops and weeds to their roots , particularly resulting from glyphosate applications later in the growing season . multiple applications of glyphosate in gr - cropping systems would ( 1 ) increase the risk of carryover , especially in regions where soils are seasonally frozen for extended periods and ( 2 ) increase the risk of leaching to tile drains or groundwater . multiple applications of glyphosate increase the time that bioavailable glyphosate is present in soil . also , plant interception of glyphosate in the field may lead to a delayed release of glyphosate into the soil following foliage decomposition . the degree of metabolic degradation of glyphosate in plants would influence how much glyphosate is released into soil by degradation of plant material . both of these processes have been investigated in laboratory experiments , but corresponding field studies are not yet available . any increased persistence is potentially important in cropping systems where glyphosate - sensitive ( gs ) crops closely follow the gr - crop . the risks associated with planting highly sensitive crops shortly after ( <3 days ) glyphosate applications were known long before the advent of gr - crops . during the course of their action on susceptible plants , herbicides eventually affect almost every physiological and biochemical process , including mineral nutrition . thus , glyphosate would be expected to affect mineral nutrition of gs plants at herbicidal doses , but not of gr plants at the same doses . recent reports of mineral deficiencies in gr crops after glyphosate application were linked with claims of increased susceptibility to plant diseases . there are conflicting papers on the effects of glyphosate on mineral nutrition on gr crops . this is a complex topic that , for clarity , we have separated into the aspects listed below . many natural metal chelators such as organic and amino acids are found in plant cytoplasm and xylem and phloem fluids . citrate is an important chelator of fe in xylem fluid , while some amino acids chelate metals in the cytoplasm where the higher ph favors chelation by amino acids compared to organic acids . synthetic chelators have been used in agriculture since 1950 to supply fe or zn to plants , and more recently to induce phytoextraction of soil metals by plants . adding high amounts of strong chelators such as edta ( ethylenediaminetetraacetate ) to soils causes sorbed metals to be released from soil and metal chelates to be formed , making the metal mobile . in order for added chelating agents to be effective in increasing metal uptake for example , induced phytoextraction of pb required the addition of 10 mmol of edta kg of soil which would cost over $ 30 000 ha . edta was only effective when , after binding other metals in the soil , there was some free edta which attached to the root membranes , causing them to become leaky . the added edta , however , also causes metal leaching , and use of edta to induce phytoextraction of soil metals is not allowed in the open environment . occasionally excessive fertilizer rates of feedta or znedta cause phytotoxicity in the field or greenhouse . our experience with metal chelation in soils and metal chelate injury of plants provides insight into whether glyphosate would be expected to affect plant uptake of micronutrient cations . if low concentrations of edta or similar strong chelators are added to soils , they can promote uptake of strongly adsorbed metals because the dissolved metal - chelate can move metals from soil particles to the root membranes , circumventing the diffusion limitations of metal uptake . thus , in general , addition of kg per ha levels of glyphosate might be expected to increase element uptake if glyphosate were a strong chelator . however , glyphosate is a relatively weak metal cation chelator compared to edta ( table 1 ) . in general , none of the research on chelating agent effects on metal uptake would indicate that a weak chelator such as glyphosate would reduce or increase uptake of micronutrient cations from soil . although protonated and deprotonated chelates also occur , the 1:1 metal - ligand chelates are listed for comparison using data from the program geochem - pc . values for edta , citrate and glycine are for 0 ionic strength , while the values for glyphosate are 0.1 m ionic strength . equilibria depend very strongly on solution ph and the pka values for the different proton binding functional groups of a chelator molecule . examination of glyphosate levels in glyphosate - treated gr soybean seeds at maturity and mineral levels in soybean seed shows that on a molar basis the metal : glyphosate ratio can be from almost 10 000 times more mn to around 100 000 times more minerals such as mg or ca compared to glyphosate . comparing glyphosate content of leaves of glyphosate - treated gr soybean with recently measured mineral content of gr soybean leaves , the ratios are smaller ( ca . 300 for ca , 30 for fe , 20 for mn , and only 2 for cu ) , but the ratio of total metal atoms to glyphosate molecules is close to 1000 . even if a substantial fraction of the minerals in the plant tissue were unavailable to glyphosate due to chelation with other compounds , sequestration , or other means , the ratio of mineral cations to glyphosate anions would still be very large . these large ratios do not support the view that the chelator properties of glyphosate would interfere substantially with plant mineral nutrition in planta . furthermore , at very high in vivo concentrations of glyphosate in the plant phloem , glyphosate has been calculated to be unable to effectively compete for fe , fe , ca , mn , mg , cu , and zn with biological chelating agents . reduction in soil mn concentrations due to glyphosate use has not been demonstrated . in practice , glyphosate which reaches soil is strongly adsorbed by fe and mn oxides and organic matter . when glyphosate is bound by soil , it can be abiotically degraded in addition to the biodegradation pathways discussed earlier . other studies tested whether glyphosate reaching soil would cause leaching of soil metals . barrett and mcbride tested leaching of metals in response to glyphosate application for several soils and found that leaching occurred only with soils highly contaminated with metals and only with high rates or repeated applications of glyphosate . in contrast with some descriptions of glyphosate as a strong chelator , the stability constants of glyphosate , edta , and citric acid with common micronutrient ions show that glyphosate is a weak chelator ( table 1 ) . the fact that the relative concentrations of metal cations in soil are several orders of magnitude greater ( in terms of moles of metals per ha vs moles of glyphosate per ha ) than the highest concentrations of glyphosate that could be expected ( discussed in detail in a previous section ) , significant effects of glyphosate on soil mineral content or availability to plants are highly unlikely . from the earliest days of glyphosate use , it was known that using water containing high levels of metal ions would significantly reduce the efficacy of the herbicide , presumably because the precipitated or chelated herbicide is not taken up by target plants as well as the free glyphosate anion and/or precipitation of glyphosate : mineral complexes ( reviewed by duke , sundaram and sundaram , and nilsson ) . if metal cations are present in a tank mix solution , and ph is raised by addition of microelement fertilizer or by hard water , precipitation of glyphosate reduces the plant uptake of glyphosate , thereby significantly reducing its herbicidal effectiveness . the solubility of 1:1 metal / glyphosate complexes decreases in the order of mg ca > mn > found ca , mn , and zn ions to reduce glyphosate efficacy on a variety of weeds when included in a tank mixture . several researchers have shown that separate application of mn fertilizer and glyphosate caused no effect or interaction , and recommend careful consideration of tank mixes . edta , being a stronger chelator than glyphosate , reverses the reduction of glyphosate herbicidal efficacy by metal cations in spray tanks . farmers have been advised to not spray glyphosate with micronutrient plant nutrition supplements unless the metal is chelated with a strong chelator such as edta or eddha . thus , studies on effects of glyphosate on mineral nutrition of plants should not be conducted with combined spray solutions of minerals and glyphosate . in short , a finding of metal ion precipitation of glyphosate in a tank mix is not relevant to questions raised about chemical interactions between glyphosate and micronutrients in plants or soils . because glyphosate is a metal ion chelator , there was speculation decades ago that this might be related to the mode of action of the herbicide . however , the finding that gr crops with only a change in their epsps are about 50-fold less sensitive to glyphosate than similar gs crops indicated that mineral nutrition is not involved in the mode of action of glyphosate . further evidence of this is the recent evolution of gr palmer amaranth ( amaranthus palmeri ) biotypes that have multiple copies of the gs epsps gene . the greater the number of copies of the gene , the more resistant these plants are . if chelating mn or any other mineral was significantly involved in the mode of action of glyphosate , this would not be the case . glyphosate can impede absorption and translocation of calcium and magnesium in gs plants ( reviewed in duke ) . nilsson found glyphosate to stimulate the accumulation of fe in gs plants , while impeding movement of zn to the same sites . this result supports the finding that subtoxic levels of glyphosate stimulate growth of iron - deficient wheat . nilsson found no effects of glyphosate on mn , zn , or cu content of gs wheat leaves . eker et al . reported that glyphosate reduced uptake and translocation of mn and fe in gs sunflower . likewise , foliar - applied glyphosate to gs soybean seedlings reduced uptake and translocation of mg and ca , reduced tissue ca content , and altered cellular ca distribution . found reduced levels of ca , mn , mg , and fe in seeds and leaves of glyphosate - treated , gs soybean . in studies with gs festuca spp . , ca , mg , mn , and fe were most reduced by glyphosate treatment compared to other minerals . such effects are readily explained by the known effect of glyphosate on root growth and function in gs plants . glyphosate from foliar sprays is rapidly translocated to roots , where it strongly inhibits root growth and other processes . nearly all of the multivalent metal cations are absorbed for translocation to shoots by young roots . reported reductions in plant shoot fe due to glyphosate application , resulting in chlorosis in both gr and gs soybean cultivars . the authors correlated the effects on fe content with effects on root ferric reductase activity , however , the methods used for measuring ferric reductase activity were inappropriate . roots grown in soil were removed , washed , and used in a bioassay of feedta reduction . broken roots , loss of fine roots and root hairs , and the presence of soil in the assay mixture confounds the measurement . ozturk et al . found inhibitory effects of glyphosate on root ferric reductase in iron - deficient gs sunflower . however , no in vitro effect of the herbicide on the enzyme was reported to determine whether it was a primary or secondary effect . high rates of phosphate fertilizer have been reported to remobilize small amounts of glyphosate bound to soil . these low soil solution concentrations of glyphosate were phytotoxic to a gs soybean cultivar on most soil types , but stimulated plant growth ( hormesis ) on one soil type . hormesis ( the stimulatory effect of a toxin at subtoxic concentrations ) at low glyphosate doses is a well - established phenomenon ( e.g. , velini et al . ) . however , the bott et al . experiment has no relevance to practical field environments , as the researchers applied extreme rates of dissolved superphosphate to the surface of glyphosate - amended soils and planted the seeds immediately . fertilizer p rates are usually applied in bands below and to the side of seeds to prevent adverse effects on seed germination . considering that the 240 mg p kg highest rate of p application would cause the amount in the surface 12 cm of the potted soil to be 1020 times higher than normally found in field applications of p , one should not extrapolate from the results with the high rates used in this study . it is questionable even whether the low rates , where no adverse effects were observed , are relevant to understanding glyphosate in the environment . the studies discussed in this subsection were done on gs plants , so separating secondary effects of inhibition of epsps and effects via any other mechanism is impossible . it may be that some of the confusion regarding glyphosate effects on mineral nutrition of gr crops is due to studies on gs plants that can not be extrapolated to gr plants . as mentioned above , gr crops are highly resistant to glyphosate , with resistance factors ( i50 ratios between gr and susceptible crops ) of about 50 for both gr canola and gr soybean . no effects on growth of gr crops are normally seen at the highest recommended field rates of glyphosate . under some environmental conditions with some cultivars , transient yellow flash symptoms in gr soybeans are seen 5 to 20 days after glyphosate application ( figure 5 ) . yellow flash has been attributed to the rapid metabolism of glyphosate to the weakly phytotoxic ampa and not to mineral nutrition effects . gr crops are not necessarily resistant to ampa , as its mode of action is not the same as that of glyphosate . the yellow flash effect is temporary and does not reduce yields , nor have yellow flash symptoms been shown to be due to disease incidence in soybean . this yellowing and interveinal chlorosis of rapidly growing young leaves in soybeans experiencing yellow flash could be confused with symptoms of fe or mn deficiencies . however , yellow flash symptoms are not accompanied by effects on mn status of the plant or on mn uptake or distribution by the plants . yellow flash symptoms have not been reported in gr crops other than soybean , perhaps because sufficient levels of ampa to cause such symptoms do not accumulate in other gr crops or there is insufficient sensitivity of these crops to ampa . little is known of ampa in gr crops , including its mechanism of phytotoxicity . example of yellow flash in gr soybeans sprayed with glyphosate in illinois . huber suggested that use of glyphosate in production of gr soybean leads to mn deficiencies by reduction of mn uptake and/or translocation efficiency , changing soil / rhizosphere microbiology , or modifying the form or availability of mn in the environment . noted that gr - soybean cultivars showed lower yield , stronger yellowing symptoms , and lower foliar mn on a mn marginal or deficient soil than two conventional cultivars ( non isolines ) . it now appears that they observed that the gr - cultivar was inherently less able to obtain soil mn than the conventional cultivars . mn deficiency can occur in soybeans grown on low mn soils such as the lake plain soils in the midwest , and the coastal plain soils on the east coast of the united states . if these soils are limed , mn becomes much less phytoavailable and soybeans may suffer severe chlorosis and yield reduction until foliar mn sprays are applied or soil ph is lowered . genetic variation for susceptibility to mn deficiency exists in soybeans ( e.g. , graham et al . ) . soybean cultivars for areas with low phytoavailable soil mn have been developed , and farmers are advised to plant more mn deficiency - resistant cultivars on such soils . as breeders worked to solve this susceptibility problem ( much like the case of fe chlorosis susceptibility of the early gr soybean cultivars ; see below ) , improved cultivars with the gr trait were also resistant to mn deficiency . this genetic variation in resistance to mn deficiency among soybeans occurs because roots change the microenvironment in their rhizosphere to reduce mn oxides to the soluble mn , or reduce chelated mn with fulvic acids to promote uptake by the roots . local acidification of the rhizosphere may also improve mn uptake by cultivars resistant to mn deficiency . plants also up - regulate metal ion transporters in their young roots to better absorb the free mn in the rhizosphere . experiments have been conducted in the field at multiple locations over multiple years which found that there was no appreciable susceptibility to mn deficiency or need for mn fertilizer to grow gr - soybean cultivars . several field trials have shown that gr - soybeans are not commonly experiencing mn deficiency . unfortunately , no study has been reported on soils which caused clear mn deficiency in soybeans in the absence of glyphosate so that any interaction with glyphosate use could be measured . there are several peer - reviewed journal claims of effects of glyphosate on mineral nutrition in gr soybean . reported that in the absence of glyphosate , a hydroponically grown gr soybean cultivar accumulated more mn than did a gs cultivar , but the two lines were not near isogenic , making interpretation of the data impossible . in addition , when both types of soybean were grown with low mn supply , there was no effect of glyphosate on shoot concentration of mn or growth . at very high application rates of glyphosate , mn concentrations in the tissue of the gr cultivar were reduced about 50% . there were no effects of glyphosate on mn and fe content of plant tissues when the plants were grown in two different soil types , although there was a reduction in insoluble foliar zn in one of the soil types . this tests whether the low molecular weight soluble chelates were formed in the tissues as occurs with excessive edta . taken together , the data of this study show no adverse effect of glyphosate on mn uptake or translocation in gr - soybeans . zobiole et al . reported that glyphosate treatment reduced essential minerals ( mg , mn , etc . ) in gr soybean tissues . they also reported dramatic reductions in photosynthesis associated with these reductions , a result that is difficult to reconcile with the high and increasing yields of these crops ( see section on yields below ) . in a more extensive study , cavalieri et al . examined the effects of 0.96 kg ha glyphosate from six different commercial formulations on n , p , k , s , b , ca , mn , mg , fe , zn , and cu in two gr soybean cultivars in the greenhouse . the results were equivocal , with both decreases and increases in metals , depending on both cultivar and glyphosate formulation . there was no clear pattern , other than reduced levels of both metal and nonmetal elements as well as plant growth by one formulation on one of the cultivars , suggesting that something other than glyphosate was involved . comparing near - isolines of soybeans , loecker et al . found no effect of the gr transgene of gr soybean on mn uptake or response to mn in the absence of glyphosate . rosolem et al . found no effects of foliar application of glyphosate on mn absorption , accumulation , or distribution in gr soybeans . serra et al . found no effect of glyphosate doses up to 2.5 kg / ha on cu , mn , and zn uptake by gr soybeans , while fe uptake increased at this high dose . no effects of glyphosate on translocation of these metal ions were seen up to 2.5 kg / ha . in this study , exogenously applied mn had no effect on any responses to glyphosate . lundry et al . found no effects of glyphosate on mineral nutrition in gr soybean seeds , compared to an untreated near - isogenic soybean line , indicating no effect from the epsps transgene or from glyphosate . found no glyphosate - induced deficiencies in macronutrients ( n , p , k , s , mg , and ca ) or micronutrients ( b , zn , mn , fe , cu , and al ) in second generation gr soybeans . the application of glyphosate to gr soybean had no effect on leaf mineral content ( mn , fe , cu , and zn ) or yield at two different sites in brazil . there was also no effect of absorption of exogenously applied mn . exogenous mn application had no effect on yield of glyphosate - treated , gr soybeans , but it did enhance mn and reduce fe content in this study . no effects 0.86 kg ha glyphosate sprayed once or twice on mn content of both greenhouse- and field - grown gr soybean leaves ( young and old ) or seed ( figure 6 ) . the results of all of these studies indicate that glyphosate does not restrict the availability of micronutrients in glyphosate - treated , gr crops . thus , the results of the three research groups that have reported glyphosate effects on mineral nutrition in gr crops are counter to those of nine other research groups . effects of two , successive glyphosate treatments ( 0.86 kg ai h at both 3 and 6 weeks after planting ) on the metal content of mature seeds of field - grown gr soybean plants . bars respresent 1 se . there were no differences among any of the paired mean values at the 95% confidence level .. in many locations in ia , mn , nd , and some other u.s . states , soybeans may suffer iron - deficiency - chlorosis ( idc ) when grown on wet calcareous soils . soybean cultivars vary widely in resistance to idc , and many factors which influence soil moisture and bicarbonate levels interact with severity of idc . idc can cause severe yield reduction on problem soils if cultivars are not highly resistant to idc . because of the susceptibility of many soybean cultivars to idc , growers with problem soils are advised to select chlorosis - resistant cultivars . unfortunately , when gr soybeans were first developed , the cultivars which were initially transformed were not highly resistant to idc , and many of the early high yielding gr soybean cultivars developed for normal soils were susceptible to idc on wet calcareous soils . soybean agronomists in states where idc is prevalent now screen genotypes for resistance to idc and report the results to growers so they can choose cultivars to match their soil idc problems . thus , gr soybean cultivars have been screened for susceptibility to idc with and without glyphosate applications in many locations . although this has not been reported in the literature , several scientists involved in soybean idc screening confirm that based on their observations in chlorosis rating field plots , glyphosate causes no adverse interaction with iron deficiency in soybean ( s.r . although duke et al . found no effect of two applications of glyphosate on nickel content of leaves or seed ( figure 6 ) of gr soybean , another report notes that glyphosate use on gr - soybeans in a brazilian study caused a significant reduction in plant n - fixation and a decline in leaf ni . the foliar ni levels , even in their controls , were far below normal soybean foliar ni levels in other research . ni deficiency of significant consequence in the field has only been observed with some low ni soils of the southeastern coastal plain where pecans suffered severe deficiency under some conditions of previous management which included raising soil ph which reduces ni phytoavailability . legumes have a higher ni requirement than nonlegumes because ni is needed for biochemical processes in nodule bacteria , as well as for certain plant biochemistry . unfortunately , zobiole et al . did not test application of foliar ni fertilizer to confirm that the measured yield reduction actually resulted from ni deficiency induced by glyphosate . furthermore , the level of ni in the brazilian soil was not reported , so whether soil ni deficiencies were involved can not be determined . that glyphosate is directly toxic to some strains of bradyrhizobium japonicum due the fact that their epsps is also sensitive to glyphosate is well - known ( see section under glyphosate effects on soil microflora below ) , and this toxicity is not related to effects on ni . studies designed to address the interactions of glyphosate and ni metabolism conducted on ni - deficient soils with and without ni supplementation would be useful in interpreting the results of zobiole et al . there are numerous studies on the compositional ( chemical and nutritional ) equivalence of gr crops with gs crops , including mineral content , although the intent of these papers was to evaluate the effect of the transgene(s ) on composition , rather than the effect of glyphosate treatment on gr crops . in most of the published studies no mention is made of whether glyphosate was used on the crop . in other studies the glyphosate application to the gr crop is not completely described . , the timing of glyphosate applications in gr corn is given , but not the rates . in another study with gr corn the only information provided for the glyphosate treatments was that they were made according to the label . a study with gr alfalfa states only that glyphosate was applied prior to each cutting . more detailed information on the glyphosate applications is provided in a study with gr corn by ridley et al . for one set of trials , the gr corn received an application of 1.08 kg ha , and in another ca . the purpose of these studies was to provide data required by regulatory agencies to determine the effect of the transgenes on the composition of the harvested crop . no effects of the gr transgenes or glyphosate application on mineral content have been found in these field studies conducted under good laboratory practices that usually involved multiple years and locations . however , these studies lacked comparisons of glyphosate - treated with untreated crops to allow evaluation of the glyphosate effect , independent of the genetic effect of the gr technology . clearly , glyphosate can have effects on mineral nutrition of gs plants through its herbicidal effects on plant roots and other parts of the plant . published data on the effects of glyphosate on mineral nutrition of gr crops are contradictory . three groups have claimed adverse effects on mineral nutrition in gr crops in peer - reviewed journals the zobiole et al . the peer - reviewed results of nine laboratories show no effect of glyphosate on mineral nutrition . these seemingly contradictory results could be entirely or in part due to differences in the soils , climatic conditions , and/or gr cultivars used . for example , one group of experiments is based almost entirely on studies with low ph soils using soybean varieties developed in brazil and evaluated in greenhouse studies . rigorous field studies on different soil types ( including those highly susceptible to inducing mn or fe deficiency in soybeans ) are needed to resolve the issue of whether glyphosate might have adverse effects on mineral nutrition of gr crops . considering the available data , growers are unlikely to need mn fertilizers just because they use glyphosate on gr soybeans . soil microflora can influence the persistence of glyphosate and its metabolites in soil . evaluation of glyphosate effects on soil microorganisms requires knowledge of the direct effects of glyphosate and its metabolites on soil microorganisms as well as effects on microorganisms through processes mediated by plants on root symbionts and rhizosphere microorganisms . the determination of relevant environmental exposure concentrations needs to be compared to known response factors . finally , short - term and long - term responses on processes and community structure need to be evaluated . as in plants , glyphosate blocks the synthesis of the aromatic amino acids phenylalanine , tyrosine , and tryptophan in some bacteria and fungi through the inhibition of epsps , which also causes accumulation and excretion of shikimate-3-phosphate and hydroxybenzoic acids in sensitive microorganisms . the sensitivity of bacterial epsps to glyphosate varies widely . divided microbial epsps into two groups : sensitive ( class i ) and relatively insensitive ( class ii ) . class ii includes agrobacterium cp4 ( the source of the gr - epsps transgene in most gr - cultivars ) in which the resistance to glyphosate results from variations in the amino acid sequence of epsps . concentrations required for 50% inhibition were 75 m for e. coli , 174 m for bacillus subtilis , and 1100 m for pseudomonas aeruginosa epsps . moorman et al . reported variation in susceptibility of strains of bradyrhizobium japonicum to glyphosate : 1000 m ( 169 mg l ) glyphosate produced 47% inhibition for strain 110 , but only 12 and 19% inhibition for strains 123 and 138 , respectively . similarly , hernandez et al . reported b. japonicum strains ranging from sensitive to glyphosate ( 50% inhibition at 30 m ) to insensitive ( 50% inhibition at > 1000 m ) . the full range of resistance or sensitivity to glyphosate within the soil microbial community is not fully known . addition of aromatic amino acids to bacterial cultures can partially or fully reverse the effects of glyphosate . some fungi are also sensitive to glyphosate , with 50% inhibition of growth at concentrations of 5 to 50 mg / l ( 0.848.4 m ) in culture . understanding the impact of glyphosate on soil microorganisms requires estimating concentrations to which the microorganisms are exposed . glyphosate applied to foliage is rapidly translocated to roots and other metaboically active tissues . glyphosate is exuded from roots of treated plants into the rhizosphere , but the resulting concentrations in the rhizosphere soil are difficult to document . glyphosate applied to gs crops can be translocated to the roots and released initially in exudates and later from decaying tissues . as much as 15% of glyphosate applied to sensitive plants could be translocated to roots laitinen et al . also showed movement of glyphosate from roots of treated plants to the soil , with the concentration of glyphosate reaching 0.07 mg kg soil in the rhizosphere at four days after application . kremer et al . compared carbohydrate and amino acid exudation from roots of gr soybeans with or without glyphosate treatment in hydroponic culture . amino acid exudation was increased by glyphosate , but carbohydrates ( measured by an anthrone reaction ) were not different . glyphosate treatment of a gs soybean variety ( williams ) also resulted in increased carbohydrate exudation . the root exudation of shikimate-3-p and protochatecuic acid have not been examined , but exudation of these compounds might be expected from gs plants after glyphosate application , as glyphosate causes marked accumulation of these compounds in sensitive plants ( e.g. , lydon and duke ) . the effects of glyphosate on microorganisms in soil have been extensively investigated using a variety of techniques . two techniques that investigate the community level responses , microbial biomass and respiration , show either no effect or a temporary inhibition of respiration due to glyphosate applied at rates less than 50 mg kg . at glyphosate application rates above 50 and up to 1500 mg kg soil , the range of concentrations used in these studies resulted from different assumptions about the penetration of sprayed glyphosate into the soil ( see lancaster et al . ) . the stimulatory effect of high glyphosate concentrations on soil respiration is partly attributed to microbial metabolism of glyphosate , but secondary effects due to n and p mineralization could also stimulate respiration . these concentrations of glyphosate seem sufficient to induce glyphosate toxicity ; a hypothetical application of 50 mg kg glyphosate soil at 25% gravimetric water content would result in a 1.18 mm aqueous concentration in a thin layer at the surface of the soil . however , rapid adsorption would reduce the concentration in the soil solution . a kd of 50 would result in approximately 2% of the applied herbicide being present in the soil solution resulting in an aqueous concentration of approximately 24 m glyphosate , which is sufficient to affect sensitive microbial species . community level measures , such as respiration or total microbial biomass , are not sufficiently sensitive to detect changes in population or activity of small subpopulations . alternatively , glyphosate impacts on soil microorganisms can be assessed using measures of community structure and in long - term studies where cumulative impacts may be determined . hart and brookes found no difference in microbial biomass , microbial respiration and n mineralization in soils after 19 years of annual glyphosate application compared to an untreated control soil . busse et al . compared ponderosa pine ( pinus ponderosa ) forest soils receiving glyphosate treatment for understory vegetation control to control treatments ( understory cover at 25100% ) . no glyphosate effects on soil respiration , n mineralization , or microbial biomass were found when these plots were evaluated after 9 to 13 years at each of the three sites . powell et al . compared a gr - soybean to a near isoline sensitive cultivar over four years in ontario . rates of soybean litter decomposition of the gr and conventional cultivars were nearly identical ; however , glyphosate reduced litter decomposition on the soil surface , but not on buried litter . the ratios of fungal biomass to bacterial biomass in the litter were only occasionally different , with an increased ratio in the gr cultivars . the rhizosphere is comprised of the root surface and the immediate soil layer ( 25 mm ) surrounding the root where microbial processes are driven by root exudation of simple and complex substrates , which include organic acids , flavonols , lignins , indole compounds , and amino acids . the rhizosphere community includes root symbionts , pathogens , plant growth - promoting rhizobacteria , phosphate - solublizing bacteria , and microoganisms active in carbon and nitrogen cycling . significant amounts of carbon are exuded from growing roots , and rhizosphere populations may be exposed to glyphosate through leaching of glyphosate from the soil surface and root exudation of glyphosate . mijangos et al . examined glyphosate effects on gs plants ( triticale and peas ) and their rhizosphere microbial communities . ammonia concentrations increased in rhizosphere soil after glyphosate treatment compared to the control ( no glyphosate , but clipped to remove above - ground biomass ) . functional diversity of the rhizosphere microbial community was examined using a multiple substrate utilization test ( biolog ecoplates ) and genetic diversity by denaturing gradient gel electrophoresis of 16s - rdna after pcr amplification . community diversity and richness were reduced at the highest rate of glyphosate application in rhizospheres of killed gs pea and gs triticale , but not in soil from triticale grown alone . the magnitude of these differences was similar to the differences due to growing triticale alone or in combination with peas . several studies using different methods have examined the impact of glyphosate on the rhizosphere of gr crops . glyphosate application to gr - soybean cultivars in the field in two growing seasons caused transient differences in dehydrogenase activity , -glucosaminidase activity , -glucosidase , and respiration . these enzyme activities are broadly distributed in soil microorganisms , and the results suggest that broad spectrum toxicity did not result from glyphosate application . subsequent studies reported increases in the ratio of mn oxidizers / mn reducers in response to glyphosate and decreases in iaa - producing rhizobacteria . the magnitude of these responses increased as the glyphosate application rate increased up to a rate equivalent to 1.2 g ha . manganese oxidation ( mn + 1/2o2 + h2o mno2 + 2h ) reduces the solubility of manganese . the observation that glyphosate affects the ratio of mn oxidizers / mn reducers in the gr soybean rhizosphere led to suggestions that glyphosate reduced plant available mn in soil and plant uptake of mn . however , the extent that this shift to a higher ratio of mn oxidizers to mn reducers has on the availability of mn to plants was not determined . manganese is most available in soil under reduced conditions and/or at low ( < 5.4 ) soil ph . a phylogenetically diverse group of both bacteria and fungi are capable of mn oxidation , but the cultural methods used to assess mn oxidation or reduction potential may not measure all the microorganisms capable of mn transformation , or their in situ activity . also , plant roots actively regulate their ability to obtain mn from soils , up - regulate mn transporters , and secrete reducing materials which would release mn from bound forms in the soil for plant uptake . additional research is needed to investigate glyphosate - induced changes in mn bioavailability in the rhizosphere . reported reduced functional diversity ( also using the multiple substrate utilization test ) in response to two glyphosate applications to gr - canola . hart et al . found that rhizosphere populations of denitrifying bacteria and fungi were not affected by glyphosate application to gr - corn compared to gr - corn treated with conventional herbicides or a gs corn isoline treated with conventional herbicides . extracted bacterial dna from gr corn rhizospheres after pre - emergence treatment with no herbicide , glyphosate , or gtz ( a mixture of the herbicides acetochlor and terbuthylazine ) . pyrrosequencing of cloned 16s - rdna showed that microbial community structure after glyphosate treatment more resembled the control ( no herbicide ) than the gtz - treated community . glyphosate reduced actinobacteria relative to the untreated control and proteobacteria were relatively unaffected . the gtz treatment reduced microbial diversity relative to the glyphosate or no - herbicide treatments . in contrast , lancaster et al . showed a variable response of actinobacteria populations to one or five applications of glyphosate to soil without a crop , while proteobacteria were increased by glyphosate applications . the concentrations of microbial fatty acid methyl - esters ( fame ) from gram - negative bacteria also increased , which is consistent with the increase in proteobacteria populations . longer - term ( 3 year ) studies identified three microbial groups dominating the gr corn rhizosphere in two fields in spain : the proteobacteria , actinobacteria , and acidobacteria . glyphosate was applied postemergence to gr - corn , and roots were sampled 7 days after glyphosate treatment and just prior to harvest . dna extraction and sequencing provided a database that was screened for 16s - rdna phylogenetic sequences . the abundance of these groups indicated little effect of glyphosate over three years ( figure 7 ) . analysis of the same data with a clustering procedure showed that the rhizosphere community was most affected by year and field and least affected by time of sampling and herbicide . acidobacteria increased over time in both fields ( figure 7 ) , while actinobacteria tended to decrease . eubacterial phyla ( 16s - rdna sequence abundance ) recovered from gr - corn rhizosphere treated with glyphosate ( g ) or without glyphosate ( c ) in two fields ( upper and lower panels ) . also used fame biomarkers to examine the effects of two postemergent glyphosate applications to gr - soybeans grown in soil with and without a history of previous glyphosate use . at 7 days after application , total fame ( an indicator of microbial biomass ) nonmetric multidimensional scaling of the fame data showed a significant effect of the soil ( history vs no - history ) on community structure , but no effect of application or sampling times on community structure . the decrease in microbial biomass at 7 days after application does not support the conjecture that glyphosate treatment increases root exudation . weaver et al . also used fame analysis to compare rhizosphere and bulk soil community structures after glyphosate application to gr - soybean in the field . after the second in - season glyphosate application , the community structure of the bulk soil differed from that of the rhizosphere , but two previous applications of glyphosate had no effect on fame . the same study included two fungal fame biomarkers ( 16:1 5c and 18:2 6c ) , and these were not affected by the glyphosate treatments . the 16:1 5c ( hexadecenoic acid ) content is a biomarker for arbuscular mycorrhizal fungi , while 18:2 6c is a more broad fungal marker , including rhizoctonia solani and fusarium oxysporum.(167 ) zablotowicz and reddy summarized the effects of glyphosate on soybean nodulation and n fixation . gr soybeans treated with glyphosate had reduced nodulation , as well as delayed n fixation , plant biomass accumulation , and n fixation , but the severity of these effects was dependent upon several factors . these included when glyphosate was applied to the soybean , the number of glyphosate applications , the glyphosate formulation , and the gr - soybean cultivar . compared nodule number and mass in six gr and three near isoline gs soybean cultivars in the absence of glyphosate . significant differences in nodulation were found among the cultivars , but these were not related to glyphosate resistance . concentrations of glyphosate in nodules and roots of soybeans were low ( < 200 ng g nodule tissue ) , although shikimate and hydroxybenzoic acids were present in three - to 4-fold greater concentrations , indicating inhibition of b. japonicum epsps . among strains of b. japonicum , glyphosate tolerance in culture multiple field studies show no effect of glyphosate on gr - soybean yield . using differences in natural abundance of n , bohm et al . estimated the percentage of soybean n derived from fixation to be 80% for gr soybean without glyphosate , 57% for the same cultivar with one glyphosate application , and 66% after two applications . suggested that the glyphosate - treated soybeans obtained more reduced n from the soil . these effects on nodulation and n fixation may be due in part to the inhibitory effects of glyphosate on b. japonicum , but may also be related to gr cultivar responses to glyphosate . additional evidence of cultivar variability was found in a field study using 20 gr soybean cultivars with and without glyphosate applied at four combinations of rates and timings . of the 20 cultivars , 9 showed no difference in nodule biomass compared to the unsprayed treatment . one gr cultivar , brs 244 rr , which had no glyphosate effect on nodule biomass in this study , was reported to have reduced nodulation after glyphosate application in a subsequent study . the survival of b. japonicum in soil without plants was not affected by concentrations equivalent to 1x or 10x field application rates of glyphosate . selection or construction of gr b. japonicum would be an effective strategy for alleviating negative effects on nodulation and n fixation . the glyphosate metabolite ampa can temporarily reduce chlorophyll content ( causing yellowing or chlorosis ) and photosynthesis in gr soybeans , particularly after foliar applications of ampa at 1.0 kg ha or high rates of glyphosate . this rate was chosen to represent the complete metabolism of a glyphosate application to ampa . this rate of ampa did not affect nodulation or nitrogenase activity , suggesting that b. japonicum is less sensitive to ampa than soybeans . the responses of gs cultivars were similar to the gr cultivars , which are explained by the fact that ampa does not affect epsps . arbuscular mycorrhizal fungi are obligate symbionts that transfer mineral nutrients to their plant hosts . savin et al . evaluated glyphosate effects on arbuscular mycorrhizal fungi ( amf ) colonization of gr cultivars of cotton , corn and soybean grown in soil under greenhouse conditions . amf colonization of roots was not affected by glyphosate , and neither were acid nor alkaline phosphatase soil enzyme activities . other research has shown that in the tripartite symbiosis of mycorrhiza , rhizobium , and soybean , no adverse effects of glyphosate use on gr cultivars was observed . these studies indicate that effects of glyphosate on plant mineral nutrition through effects on amf are unlikely . plants use a variety of preformed and postinfection - induced defenses to resist pathogens . these include phenolic compounds which are considered to be major components of defense across the plant kingdom . phenolic compounds may act in defense as preformed antibiotics , pathogen - induced phytoalexins , or as structural barriers in the form of lignin . thus , it is not surprising that any alteration in phenolic metabolism may have an impact on the expression of disease . for example , treatment with inhibitors of phenylalanine ammonia lyase have been shown to enhance disease susceptibility ( e.g. , holliday and keen ) , while treatment with compounds such as microbial elicitors or even herbicides such as the protoporphyrinogen oxidase ( ppo ) inhibitor lactofen can stimulate accumulation of phenolic compounds and enhance disease resistance . because glyphosate inhibits epsps , a key enzyme in the shikimic acid pathway , it also inhibits the biosynthesis of phenyalanine - derived phenolic compounds and should also result in lack of synthesis of salicylic acid from isochorismate . hence , this herbicide may enhance susceptibility to diseases in plants that are susceptible to glyphosate . in addition to phenolic compounds , glyphosate should also prevent synthesis of anthranilic acid , an intermediate needed for the synthesis of the indole - based glucosinolates and phytoalexins in crucifers and the avenalumin phytoanticipins and avenanthramide phytoalexins in oats . finally , many plants respond to infection by strengthening their cell walls with hydroxyproline - rich glycoproteins . because these proteins contain a significant amount of tyrosine , and formation of isodityrosine cross - links is important in cell wall reinforcement , it would seem likely that glyphosate may also impact this aspect of plant defense . however , the effect of glyphosate on these nonphenylpropanoid based defenses has not been reported . a summary of possible effects of glyphosate on plant defenses derived from the shikimic acid pathway in shown in figure 8 . based on both known and potential effects of glyphosate on disease defense compounds , it is not surprising to find reports in the published literature that show the disease - enhancing effects of glyphosate on gs plants ( e.g. , reviewed in johal and huber and duke et al . ) . possible effects of glyphosate treatment of glyphosate - senstive plants on shikimic acid pathway metabolites considered to be important in defense . products of the shikimic acid pathway involved in plant defenses are outlined by boxes . metabolites and metabolic groups in red have been demonstrated or are hypothesized to be reduced in gs plants after glyphosate treatment . of these , only isoflavonoid phytoalexins from bean and soybean and lignin deposition in bean have been examined for effects of glyphosate ( and only in gs plants ) . protocatechuic acid has been demonstrated to increase in gs plant tissue after glyphosate treatment . * * epsps : 5-enolpyruvylshikimic acid-3-phosphate synthase , the site of glyphosate action . the effect of glyphosate on disease resistance of gs plants was initially reported by keen and co - workers in 1982 . they reported that treatment of gs soybean hypocotyls with glyphosate decreased the resistance to phytophthora megsaperma and reduced the accumulation of the isoflavonoid phytoalexin glyceollin . in a subsequent study , ward ( 193 ) confirmed the results of keen et al . and also showed that glyphosate also reduced the efficacy of metalaxyl , a fungicide specific for oomycetes . the resistance breaking effect of glyphosate has also been tested in two gs bean pathosystems . pretreatment of bean hypocotyls with glyphosate resulted in only a subset of the plants becoming more susceptible to infection with an incompatible ( avirulent ) race of colletotrichum lindemuthianum . the increase in susceptibility was associated with a decrease in phytoalexin production , but it is important to note that the response was not uniform . a much different result was found in the bean pythium interaction . in this case , this change in host reaction was associated with reduced accumulation of phenolic phytoalexins and deposition of lignin . although treatment of gs plants with glyphosate can result in increased susceptibility to pathogens , it is important to know if gr crops can be predisposed to susceptibility by treatment with glyphosate . biochemically , it would seem unlikely that gr plants would become more susceptible after glyphosate treatment , but is that the case ? johal and huber and kremer and means reviewed glyphosate effects on gs and gr cultivars and suggested that fungal root diseases were increased by the adoption of gr - cultivars and the increased use of glyphosate . several studies have addressed whether or not gr plants are more susceptible to disease , with much of the work focusing on gr soybeans . tested two near - isogenic lines of soybean gl2415 ( gs ) and gl2600rr ( gr ) for susceptibility to sclerotinia sclerotiorum , the cause of the sclerotinia stem rot or white mold disease . using a detached leaf assay , there was no significant difference in lesion development in nontreated gl2415 as compared to gl2600rr . the formulation blank for the glyphosate product used in the work also had no effect on disease . most important was the observation that treatment of gl2600rr with three different rates of glyphosate did not increase the severity of disease in that line as compared to the untreated controls for both gl2600rr and the gs line gl2415 . thus , the conclusions for this work were that the gr gene had no impact on disease and treatment of the gr plants with glyphosate did not enhance susceptibility . nelson et al . provided further evidence that the gr trait did not impact host reaction to s. sclerotiorum in field studies . comparing four lines of soybean that were near - isogenic for the gr trait , they found that , with one exception , the glyphosate resistance trait had no effect on disease reaction . glyphosate treatment of two gr lines increased disease as compared to the untreated , while the opposite effect was observed with two others . however , the two gr lines that showed increased disease after glyphosate treatment and their near - isogenic lines were significantly more susceptible to sclerotinia as compared to the two other lines in which glyphosate had no effect on disease . interestingly , treatment of the two most white mold susceptible gr lines with another soybean herbicide ( thifensulfuron ) resulted in enhanced disease development comparable to the plants treated with glyphosate . lactofen , a ppo inhibitor known to induce resistance to s. sclerotiorum , was able to reduce disease severity in all lines regardless of the presence or absence of the gr gene . perhaps most significant was the observation that glyphosate treatment of gr lines had no effect on yield regardless of the amount of disease . also addressed the issue of cultivar differences in sclerotinia stem rot susceptibility by examining management options . this work further illustrated that issues related to greater susceptibility of gr soybeans was not related to glyphosate resistance trait , but rather to the susceptibility of the cultivars that were used for transformation . a lack of impact on defense responses in gr soybean was supported by analysis of glyceollin accumulation in resistant as compared to susceptible plants . using silver nitrate as an elicitor colonization increased from 20 to 30 infections per 100 cm of untreated soybean root to as little as 30 infections or as much as 120 infections per 100 cm root , depending upon glyphosate dose and soybean growth stage and cultivar . in the same studies , decreases in populations of pseudomonas spp . and indole acetic acid ( iaa)-producing bacteria , as well as a reduction in the ratio of mn - reducing to mn oxidizing microorganisms were observed . fluorescent pseudomonas populations in the gr - rhizosphere were decreased by glyphosate application and negatively correlated with fusarium root colonization . these fusarium infections of soybeans roots developed from soil - borne inoculum , and the species distribution of fusarium was not determined . the mechanisms of these glyphosate - mediated increases in fusarium root infection in gr soybeans are not established . results of studies on translocation of c - glyphosate from treated leaves into roots and rhizospheres indicate that beneficial microorganism - inhibiting glyphosate concentrations could occur . kremer et al . showed that root exudates in general increased from glyphosate - treated plants grown in soil - free conditions . however , these studies did not establish that these root exudates specifically stimulated the growth of fusarium spp . still , glyphosate - mediated changes in quantity and quality of root exudates into the rhizosphere has not been sufficiently evaluated as an influence on plant disease . the effects of glyphosate and glyphosate resistance in relation to sudden death syndrome ( sds ) , in soybean caused by fusarium virguliforme ( formerly fusarium solani f. sp . glycines ) has also been examined . sanogo et al . reported on the effects of glyphosate and two other soybean herbicides ( lactofen and imazethapyr ) on sds response in two gr soybean lines ( pioneer 9344 and asgrow 3701 ) and one gs line ( bsr101 ) . two of the lines , pioneer 9344 and bsr 101 , are susceptible to sds while the other line was noted by the authors as having above average tolerance . in growth chamber tests , the foliar symptom severity of glyphosate - treated plants was no different than the untreated control or plants treated with imazethapyr . , the severity of foliar and root symptoms of sds was increased by both glyphosate and imazethapyr ( with the exception of foliar severity in bsr 101 in which glyphosate treatment resulted in no difference from the control ) . these results suggested that the glyphosate resistance trait did not impact sds response and that all three lines reacted to infection by fusarium in a similar manner after herbicide treatments . later reported on the field reaction of the same three soybean lines to f. virguliforme and treatment with the same three herbicides plus acifluorfen . they examined both foliar symptoms and frequency of fusarium isolation from roots , and found no significant cultivar - herbicide interaction . there was also a lower amount of disease in the more resistant line regardless of herbicide type as compared to the two susceptible lines , and treatment with glyphosate , acifluorfen and imazethapyr all increased disease severity in susceptible lines as compared to controls . lactofen , in general , had no effect on disease severity compared to the controls . the authors concluded that there was no change in host resistance to sds as a result of glyphosate treatment . the effects of glyphosate treatments on sds were examined in ten gr soybean lines from a variety of maturity groups . in work similar that of sanogo et al . , njiti et al . reported no effect of glyphosate treatment on yield , foliar symptoms , or root infection . the overall conclusion from this study is that the host genotype , and not glyphosate resistance or treatment with glyphosate , was the most important factor in determining the reaction of a cultivar to sds . found that glyphosate sprayed on mixed populations of weed species caused increased fusarium spp . infection in some weed species , but not in others . the number of colony - forming units of fusarium spp . per gram of dried soil increased after application of glyphosate , but gs crops ( corn , pea , cucumber , and bean ) subsequently grown on in the field were not affected . powell and swanton concluded that there was insufficient evidence to prove a link between glyphosate and plant diseases associated with fusarium spp . harikrishnan and yang et al . examined the effect of glyphosate and other herbicides on reaction of bsr 101 ( gs ) and pioneer 93b01 ( gr ) to rhizoctonia solani . in greenhouse studies , the severity of rhizoctonia infection in autoclaved soil was not increased by glyphosate in pioneer 93b01 compared to the inoculated control . in fact , statistically similar levels of severity were also observed after imazethapyr treatment . in nonautoclaved soil , glyphosate actually reduced the severity of rhizoctonia as compared to plants that were inoculated but not treated with a herbicide . in two years of field trials , glyphosate treatment of pioneer 9344 resulted in no difference in response to rhizoctonia infection based on shoot dry weight , rhizoctonia severity and plant stand . gr soybeans are a rotation crop with cereals , and recent reports have suggested that glyphosate treatment of soybeans increases the occurrence of fusarium head blight of wheat and barley . because of these observations , brub et al . tested the effects of tillage and glyphosate treatment of gr soybean on fusarium head blight development in a subsequent planting of wheat and barley . there was no measurable effect of treating gr soybeans with glyphosate on development of head blight and accumulation of mycotoxins in barley or wheat . in sugar beet , gr varieties were tested for the effects of glyphosate treatment on expression of disease caused by rhizoctonia solani and fusarium oxysporum f. sp . betae . inoculation with r. solani isolate r-1411 ( ag-4 ) resulted in comparable amounts of disease in both b4rr and h16 whether or not they were treated with glyphosate or a surfactant . however , inoculation with r. solani r-9 ( ag-2 - 2 ) revealed that glyphosate treatment resulted in increased disease in b4rr as compared to h16 . inoculation with f. oxysporum isolate fob13 resulted in increased disease in glyphosate - treated b4rr and h16 as compared to nontreated controls . there was no effect of glyphosate treatment on infection of the two sugar beet lines by f. oxysporum isolate f19 . in a two year field study with gr sugar beet , barnett et al . reported that glyphosate had no effect on expression of rhizoctonia crown and root rot in four gr lines ( hilleshg 9027rr , hilleshg 9029rr , hilleshg 9028rr and crystal ) . they also reported that glyphosate treatments did not impact efficacy of the fungicide azoxystrobin . using field and greenhouse evaluations , a follow - up study by barnett et al . confirmed that glyphosate treatment of gr lines had no effect on reaction to rhizoctonia . their recommendation to growers was to use gr sugar beet varieties with the greatest amount of rhizoctonia resistance . two wheat lines that were near - isogenic for glyphosate resistance were tested for the effect of glyphosate on disease caused by rhizoctonia oryzae , r. solani , pythium ultimum and gaeumannomyces graminis var . the gr lines were not more susceptible to any of these pathogens than the lines from which they were derived . however , this study reported that volunteer gs wheat , if killed by a foliar treatment with glyphosate resulted in increased infection by r. solani and g. graminis var . tritici , possibly as a result of increased amounts of pathogen inoculum produced in the crop residue . the reaction of gr cotton seedlings to rhizoctonia solani after treatment with several pre- emergent herbicides and glyphosate as a foliar treatment was tested in field and greenhouse experiments . glyphosate applied at the cotyledon or four leaf stage of gr cotton reduced rhizoctonia infection of hypocotyls in the field . in greenhouse studies , several pre - emergent herbicides predisposed cotton seedlings to greater hypocotyl infection by r. solani , but subsequent application of glyphosate did not increase severity of the disease . baird et al . found that four varieties of gr cotton ( pm 1220 , dpl 5690 , dpl 5415 , and dpl 50 ) had similar seedling stand count , height , and dry weight when compared to gs varieties from the same lineage group , regardless of glyphosate application . when differences did occur , no consistent trends could be determined within the lineage groups tested . glyphosate was shown to have both preventive and curative activities against both stripe rust ( puccinia striiformis f. sp . tritici ) and leaf rust ( puccinia triticina ) on gr wheat . in these cases , it appears that glyphosate is acting directly as a fungicide . some efficacy against phakopsora pachyrhizi , the cause of asian soybean rust , was reported in both greenhouse and in the field on gr soybeans . tuffi santos at al . showed that glyphosate reduced the severity of rust caused by puccinia pdisii on eucalyptus grandis . they found that there was a systemic effect of glyphosate on rust development as illustrated by reduced urediniospore germination and appressorium formation on tissues that were not directly treated with the herbicide . similar to soybean , glyphosate has recently been reported to protect gr alfalfa against the rust uromyces striatus when applied prior to or up to 10 days after inoculation . in this study , these latter two results are interesting as these pathogens , unlike biotrophic rusts , are hemibiotrophic and necrotophic in their attack of their hosts . holliday and keen examined the effect of glyphosate on the response of gs soybean leaves to the bacterial pathogen pseudomonas syringae pv glycinea . in this case , the effect of glyphosate on resistance was less conclusive . although glyphosate treatment significantly decreased glyceollin accumulation , it had no effect on the expression of the hypersensitive response . glyphosate treatment also resulted in only a relatively small increase in bacterial growth in the treated plants . this suggests that in gs plants resistance to bacterial blight is not greatly reduced after treatment with glyphosate . several hundred gr soybean lines were screened for resistance to bacterial pustule , caused by xanthomonas axonopodis pv glycines . the authors report that resistance to the disease occurs in gr soybeans , but that not all genotypes were resistant . although they did not test the effect of glyphosate on the host response to xanthomonas , the authors did recommend that growers assess the risk for this disease and plant resistant cultivars when the disease is likely to occur . goss s wilt and leaf blight of corn , caused by the gram positive bacterium clavibacter michiganensis subsp . nebraskensis , has increased over the last 5 years in corn - producing states , as has increased planting of gr corn ( figure 1 ) . there are some logical explanations for the recent increase in the occurrence of goss s wilt and leaf blight in corn growing areas that do not implicate the use of gr corn or glyphosate . . both of these practices will allow the buildup of pathogen inoculum over time , and reduced tillage practices allow the pathogen to survive . reduced tillage practices that reduce residue decomposition will also increase pathogen inoculum , although one of the perceived benefits of gr crops has been the ability to manage weeds in reduced tillage . another factor that may contribute to increased disease development are reduced efforts to select for resistant hybrids and/or failure to promote resistant hybrids by seed companies . finally , weather events ( such as early season hail damage ) will promote infection in even the most resistant hybrids . nebraskensis genotypes might also be a factor , but further work is needed to determine if this has occurred . there was no mention in any of the recently published reports that the gr trait or glyphosate application is a contributing factor to the increase in goss s wilt . the report by ruhl et al . noted that the first indiana finds were on both field corn and popcorn . since popcorn is not gr , this would further implicate other factors in the recent outbreaks of the disease . considering that most corn produced in the us is now gr ( figure 1 ) , it is likely that inoculum buildup and use of gr corn that is not resistant to goss s wilt are the reasons for increases in this disease . in addition , there are no reports in the published literature that suggest that glyphosate resistance or treatment of gr varieties with the herbicide will increase the risk of other diseases in this crop . yang et al . examined the effect of glyphosate on soybean cyst nematode ( scn , heterodora glycines ) infection of the gr and scn - resistant variety countrymark 316 . greenhouse tests demonstrated no effect of glyphosate on scn development on this genotype as compared to untreated controls . noel and wax compared the reactions of gr soybean lines dr 320 ( scn susceptible ) and dsr 327 ( scr resistant ) to glyphosate treatment and inoculation with h. glycines . they reported that glyphosate did result in increased numbers of the nematode on the susceptible line , but not the resistant line . even with the increase in nematode populations , there was no impact on yield . this study , like those with other soybean diseases , suggests that genotypic resistance or susceptibility , rather than glyphosate resistance , is the most important factor related to disease severity . although it is clear that glyphosate does increase severity of disease on gs plants , the published evidence for its effects on gr plants presents a different story . overall , it appears that in gr crops the baseline disease resistance or susceptibility of the host plant , not the presence of the glyphosate resistance gene or treatment with glyphosate , is the major contributor to susceptibility . in the u.s . , gr soybeans , cotton , and corn were introduced in 1996 , 1997 , and 1998 , respectively . adoption of the crops has been rapid and overwhelming , with more than 90% of soybeans , ca . 80% of cotton , and about 70% of corn currently grown being gr ( figure 1 ) . after the introduction of gr sugar beets in 2008 , the adoption rate was essentially 100% in 2009 . thus , one might expect that if there were any significant mineral nutrition and/or disease problems with these crops , the problems would be manifested in yield reductions and farmer dissatisfaction . yield data from the years before introduction of gr crops , continuing to the present show that the same yield trends before introduction continued after introduction ( e.g. , figure 9 ) . while there could be isolated pockets of adverse effects of glyphosate on gr crops that would be masked by their general success , such cases have not been conclusively documented . there were initial concerns with transgenic crops in general that there would be yield drags due to factors not associated with disease or mineral nutrition , but to suboptimal cultivars and potential pleiotrophic effects of the transgenes . these problems have not materialized . to summarize , yield data for crops that are now predominantly gr cultivars do not support the view that there are significant mineral nutrition or disease problems with gr crops . yields of the three crops over the past 30 years that are now grown mostly as gr cultivars . data are from the usda , national agricultural statistics service data and statistics web site : http://www.nass.usda.gov/data_and_statistics/quick_stats/ ( accessed september 12 , 2012 ) . scientific accounts about increased plant disease and mineral nutrition problems in gr crops are based on publications from a limited number of researchers . in the context of the entire body of relevant science still , considering the enormous importance of and reliance on gr crops and glyphosate , there has been a paucity of publically funded research into potential problems with this weed management technology . farmers have generally embraced this technology , so that there has been no widespread call for studies of potential problems with gr crops other than those associated with gr weeds , a growing problem that is well documented . furthermore , publication of negative ( no effect ) results is generally unattractive to journals , and , therefore , to scientists whose success depends on publications . so , the no effect papers that have been published may not represent all such data that have been generated . reports of significant adverse effects of glyphosate on mineral nutrition and diseases of gr crops are perplexing in light of the considerable body of literature and yield data that contradict such claims . nevertheless , there might be effects of glyphosate in gr crops on mineral nutrition and/or disease under particular but uncommon conditions ( e.g. , specific soil , environmental conditions , particular gr crop cultivars , and/or glyphosate formulations ) .

claims have been made recently that glyphosate - resistant ( gr ) crops sometimes have mineral deficiencies and increased plant disease . this review evaluates the literature that is germane to these claims . our conclusions are : ( 1 ) although there is conflicting literature on the effects of glyphosate on mineral nutrition on gr crops , most of the literature indicates that mineral nutrition in gr crops is not affected by either the gr trait or by application of glyphosate ; ( 2 ) most of the available data support the view that neither the gr transgenes nor glyphosate use in gr crops increases crop disease ; and ( 3 ) yield data on gr crops do not support the hypotheses that there are substantive mineral nutrition or disease problems that are specific to gr crops .

Introduction Glyphosate in Soil: Bioavailabiltiy, Degradation, and Persistence Glyphosate Degradation, Persistence, and Leaching Glyphosate and Mineral Nutrition of Plants Glyphosate Effects on Soil Microflora Effects of Glyphosate on Plant Disease in Glyphosate-Resistant Crops Yields of Glyphosate-Resistant Crops

since the herbicide glyphosate ( n-(phosphonomethyl)glycine ) was commercialized in 1974 , it has become the most widely used herbicide in the world , due largely to the wide scale adoption of transgenic , glyphosate - resistant ( gr ) crops after their introduction in 1996 ( figure 1 ) . in gr crops , this relatively high use rate herbicide ( commonly 0.5 to 2.0 kg / ha / application ) is often used multiple times in a growing season . use of other herbicides declined steadily , while glyphosate use increased in the three major gr crops ( figure 2 ) . the increasing incidence of evolved , gr weeds , as well as weed shifts to naturally glyphosate - tolerant weed species , has resulted in increased use rates and numbers of applications of glyphosate , as well as other herbicides , per growing season in gr crops . adoption of the three most widely grown , herbicide - resistant ( hr ) crops in the united states . glyphosate is a nonselective herbicide , that is , it can kill all plant species , although there is variation between species with regard to levels of natural tolerance . during its several decades of use over vast areas , no significant adverse secondary effects of the herbicide have been established , other than the intense selection pressure that has resulted in the evolution of gr weeds . in fact , its use in gr crops has been associated with several environmental benefits . several papers have been published recently that conclude that glyphosate adversely affects mineral nutrition in gr crops , leading to several adverse effects , including increased plant disease . this review addresses these concerns in the context of the available literature on glyphosate ( ca . in order to understand possible effects of glyphosate on mineral nutrition of plants , it is necessary to understand the processes that affect glyphosate in soil . this topic is discussed in more detail in the section below on the effects of glyphosate on mineral nutrition of plants . in bioassay experiments using tomato plants and white spruce seedlings , soils containing saturated solutions of glyphosate - metal complexes had little or no effect on the plants . since some microorganisms are sensitive to glyphosate ( see this review ) , the degradation of glyphosate in situ represents the activity of the glyphosate - degrading microbial community modulated by relative resistance or sensitivity to the herbicide . in addition to the direct application of glyphosate to soil , any glyphosate remaining in gr crop residues will be released to the soil as those crop residues degrade . compared the bound residue formed from several pesticides and found that glyphosate - bound residues generally accounted for less than 20% of the initial c added , equivalent to trifluralin , but less than those formed after application of the c - labeled herbicides metazachlor , metamitron , and sulcotrione . multiple applications of glyphosate in gr - cropping systems would ( 1 ) increase the risk of carryover , especially in regions where soils are seasonally frozen for extended periods and ( 2 ) increase the risk of leaching to tile drains or groundwater . any increased persistence is potentially important in cropping systems where glyphosate - sensitive ( gs ) crops closely follow the gr - crop . recent reports of mineral deficiencies in gr crops after glyphosate application were linked with claims of increased susceptibility to plant diseases . there are conflicting papers on the effects of glyphosate on mineral nutrition on gr crops . examination of glyphosate levels in glyphosate - treated gr soybean seeds at maturity and mineral levels in soybean seed shows that on a molar basis the metal : glyphosate ratio can be from almost 10 000 times more mn to around 100 000 times more minerals such as mg or ca compared to glyphosate . these large ratios do not support the view that the chelator properties of glyphosate would interfere substantially with plant mineral nutrition in planta . in contrast with some descriptions of glyphosate as a strong chelator , the stability constants of glyphosate , edta , and citric acid with common micronutrient ions show that glyphosate is a weak chelator ( table 1 ) . the fact that the relative concentrations of metal cations in soil are several orders of magnitude greater ( in terms of moles of metals per ha vs moles of glyphosate per ha ) than the highest concentrations of glyphosate that could be expected ( discussed in detail in a previous section ) , significant effects of glyphosate on soil mineral content or availability to plants are highly unlikely . from the earliest days of glyphosate use , it was known that using water containing high levels of metal ions would significantly reduce the efficacy of the herbicide , presumably because the precipitated or chelated herbicide is not taken up by target plants as well as the free glyphosate anion and/or precipitation of glyphosate : mineral complexes ( reviewed by duke , sundaram and sundaram , and nilsson ) . thus , studies on effects of glyphosate on mineral nutrition of plants should not be conducted with combined spray solutions of minerals and glyphosate . however , the finding that gr crops with only a change in their epsps are about 50-fold less sensitive to glyphosate than similar gs crops indicated that mineral nutrition is not involved in the mode of action of glyphosate . nilsson found no effects of glyphosate on mn , zn , or cu content of gs wheat leaves . found inhibitory effects of glyphosate on root ferric reductase in iron - deficient gs sunflower . it may be that some of the confusion regarding glyphosate effects on mineral nutrition of gr crops is due to studies on gs plants that can not be extrapolated to gr plants . gr crops are not necessarily resistant to ampa , as its mode of action is not the same as that of glyphosate . yellow flash symptoms have not been reported in gr crops other than soybean , perhaps because sufficient levels of ampa to cause such symptoms do not accumulate in other gr crops or there is insufficient sensitivity of these crops to ampa . little is known of ampa in gr crops , including its mechanism of phytotoxicity . soybean cultivars for areas with low phytoavailable soil mn have been developed , and farmers are advised to plant more mn deficiency - resistant cultivars on such soils . as breeders worked to solve this susceptibility problem ( much like the case of fe chlorosis susceptibility of the early gr soybean cultivars ; see below ) , improved cultivars with the gr trait were also resistant to mn deficiency . there are several peer - reviewed journal claims of effects of glyphosate on mineral nutrition in gr soybean . at very high application rates of glyphosate , mn concentrations in the tissue of the gr cultivar were reduced about 50% . there were no effects of glyphosate on mn and fe content of plant tissues when the plants were grown in two different soil types , although there was a reduction in insoluble foliar zn in one of the soil types . taken together , the data of this study show no adverse effect of glyphosate on mn uptake or translocation in gr - soybeans . found no effect of the gr transgene of gr soybean on mn uptake or response to mn in the absence of glyphosate . found no effects of foliar application of glyphosate on mn absorption , accumulation , or distribution in gr soybeans . no effects of glyphosate on translocation of these metal ions were seen up to 2.5 kg / ha . found no effects of glyphosate on mineral nutrition in gr soybean seeds , compared to an untreated near - isogenic soybean line , indicating no effect from the epsps transgene or from glyphosate . the application of glyphosate to gr soybean had no effect on leaf mineral content ( mn , fe , cu , and zn ) or yield at two different sites in brazil . the results of all of these studies indicate that glyphosate does not restrict the availability of micronutrients in glyphosate - treated , gr crops . thus , the results of the three research groups that have reported glyphosate effects on mineral nutrition in gr crops are counter to those of nine other research groups . found no effect of two applications of glyphosate on nickel content of leaves or seed ( figure 6 ) of gr soybean , another report notes that glyphosate use on gr - soybeans in a brazilian study caused a significant reduction in plant n - fixation and a decline in leaf ni . there are numerous studies on the compositional ( chemical and nutritional ) equivalence of gr crops with gs crops , including mineral content , although the intent of these papers was to evaluate the effect of the transgene(s ) on composition , rather than the effect of glyphosate treatment on gr crops . in most of the published studies no mention is made of whether glyphosate was used on the crop . no effects of the gr transgenes or glyphosate application on mineral content have been found in these field studies conducted under good laboratory practices that usually involved multiple years and locations . however , these studies lacked comparisons of glyphosate - treated with untreated crops to allow evaluation of the glyphosate effect , independent of the genetic effect of the gr technology . clearly , glyphosate can have effects on mineral nutrition of gs plants through its herbicidal effects on plant roots and other parts of the plant . published data on the effects of glyphosate on mineral nutrition of gr crops are contradictory . three groups have claimed adverse effects on mineral nutrition in gr crops in peer - reviewed journals the zobiole et al . the peer - reviewed results of nine laboratories show no effect of glyphosate on mineral nutrition . rigorous field studies on different soil types ( including those highly susceptible to inducing mn or fe deficiency in soybeans ) are needed to resolve the issue of whether glyphosate might have adverse effects on mineral nutrition of gr crops . considering the available data , growers are unlikely to need mn fertilizers just because they use glyphosate on gr soybeans . evaluation of glyphosate effects on soil microorganisms requires knowledge of the direct effects of glyphosate and its metabolites on soil microorganisms as well as effects on microorganisms through processes mediated by plants on root symbionts and rhizosphere microorganisms . addition of aromatic amino acids to bacterial cultures can partially or fully reverse the effects of glyphosate . the effects of glyphosate on microorganisms in soil have been extensively investigated using a variety of techniques . these concentrations of glyphosate seem sufficient to induce glyphosate toxicity ; a hypothetical application of 50 mg kg glyphosate soil at 25% gravimetric water content would result in a 1.18 mm aqueous concentration in a thin layer at the surface of the soil . rates of soybean litter decomposition of the gr and conventional cultivars were nearly identical ; however , glyphosate reduced litter decomposition on the soil surface , but not on buried litter . several studies using different methods have examined the impact of glyphosate on the rhizosphere of gr crops . found that rhizosphere populations of denitrifying bacteria and fungi were not affected by glyphosate application to gr - corn compared to gr - corn treated with conventional herbicides or a gs corn isoline treated with conventional herbicides . also used fame biomarkers to examine the effects of two postemergent glyphosate applications to gr - soybeans grown in soil with and without a history of previous glyphosate use . the decrease in microbial biomass at 7 days after application does not support the conjecture that glyphosate treatment increases root exudation . (167 ) zablotowicz and reddy summarized the effects of glyphosate on soybean nodulation and n fixation . among strains of b. japonicum , glyphosate tolerance in culture multiple field studies show no effect of glyphosate on gr - soybean yield . these effects on nodulation and n fixation may be due in part to the inhibitory effects of glyphosate on b. japonicum , but may also be related to gr cultivar responses to glyphosate . the survival of b. japonicum in soil without plants was not affected by concentrations equivalent to 1x or 10x field application rates of glyphosate . other research has shown that in the tripartite symbiosis of mycorrhiza , rhizobium , and soybean , no adverse effects of glyphosate use on gr cultivars was observed . these studies indicate that effects of glyphosate on plant mineral nutrition through effects on amf are unlikely . a summary of possible effects of glyphosate on plant defenses derived from the shikimic acid pathway in shown in figure 8 . based on both known and potential effects of glyphosate on disease defense compounds , it is not surprising to find reports in the published literature that show the disease - enhancing effects of glyphosate on gs plants ( e.g. possible effects of glyphosate treatment of glyphosate - senstive plants on shikimic acid pathway metabolites considered to be important in defense . of these , only isoflavonoid phytoalexins from bean and soybean and lignin deposition in bean have been examined for effects of glyphosate ( and only in gs plants ) . tested two near - isogenic lines of soybean gl2415 ( gs ) and gl2600rr ( gr ) for susceptibility to sclerotinia sclerotiorum , the cause of the sclerotinia stem rot or white mold disease . the effects of glyphosate and glyphosate resistance in relation to sudden death syndrome ( sds ) , in soybean caused by fusarium virguliforme ( formerly fusarium solani f. sp . reported on the effects of glyphosate and two other soybean herbicides ( lactofen and imazethapyr ) on sds response in two gr soybean lines ( pioneer 9344 and asgrow 3701 ) and one gs line ( bsr101 ) . the effects of glyphosate treatments on sds were examined in ten gr soybean lines from a variety of maturity groups . per gram of dried soil increased after application of glyphosate , but gs crops ( corn , pea , cucumber , and bean ) subsequently grown on in the field were not affected . examined the effect of glyphosate and other herbicides on reaction of bsr 101 ( gs ) and pioneer 93b01 ( gr ) to rhizoctonia solani . in sugar beet , gr varieties were tested for the effects of glyphosate treatment on expression of disease caused by rhizoctonia solani and fusarium oxysporum f. sp . in greenhouse studies , several pre - emergent herbicides predisposed cotton seedlings to greater hypocotyl infection by r. solani , but subsequent application of glyphosate did not increase severity of the disease . they found that there was a systemic effect of glyphosate on rust development as illustrated by reduced urediniospore germination and appressorium formation on tissues that were not directly treated with the herbicide . holliday and keen examined the effect of glyphosate on the response of gs soybean leaves to the bacterial pathogen pseudomonas syringae pv glycinea . although they did not test the effect of glyphosate on the host response to xanthomonas , the authors did recommend that growers assess the risk for this disease and plant resistant cultivars when the disease is likely to occur . there was no mention in any of the recently published reports that the gr trait or glyphosate application is a contributing factor to the increase in goss s wilt . considering that most corn produced in the us is now gr ( figure 1 ) , it is likely that inoculum buildup and use of gr corn that is not resistant to goss s wilt are the reasons for increases in this disease . in addition , there are no reports in the published literature that suggest that glyphosate resistance or treatment of gr varieties with the herbicide will increase the risk of other diseases in this crop . examined the effect of glyphosate on soybean cyst nematode ( scn , heterodora glycines ) infection of the gr and scn - resistant variety countrymark 316 . they reported that glyphosate did result in increased numbers of the nematode on the susceptible line , but not the resistant line . overall , it appears that in gr crops the baseline disease resistance or susceptibility of the host plant , not the presence of the glyphosate resistance gene or treatment with glyphosate , is the major contributor to susceptibility . thus , one might expect that if there were any significant mineral nutrition and/or disease problems with these crops , the problems would be manifested in yield reductions and farmer dissatisfaction . yield data from the years before introduction of gr crops , continuing to the present show that the same yield trends before introduction continued after introduction ( e.g. while there could be isolated pockets of adverse effects of glyphosate on gr crops that would be masked by their general success , such cases have not been conclusively documented . there were initial concerns with transgenic crops in general that there would be yield drags due to factors not associated with disease or mineral nutrition , but to suboptimal cultivars and potential pleiotrophic effects of the transgenes . to summarize , yield data for crops that are now predominantly gr cultivars do not support the view that there are significant mineral nutrition or disease problems with gr crops . scientific accounts about increased plant disease and mineral nutrition problems in gr crops are based on publications from a limited number of researchers . in the context of the entire body of relevant science still , considering the enormous importance of and reliance on gr crops and glyphosate , there has been a paucity of publically funded research into potential problems with this weed management technology . farmers have generally embraced this technology , so that there has been no widespread call for studies of potential problems with gr crops other than those associated with gr weeds , a growing problem that is well documented . reports of significant adverse effects of glyphosate on mineral nutrition and diseases of gr crops are perplexing in light of the considerable body of literature and yield data that contradict such claims . nevertheless , there might be effects of glyphosate in gr crops on mineral nutrition and/or disease under particular but uncommon conditions ( e.g.

respiratory syncytial virus ( rsv ) is currently considered the single most important cause of childhood respiratory infection.1,2 the world health organization reports four million deaths annually worldwide among children aged < 5 years due to the virus.3 the primary cause of severe lower respiratory tract infection ( lrti ) globally among infants aged < 2 years , the virus is the leading cause of hospitalizations for a lrti during respiratory virus season . premature infants with underdeveloped lungs and an immature immune system are particularly vulnerable to serious infections from rsv.1,2,46 the transmission of rsv is difficult to prevent because it is easily transmitted by unprotected coughing or sneezing , or contact with contaminated hands.7 as many as 60% of all children are infected with rsv in their first year of life and almost 100% within their second year of life.8 rsv does not stimulate a robust immunological response , therefore reinfection is common.9 unfortunately , once children are infected with the virus , there are no etiopathogenetic treatment options available . routine use of bronchodilators , epinephrine , steroids , and ribavirin has proven to be of no significant benefit ; therefore , treatment has been limited to supportive measures.10 palivizumab ( synagis ; medimmune inc , gaithersburg , md ; marketed outside the usa by abbott laboratories , abbott park , il ) is a humanized igg1 monoclonal antibody targeting the f protein of rsv . when administered monthly while rsv is circulating in the community , palivizumab has been shown to reduce severe rsv disease requiring hospitalization in high - risk children ( ie , those born 35 weeks gestational age ; those with bronchopulmonary dysplasia ; and infants with hemodynamically significant congenital heart disease who were less than two years of age at the start of rsv season ) , by approximately 50% compared to placebo.11,12 approved in the united states in 1998 , palivizumab received european approval in 1999 , and its approval in the russian federation was received in february 2010 . a review of literature published over a 30-year period regarding rsv seasonality and epidemiology disclosed that rsv season in the northern hemisphere roughly starts in september and ends in april.5,6 european countries , including those countries near russia , with published data , had similar rsv seasons that started as early as september , and ended as late as july.1324 in the nordic countries of finland , denmark , norway , and sweden , rsv season begins in october or november and continues through march or april , with peak activity in december / january.5,18,25 in norway , rsv has been reported through june , with later peak activity in january/ february.14 although the russian federation covers a very large territory , including arctic areas and subtropics , most of its population occupies temperate regions . the objective of this study was to describe the prevalence and seasonality of rsv infection in children aged 2 years , hospitalized for lrti between september 2008 and april 2009 , in three temperate regions of the russian federation . this prospective , multicenter , observational study was conducted at nine participating hospital centers located in three regions : seven in moscow ( central ) , one in saint petersburg ( northwest ) , and one in tomsk ( east ) . the study included parental written informed consent and received approval from the institutional ethics boards of all participating centers . the timing of the study , from september 2008 through april 2009 , allowed essential epidemiological information on rsv to be obtained during the cold season in the northern hemisphere when rsv is most prevalent . the plan was to enroll approximately 500 eligible children into the study over the period . the determination of lrti was based on a medical diagnosis of acute bronchiolitis , bronchitis , and/or pneumonia , and based on the presence of symptoms ( cough , coryza , rhinorrhea , fever , or retractions ) and signs ( wheezing , crackles , or rales ) . children with at least one symptom and a chest radiograph diagnostic of bronchiolitis , pneumonia , or bronchitis were also designated as having an lrti . additionally , apneic events with accompanying auscultatory findings , chest radiograph of lrti , clinical symptoms of fever , or coryza were designated an lrti episode . eligibility requirements also included age 2 years at the time of hospital admission and hospitalization < 24 hours prior to rsv testing . enrollment was prohibited if the child had received any product containing rsv - neutralizing antibodies ( including hyperimmunoglobulins ) within 100 days of enrollment , or had participated in a clinical trial for rsv treatment or prophylaxis prior to or during the hospitalization . physical examination was performed and a parent provided information regarding the child s personal and family medical and social history during a single screening and enrollment visit prior to rsv testing . the rsv diagnostic test method utilized was approved for use by regulatory authorities in the russian federation . nasopharyngeal lavage was collected and evaluated within 24 hours of hospitalization , using a rapid immunochromatographic rsv detection technique , quickstripe rsv ( savyon diagnositics ltd , ashdod , israel ) . two retests could be conducted within 24 hours of enrollment if the initial test results were inconclusive . a classification of children at high - risk for serious rsv infection was made based on a medical history of premature birth ( 35 weeks of gestational age ) ; chronic lung disease ( cld ) , including bronchopulmonary dysplasia ( bpd ) ; or congenital heart disease ( chd ) . the details of hospital stay and discharge were recorded from the hospital medical records using study - specific case report forms only for rsv - positive ( rsv+ ) children . no subsequent visits were required for any enrolled child and no interventions were assessed in the study . all children enrolled in the study formed the analysis population for primary and secondary analyses , irrespective of rsv diagnosis . the primary analysis , rsv prevalence , was determined by the number and proportion of rsv+ children ( 95% exact confidence interval [ ci ] for the proportion ) , calculated by dividing the total number of rsv+ children by the total number of children tested for rsv . the number and proportion of rsv+ children enrolled was calculated at weekly intervals and across the entire study period . calculations were performed for the total enrollment overall and for children from each of the three individual geographic areas . means and standard deviations were calculated for continuous variables . for categorical variables , the number and percentage of enrollees in each category within an assessment were calculated . length of hospital stay , use and duration of oxygen supplementation , use of mechanical ventilation , use and duration of continuous positive airway pressure ( cpap ) , admission to an intensive care unit ( icu ) , duration of stay at icu , and mortality were summarized descriptively . for continuous variables , comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection ( based on medical history as described in methods ) were made using a one - way analysis of variance ( anova ) with group as the only factor . for categorical variables , the number and percentage of children in each category within an assessment were calculated for non - missing data , and comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection were made using fisher s exact test . the determination of lrti was based on a medical diagnosis of acute bronchiolitis , bronchitis , and/or pneumonia , and based on the presence of symptoms ( cough , coryza , rhinorrhea , fever , or retractions ) and signs ( wheezing , crackles , or rales ) . children with at least one symptom and a chest radiograph diagnostic of bronchiolitis , pneumonia , or bronchitis were also designated as having an lrti . additionally , apneic events with accompanying auscultatory findings , chest radiograph of lrti , clinical symptoms of fever , or coryza were designated an lrti episode . eligibility requirements also included age 2 years at the time of hospital admission and hospitalization < 24 hours prior to rsv testing . enrollment was prohibited if the child had received any product containing rsv - neutralizing antibodies ( including hyperimmunoglobulins ) within 100 days of enrollment , or had participated in a clinical trial for rsv treatment or prophylaxis prior to or during the hospitalization . physical examination was performed and a parent provided information regarding the child s personal and family medical and social history during a single screening and enrollment visit prior to rsv testing . the rsv diagnostic test method utilized was approved for use by regulatory authorities in the russian federation . nasopharyngeal lavage was collected and evaluated within 24 hours of hospitalization , using a rapid immunochromatographic rsv detection technique , quickstripe rsv ( savyon diagnositics ltd , ashdod , israel ) . two retests could be conducted within 24 hours of enrollment if the initial test results were inconclusive . a classification of children at high - risk for serious rsv infection was made based on a medical history of premature birth ( 35 weeks of gestational age ) ; chronic lung disease ( cld ) , including bronchopulmonary dysplasia ( bpd ) ; or congenital heart disease ( chd ) . the details of hospital stay and discharge were recorded from the hospital medical records using study - specific case report forms only for rsv - positive ( rsv+ ) children . no subsequent visits were required for any enrolled child and no interventions were assessed in the study . all children enrolled in the study formed the analysis population for primary and secondary analyses , irrespective of rsv diagnosis . the primary analysis , rsv prevalence , was determined by the number and proportion of rsv+ children ( 95% exact confidence interval [ ci ] for the proportion ) , calculated by dividing the total number of rsv+ children by the total number of children tested for rsv . the number and proportion of rsv+ children enrolled was calculated at weekly intervals and across the entire study period . calculations were performed for the total enrollment overall and for children from each of the three individual geographic areas . length of hospital stay , use and duration of oxygen supplementation , use of mechanical ventilation , use and duration of continuous positive airway pressure ( cpap ) , admission to an intensive care unit ( icu ) , duration of stay at icu , and mortality were summarized descriptively . for continuous variables , comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection ( based on medical history as described in methods ) were made using a one - way analysis of variance ( anova ) with group as the only factor . for categorical variables , the number and percentage of children in each category within an assessment were calculated for non - missing data , and comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection were made using fisher s exact test . the number of centers increased gradually to nine by february 2009 , and continued through to the end of enrollment on april 26 , 2009 . three centers ( two in moscow and one in tomsk ) had two enrolling locations each . of 593 children aged 2 years assessed for inclusion in the study , 520 were enrolled . table 1 summarizes the demographic characteristics of all children enrolled , of the rsv+ subset , and of high - risk rsv+ individuals by location . children with rsv+ were predominantly full - term , white , males , of an appropriate weight for gestational age , and 7.5 months of age , on average , at hospitalization . these characteristics were similar to those of the total set ; however , the mean age at hospitalization in the rsv subset was 9.7 months . in physical examination conducted at enrollment , vital signs ( mean sd ) were similar among the subsets : respiratory rate ( 40.9 10.60 breathsmin ) and heart rate ( 130.5 12.88 bmin ) , oxygen saturation ( 96.0 2.60% ) , temperature ( 37.0 0.58c ) , length ( 68.9 10.09 cm ) and body weight ( 8.5 2.69 kg ) . during the period of september 11 , 2008 through april 26 , 2009 , 197 of 519 children enrolled were rsv+ ( 38.0% , 95% ci : 33.842.3 ) . the prevalence of rsv was highest in moscow , where 151 of 362 children enrolled were rsv+ ( 41.7% , 95% ci : 36.647.0 ) . in st . petersburg , rsv was identified in 19 of 50 children enrolled ( 38.0% , 95% ci : 24.752.8 ) , and 27 of 107 children enrolled were rsv+ ( 25.2% , 95% ci : 17.334.6 ) in tomsk . the onset of rsv occurred in moscow during week 44 ( late october ) of 2008 ( figure 1 ) . peak rsv activity occurred during week 14 of 2009 ( early april ) , when 62% of children with lrti enrolled were rsv+ ( 31/50 , 95% ci : 47.275.4 ) . during the following two weeks , at the end of the recruitment phase , the proportion of rsv+ children among those enrolled decreased to 39% ( 11/28 , 95% ci : 21.559.4 ) . the protocol - defined duration of the study did not allow for a determination of season offset . among high - risk rsv+ children , the length of hospital stay ranged from 413 days vs 137 days among rsv+ children without high - risk conditions . more of the rsv+ children at high risk for serious rsv infection required oxygen supplementation than the other rsv+ children not at high risk ( 28% v 10% , p = 0.04 ) . no mechanical ventilation , cpap , or surgeries were required and there were no deaths . in addition to those already presented , family and social history factors were identified and are described in table 2 . in the rsv+ subset at high risk for serious rsv infection , 14 children were born premature , two had a history of chd , and two had a history of cld / bpd . two criteria were identified for one high - risk child with rsv+ who was born premature and had a history of bpd . medical histories revealed one or more hospitalizations in the high - risk rsv+ subset ( 50% ) , compared with the rsv+ ( 25% ) and rsv ( 37% ) subsets not at high risk . similarly , in the high - risk rsv+ subset , 50% of the children had been admitted to an icu at birth , compared with 4% in the rsv+ and 7% in the rsv subsets not at high risk . the number of centers increased gradually to nine by february 2009 , and continued through to the end of enrollment on april 26 , 2009 . three centers ( two in moscow and one in tomsk ) had two enrolling locations each . of 593 children aged 2 years assessed for inclusion in the study , 520 were enrolled . table 1 summarizes the demographic characteristics of all children enrolled , of the rsv+ subset , and of high - risk rsv+ individuals by location . children with rsv+ were predominantly full - term , white , males , of an appropriate weight for gestational age , and 7.5 months of age , on average , at hospitalization . these characteristics were similar to those of the total set ; however , the mean age at hospitalization in the rsv subset was 9.7 months . in physical examination conducted at enrollment , vital signs ( mean sd ) were similar among the subsets : respiratory rate ( 40.9 10.60 breathsmin ) and heart rate ( 130.5 12.88 bmin ) , oxygen saturation ( 96.0 2.60% ) , temperature ( 37.0 0.58c ) , length ( 68.9 10.09 cm ) and body weight ( 8.5 2.69 kg ) . during the period of september 11 , 2008 through april 26 , 2009 , 197 of 519 children enrolled were rsv+ ( 38.0% , 95% ci : 33.842.3 ) . the prevalence of rsv was highest in moscow , where 151 of 362 children enrolled were rsv+ ( 41.7% , 95% ci : 36.647.0 ) . in st . petersburg , rsv was identified in 19 of 50 children enrolled ( 38.0% , 95% ci : 24.752.8 ) , and 27 of 107 children enrolled were rsv+ ( 25.2% , 95% ci : 17.334.6 ) in tomsk . the onset of rsv occurred in moscow during week 44 ( late october ) of 2008 ( figure 1 ) . peak rsv activity occurred during week 14 of 2009 ( early april ) , when 62% of children with lrti enrolled were rsv+ ( 31/50 , 95% ci : 47.275.4 ) . during the following two weeks , at the end of the recruitment phase , the proportion of rsv+ children among those enrolled decreased to 39% ( 11/28 , 95% ci : 21.559.4 ) . the protocol - defined duration of the study did not allow for a determination of season offset . among high - risk rsv+ children , the length of hospital stay ranged from 413 days vs 137 days among rsv+ children without high - risk conditions . more of the rsv+ children at high risk for serious rsv infection required oxygen supplementation than the other rsv+ children not at high risk ( 28% v 10% , p = 0.04 ) . no mechanical ventilation , cpap , or surgeries were required and there were no deaths . in addition to those already presented , family and social history factors were identified and are described in table 2 . in the rsv+ subset at high risk for serious rsv infection , 14 children were born premature , two had a history of chd , and two had a history of cld / bpd . two criteria were identified for one high - risk child with rsv+ who was born premature and had a history of bpd . medical histories revealed one or more hospitalizations in the high - risk rsv+ subset ( 50% ) , compared with the rsv+ ( 25% ) and rsv ( 37% ) subsets not at high risk . similarly , in the high - risk rsv+ subset , 50% of the children had been admitted to an icu at birth , compared with 4% in the rsv+ and 7% in the rsv subsets not at high risk . the prevalence of rsv during the 20082009 season in the russian federation was consistent with that expected in a northern temperate zone . both the prevalence of rsv and the peak of rsv activity were similar in the three participating cities . an abnormally warm autumn and a late winter cold - weather onset were recorded in temperate regions of the russian federation during the 20082009 season.26 this unexpected factor serves as a reminder that a single season may not be representative of the rsv season from year to year . considering the variability reported in published epidemiological studies , russia may have an epidemiology similar to that of finland , sweden , and norway , where later seasons are a regular occurrence not associated with the weather , and rsv peaks in march april.18 such variability is not only found in far north or northeastern european countries . in croatia , rsv seasons have two - year cycles , with seasons occurring from october to april one year and from december to june the next year , and with corresponding peaks in december january and march may.13,21 in ireland , rsv onset occurs in november and year - to - year , the rsv peaks vary in occurrence from december through february.27 the rsv peak reported in germany appears to be dependent upon the timing of rsv season onset , such that when onset is in december , peak activity has been reported in march.22,23 in italy , rsv onset occurs in december , peaks in march , and offsets in april.28,29 in turkey , rsv peaks in march , with onset in september and offset in may.24 the prospective design of this study contributed to the generalizability of results . all children with lrti hospitalized in the participating centers were regarded as potential candidates for the study ; therefore , the study was designed to represent the general population and to evaluate the prevalence of risk factors for rsv+ lrti . by design , demographic data , risk and protective factors , and other descriptive information of the study population were collected without bias before the rsv test was administered . demographic characteristics and risk factors for severe rsv infection identified in the russian federation were consistent with those in other developed countries where children most at risk are those born premature , with chronic lung disease , or with congenital heart disease.19,30 risk factors for hospitalization , such as sex ( male ) ; low birth weight ( < 10th percentile ) ; exposure to school - age children in the home ; daycare attendance ; failure to breast feed beyond two months ; cigarette smoke exposure ; crowded living conditions ; chronologic age < six months at the time of rsv exposure ; and being underweight at birth,31,32 were also factors in the children with rsv+ lrti . it is important to keep in mind that in previous studies , children born shortly before or during the rsv season were significantly more likely to be hospitalized for rsv+ lrti than children born during other periods of the year.31,32 these implications are key in that children who are at the highest risk of infection and complications leading to hospitalization are those most in need of prophylaxis . limitations of the study that may have influenced the results include the sensitivity of the rapid immunochromatographic rsv detection method ( 70%90% ) , leaving a potential 10%30% margin of error in identifying rsv infections . enrollments in st petersburg and tomsk were relatively low and each city had only one participating site , therefore the prevalence of rsv in those cities may not have been adequately captured . additional studies would be needed to provide a more precise definition of the rsv season than those provided by data collected during a single season . although it can be argued that such factors can be generalizable for all regions of the russian federation as a whole , the epidemiological data collected in this study remain both valuable and important as they are consistent with what has been documented globally and in europe concerning rsv . this study confirms that rsv is widespread among populations in three major temperate regions of the russian federation and is a significant cause of lrti leading to the hospitalization of young children . during one rsv season , september 2008 through april 2009 , enough data were obtained to evaluate the burden and need for rsv prophylaxis in high - risk infants . inclusion criteria ensured homogeneity of the population and objective , evidence - based medical diagnosis procedures were adequate to support a conclusion regarding the disease prevalence and seasonality . all of these factors contribute to the generalizability of the results for the purposes intended ; specifically , the preparation of health facilities to meet the burden of care and the effective timing of rsv prophylaxis with palivizumab . rsv was a major contributor to lrti hospitalization in the russian federation from october 2008 through april 2009 . its prevalence among hospitalized children two years of age was consistent with that in other developed countries . the 20082009 rsv season in the russian federation had an onset similar to other northern temperate zones . although the period of peak activity occurred in march april , later than previously reported in temperate regions of the northern hemisphere ( december january ) , it was not exceptional or inconsistent with ranges reported overall . the seasonality of rsv was similar with respect to onset and peak rsv activity between each of the three participating cities ( regions ) . the demographics and risk factors identified among children hospitalized with rsv were similar to those described outside of the russian federation . these data support the consideration of an effective time period for rsv control measures , including rsv prophylaxis in children at high risk for serious rsv infection requiring hospitalization .

backgroundrespiratory syncytial virus ( rsv ) is the leading cause of severe lower respiratory tract infections among infants and young children , and is responsible for an estimated four million deaths per year globally . a monthly injection of palivizumab has been used for prophylaxis of serious rsv infections among high - risk children in 71 countries since 1998 and approval for use in the russian federation was obtained in february 2010 . a recommendation for rsv prophylaxis in the russian federation would require knowledge of the prevalence and seasonality of rsv in that country.methodsin a prospective , multicenter , epidemiological study of the prevalence , seasonality , and peak occurrence of rsv infection , children aged 2 years hospitalized for lower respiratory tract infections in three regions of the russian federation , from september 2008 through april 2009 , were screened and tested for rsv using rapid immunochromatography of nasopharyngeal lavage . for subjects who were tested positive , hospitalization data were collected.resultsof 519 children aged 2 years enrolled from september 11 , 2008 through april 26 , 2009 , 197 tested positive for rsv ( 38.0% , 95% ci : 33.8 , 42.3 ) . the onset of the 20082009 rsv season in the russian federation occurred in late october 2008 , similar to what is observed in other northern temperate zones . peak activity occurred in early april 2009 , when 62% of children enrolled tested positive for rsv.conclusionthe prevalence of serious rsv infections in the russian federation is similar to the prevalence previously identified in other temperate zones of the northern hemisphere . the seasonality of disease shifted towards early spring , with peak activity later in the season , within a range reported in other countries . these data provide further evidence of serious rsv infection in children in the russian federation , as well as guidance for timing of seasonal rsv prophylaxis , especially among individuals at high risk for serious rsv infection .

Introduction Methods Study population Procedures Data analysis Results Enrollment Prevalence and seasonality of RSV Morbidity and mortality Risk and protective factors Discussion Conclusion

respiratory syncytial virus ( rsv ) is currently considered the single most important cause of childhood respiratory infection.1,2 the world health organization reports four million deaths annually worldwide among children aged < 5 years due to the virus.3 the primary cause of severe lower respiratory tract infection ( lrti ) globally among infants aged < 2 years , the virus is the leading cause of hospitalizations for a lrti during respiratory virus season . premature infants with underdeveloped lungs and an immature immune system are particularly vulnerable to serious infections from rsv.1,2,46 the transmission of rsv is difficult to prevent because it is easily transmitted by unprotected coughing or sneezing , or contact with contaminated hands.7 as many as 60% of all children are infected with rsv in their first year of life and almost 100% within their second year of life.8 rsv does not stimulate a robust immunological response , therefore reinfection is common.9 unfortunately , once children are infected with the virus , there are no etiopathogenetic treatment options available . routine use of bronchodilators , epinephrine , steroids , and ribavirin has proven to be of no significant benefit ; therefore , treatment has been limited to supportive measures.10 palivizumab ( synagis ; medimmune inc , gaithersburg , md ; marketed outside the usa by abbott laboratories , abbott park , il ) is a humanized igg1 monoclonal antibody targeting the f protein of rsv . when administered monthly while rsv is circulating in the community , palivizumab has been shown to reduce severe rsv disease requiring hospitalization in high - risk children ( ie , those born 35 weeks gestational age ; those with bronchopulmonary dysplasia ; and infants with hemodynamically significant congenital heart disease who were less than two years of age at the start of rsv season ) , by approximately 50% compared to placebo.11,12 approved in the united states in 1998 , palivizumab received european approval in 1999 , and its approval in the russian federation was received in february 2010 . a review of literature published over a 30-year period regarding rsv seasonality and epidemiology disclosed that rsv season in the northern hemisphere roughly starts in september and ends in april.5,6 european countries , including those countries near russia , with published data , had similar rsv seasons that started as early as september , and ended as late as july.1324 in the nordic countries of finland , denmark , norway , and sweden , rsv season begins in october or november and continues through march or april , with peak activity in december / january.5,18,25 in norway , rsv has been reported through june , with later peak activity in january/ february.14 although the russian federation covers a very large territory , including arctic areas and subtropics , most of its population occupies temperate regions . the objective of this study was to describe the prevalence and seasonality of rsv infection in children aged 2 years , hospitalized for lrti between september 2008 and april 2009 , in three temperate regions of the russian federation . this prospective , multicenter , observational study was conducted at nine participating hospital centers located in three regions : seven in moscow ( central ) , one in saint petersburg ( northwest ) , and one in tomsk ( east ) . the timing of the study , from september 2008 through april 2009 , allowed essential epidemiological information on rsv to be obtained during the cold season in the northern hemisphere when rsv is most prevalent . the rsv diagnostic test method utilized was approved for use by regulatory authorities in the russian federation . nasopharyngeal lavage was collected and evaluated within 24 hours of hospitalization , using a rapid immunochromatographic rsv detection technique , quickstripe rsv ( savyon diagnositics ltd , ashdod , israel ) . a classification of children at high - risk for serious rsv infection was made based on a medical history of premature birth ( 35 weeks of gestational age ) ; chronic lung disease ( cld ) , including bronchopulmonary dysplasia ( bpd ) ; or congenital heart disease ( chd ) . no subsequent visits were required for any enrolled child and no interventions were assessed in the study . all children enrolled in the study formed the analysis population for primary and secondary analyses , irrespective of rsv diagnosis . the primary analysis , rsv prevalence , was determined by the number and proportion of rsv+ children ( 95% exact confidence interval [ ci ] for the proportion ) , calculated by dividing the total number of rsv+ children by the total number of children tested for rsv . the number and proportion of rsv+ children enrolled was calculated at weekly intervals and across the entire study period . for continuous variables , comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection ( based on medical history as described in methods ) were made using a one - way analysis of variance ( anova ) with group as the only factor . for categorical variables , the number and percentage of children in each category within an assessment were calculated for non - missing data , and comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection were made using fisher s exact test . the determination of lrti was based on a medical diagnosis of acute bronchiolitis , bronchitis , and/or pneumonia , and based on the presence of symptoms ( cough , coryza , rhinorrhea , fever , or retractions ) and signs ( wheezing , crackles , or rales ) . the rsv diagnostic test method utilized was approved for use by regulatory authorities in the russian federation . nasopharyngeal lavage was collected and evaluated within 24 hours of hospitalization , using a rapid immunochromatographic rsv detection technique , quickstripe rsv ( savyon diagnositics ltd , ashdod , israel ) . a classification of children at high - risk for serious rsv infection was made based on a medical history of premature birth ( 35 weeks of gestational age ) ; chronic lung disease ( cld ) , including bronchopulmonary dysplasia ( bpd ) ; or congenital heart disease ( chd ) . the details of hospital stay and discharge were recorded from the hospital medical records using study - specific case report forms only for rsv - positive ( rsv+ ) children . all children enrolled in the study formed the analysis population for primary and secondary analyses , irrespective of rsv diagnosis . the primary analysis , rsv prevalence , was determined by the number and proportion of rsv+ children ( 95% exact confidence interval [ ci ] for the proportion ) , calculated by dividing the total number of rsv+ children by the total number of children tested for rsv . for continuous variables , comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection ( based on medical history as described in methods ) were made using a one - way analysis of variance ( anova ) with group as the only factor . for categorical variables , the number and percentage of children in each category within an assessment were calculated for non - missing data , and comparisons of rsv+ children that were at high risk and not at high risk for serious rsv infection were made using fisher s exact test . the number of centers increased gradually to nine by february 2009 , and continued through to the end of enrollment on april 26 , 2009 . of 593 children aged 2 years assessed for inclusion in the study , 520 were enrolled . table 1 summarizes the demographic characteristics of all children enrolled , of the rsv+ subset , and of high - risk rsv+ individuals by location . children with rsv+ were predominantly full - term , white , males , of an appropriate weight for gestational age , and 7.5 months of age , on average , at hospitalization . these characteristics were similar to those of the total set ; however , the mean age at hospitalization in the rsv subset was 9.7 months . during the period of september 11 , 2008 through april 26 , 2009 , 197 of 519 children enrolled were rsv+ ( 38.0% , 95% ci : 33.842.3 ) . the prevalence of rsv was highest in moscow , where 151 of 362 children enrolled were rsv+ ( 41.7% , 95% ci : 36.647.0 ) . petersburg , rsv was identified in 19 of 50 children enrolled ( 38.0% , 95% ci : 24.752.8 ) , and 27 of 107 children enrolled were rsv+ ( 25.2% , 95% ci : 17.334.6 ) in tomsk . the onset of rsv occurred in moscow during week 44 ( late october ) of 2008 ( figure 1 ) . peak rsv activity occurred during week 14 of 2009 ( early april ) , when 62% of children with lrti enrolled were rsv+ ( 31/50 , 95% ci : 47.275.4 ) . during the following two weeks , at the end of the recruitment phase , the proportion of rsv+ children among those enrolled decreased to 39% ( 11/28 , 95% ci : 21.559.4 ) . among high - risk rsv+ children , the length of hospital stay ranged from 413 days vs 137 days among rsv+ children without high - risk conditions . more of the rsv+ children at high risk for serious rsv infection required oxygen supplementation than the other rsv+ children not at high risk ( 28% v 10% , p = 0.04 ) . in the rsv+ subset at high risk for serious rsv infection , 14 children were born premature , two had a history of chd , and two had a history of cld / bpd . two criteria were identified for one high - risk child with rsv+ who was born premature and had a history of bpd . medical histories revealed one or more hospitalizations in the high - risk rsv+ subset ( 50% ) , compared with the rsv+ ( 25% ) and rsv ( 37% ) subsets not at high risk . similarly , in the high - risk rsv+ subset , 50% of the children had been admitted to an icu at birth , compared with 4% in the rsv+ and 7% in the rsv subsets not at high risk . the number of centers increased gradually to nine by february 2009 , and continued through to the end of enrollment on april 26 , 2009 . of 593 children aged 2 years assessed for inclusion in the study , 520 were enrolled . table 1 summarizes the demographic characteristics of all children enrolled , of the rsv+ subset , and of high - risk rsv+ individuals by location . children with rsv+ were predominantly full - term , white , males , of an appropriate weight for gestational age , and 7.5 months of age , on average , at hospitalization . these characteristics were similar to those of the total set ; however , the mean age at hospitalization in the rsv subset was 9.7 months . during the period of september 11 , 2008 through april 26 , 2009 , 197 of 519 children enrolled were rsv+ ( 38.0% , 95% ci : 33.842.3 ) . the prevalence of rsv was highest in moscow , where 151 of 362 children enrolled were rsv+ ( 41.7% , 95% ci : 36.647.0 ) . petersburg , rsv was identified in 19 of 50 children enrolled ( 38.0% , 95% ci : 24.752.8 ) , and 27 of 107 children enrolled were rsv+ ( 25.2% , 95% ci : 17.334.6 ) in tomsk . the onset of rsv occurred in moscow during week 44 ( late october ) of 2008 ( figure 1 ) . peak rsv activity occurred during week 14 of 2009 ( early april ) , when 62% of children with lrti enrolled were rsv+ ( 31/50 , 95% ci : 47.275.4 ) . during the following two weeks , at the end of the recruitment phase , the proportion of rsv+ children among those enrolled decreased to 39% ( 11/28 , 95% ci : 21.559.4 ) . the protocol - defined duration of the study did not allow for a determination of season offset . among high - risk rsv+ children , the length of hospital stay ranged from 413 days vs 137 days among rsv+ children without high - risk conditions . more of the rsv+ children at high risk for serious rsv infection required oxygen supplementation than the other rsv+ children not at high risk ( 28% v 10% , p = 0.04 ) . in the rsv+ subset at high risk for serious rsv infection , 14 children were born premature , two had a history of chd , and two had a history of cld / bpd . two criteria were identified for one high - risk child with rsv+ who was born premature and had a history of bpd . medical histories revealed one or more hospitalizations in the high - risk rsv+ subset ( 50% ) , compared with the rsv+ ( 25% ) and rsv ( 37% ) subsets not at high risk . similarly , in the high - risk rsv+ subset , 50% of the children had been admitted to an icu at birth , compared with 4% in the rsv+ and 7% in the rsv subsets not at high risk . the prevalence of rsv during the 20082009 season in the russian federation was consistent with that expected in a northern temperate zone . both the prevalence of rsv and the peak of rsv activity were similar in the three participating cities . an abnormally warm autumn and a late winter cold - weather onset were recorded in temperate regions of the russian federation during the 20082009 season.26 this unexpected factor serves as a reminder that a single season may not be representative of the rsv season from year to year . considering the variability reported in published epidemiological studies , russia may have an epidemiology similar to that of finland , sweden , and norway , where later seasons are a regular occurrence not associated with the weather , and rsv peaks in march april.18 such variability is not only found in far north or northeastern european countries . in croatia , rsv seasons have two - year cycles , with seasons occurring from october to april one year and from december to june the next year , and with corresponding peaks in december january and march may.13,21 in ireland , rsv onset occurs in november and year - to - year , the rsv peaks vary in occurrence from december through february.27 the rsv peak reported in germany appears to be dependent upon the timing of rsv season onset , such that when onset is in december , peak activity has been reported in march.22,23 in italy , rsv onset occurs in december , peaks in march , and offsets in april.28,29 in turkey , rsv peaks in march , with onset in september and offset in may.24 the prospective design of this study contributed to the generalizability of results . all children with lrti hospitalized in the participating centers were regarded as potential candidates for the study ; therefore , the study was designed to represent the general population and to evaluate the prevalence of risk factors for rsv+ lrti . by design , demographic data , risk and protective factors , and other descriptive information of the study population were collected without bias before the rsv test was administered . demographic characteristics and risk factors for severe rsv infection identified in the russian federation were consistent with those in other developed countries where children most at risk are those born premature , with chronic lung disease , or with congenital heart disease.19,30 risk factors for hospitalization , such as sex ( male ) ; low birth weight ( < 10th percentile ) ; exposure to school - age children in the home ; daycare attendance ; failure to breast feed beyond two months ; cigarette smoke exposure ; crowded living conditions ; chronologic age < six months at the time of rsv exposure ; and being underweight at birth,31,32 were also factors in the children with rsv+ lrti . it is important to keep in mind that in previous studies , children born shortly before or during the rsv season were significantly more likely to be hospitalized for rsv+ lrti than children born during other periods of the year.31,32 these implications are key in that children who are at the highest risk of infection and complications leading to hospitalization are those most in need of prophylaxis . limitations of the study that may have influenced the results include the sensitivity of the rapid immunochromatographic rsv detection method ( 70%90% ) , leaving a potential 10%30% margin of error in identifying rsv infections . enrollments in st petersburg and tomsk were relatively low and each city had only one participating site , therefore the prevalence of rsv in those cities may not have been adequately captured . additional studies would be needed to provide a more precise definition of the rsv season than those provided by data collected during a single season . although it can be argued that such factors can be generalizable for all regions of the russian federation as a whole , the epidemiological data collected in this study remain both valuable and important as they are consistent with what has been documented globally and in europe concerning rsv . this study confirms that rsv is widespread among populations in three major temperate regions of the russian federation and is a significant cause of lrti leading to the hospitalization of young children . during one rsv season , september 2008 through april 2009 , enough data were obtained to evaluate the burden and need for rsv prophylaxis in high - risk infants . inclusion criteria ensured homogeneity of the population and objective , evidence - based medical diagnosis procedures were adequate to support a conclusion regarding the disease prevalence and seasonality . all of these factors contribute to the generalizability of the results for the purposes intended ; specifically , the preparation of health facilities to meet the burden of care and the effective timing of rsv prophylaxis with palivizumab . rsv was a major contributor to lrti hospitalization in the russian federation from october 2008 through april 2009 . the 20082009 rsv season in the russian federation had an onset similar to other northern temperate zones . although the period of peak activity occurred in march april , later than previously reported in temperate regions of the northern hemisphere ( december january ) , it was not exceptional or inconsistent with ranges reported overall . the seasonality of rsv was similar with respect to onset and peak rsv activity between each of the three participating cities ( regions ) . the demographics and risk factors identified among children hospitalized with rsv were similar to those described outside of the russian federation . these data support the consideration of an effective time period for rsv control measures , including rsv prophylaxis in children at high risk for serious rsv infection requiring hospitalization .

body toxicity has been viewed as a health risk since the time of ancient egypt , when it was believed that stool putrefaction would lead to systemic disease . the ancient greek humoral theory of disease extended the concept of putrefaction to bile , phlegm , blood , and residues of food . in the late 1800s , metchnikoff hypothesized that intestinal toxins shortened lifespan . modern medicine embraced toxicity until at least the 1920s , when a colectomy was viewed as a cure for autointoxication . clearly , the notion of body toxicity as a health risk for disease development has been part of patient care for a long time . it therefore might be expected that various sectors of health care , and individuals within health care , might still embrace and promote the issue of body toxicity as a treatment target . the purpose of this commentary is to discuss the issue of toxicity in the context of chiropractic practice , including daniel david palmer s impression of toxicity , a consideration of toxicity regarding the liver and colon , bacterial endotoxemia , and a rational approach to dietary detoxification in the context of addressing bacterial endotoxemia . chiropractors routinely cite palmer in this regard : in his 1910 text , the chiropractor s adjuster , dd palmer identified the causes of vertebral subluxation as the three ts thoughts , trauma and toxins . although this statement is commonly attributed to palmer , did he actually mention the so - called 3 t 's ? senzon explains that thoughts , trauma , and toxins have been described for years as the three causative agents of vertebral subluxation . however , we are further told by senzon that palmer never actually stated that subluxation is caused by the 3 t 's . senzon properly quotes palmer s original statement , which had nothing to do with subluxation specifically . palmer stated that the determining cause of disease are traumatism , poison , and autosuggestion . palmer also made the following statement : impingements , poisons and intense thinking , auto - suggestion , unrelieved change of thot , insufficient rest and sleep , increase or decrease the momentum of impulses . in the study of pathology we should look to the etiological factors which , by their exciting or debilitating effects , retard or liberate stored up energy , resulting in abnormal functioning and morbid structure . palmer defined poison as follows : poison is any animal , vegetable , or mineral substance which when applied externally or taken into the body by ingestion or injection , causes such a change in the animal economy as to produce abnormal functioning , disease or death . palmer uses the terms poison and toxin interchangeably in several places in his text ; however , in the index he cites only 1 page for toxin , wherein he describes strychnine as a poison or toxin . the commentary from each page is quoted here : page 77 : why and how luxations of the vertebrae are caused by poisons , vaccine virus , tobacco , alcohol , and ptomaine poison , is taught by actual clinical work.page 116 : poisons are substances which , when introduced into the body , either impair the function of one or more of its organs or destroy life . all poisons lead to a disturbance of the nervous system , no matter by what avenue it is introduced . the lesions due to poisons may be local , as those from corrosives or caustics . in others the place of entrance is not affected , the pathological manifestations being due to nervous disturbance the effect of a poison depends upon the nature of the substance , the amount and the individual.page 124 : poison destroys that which intelligent life has accomplished ; changes physiological to pathological action.page 147 : in the context of abnormal tone in the nervous system , palmer states that this prerequisite to disease is due to pressure , poison or lack of control of the emotions and acts of the patient , a morbid self-consciousness.page 554 : toxicosis is a disease caused by poisons ; a toxic or poisoned state ; the conditions of disease induced by poisons . palmer then gives examples of poisons as being chloroform , chloral , alcohol , digitalis , strychnine , nicotine , picrotoxin , veratrum , and laudanum.page 675 : poisons are not gathered in the tissues or cavities of the body . poison is any animal , vegetable , or mineral substance which when applied externally , or taken into the stomach , or injected into the body by physician or reptile , causes such a change in the animal economy as to produce abnormal functioning , disease or death . a chemical may be poisonous or otherwise.page 835 : in the context of developing typhoid fever , palmer states , the predisposing cause is poison from some decaying animal or vegetable matter . nerves sense this poison and as a consequence vertebrae are drawn out of alignment impinging upon the ganglionic chain of the sympathetic nervous system.page 955 : irritant or acrid poisons are those which produce irritation or inflammation . sedative poisons are those which directly reduce vital powers.page 974 : the osteopath and allopath believe poisons to be the cause of fevers . the chiropractor finds bone pressure on nerves the direct cause ; however , these bones may have been drawn out of alignment by poisons . page 77 : why and how luxations of the vertebrae are caused by poisons , vaccine virus , tobacco , alcohol , and ptomaine poison , is taught by actual clinical work . page 116 : poisons are substances which , when introduced into the body , either impair the function of one or more of its organs or destroy life . all poisons lead to a disturbance of the nervous system , no matter by what avenue it is introduced . the lesions due to poisons may be local , as those from corrosives or caustics . in others the place of entrance is not affected , the pathological manifestations being due to nervous disturbance the effect of a poison depends upon the nature of the substance , the amount and the individual . page 124 : life is action . page 147 : in the context of abnormal tone in the nervous system , palmer states that this prerequisite to disease is due to pressure , poison or lack of control of the emotions and acts of the patient , a morbid self - consciousness . toxicosis is a disease caused by poisons ; a toxic or poisoned state ; the conditions of disease induced by poisons . palmer then gives examples of poisons as being chloroform , chloral , alcohol , digitalis , strychnine , nicotine , picrotoxin , veratrum , and laudanum . page 675 : poisons are not gathered in the tissues or cavities of the body . poison is any animal , vegetable , or mineral substance which when applied externally , or taken into the stomach , or injected into the body by physician or reptile , causes such a change in the animal economy as to produce abnormal functioning , disease or death page 835 : in the context of developing typhoid fever , palmer states , the predisposing cause is poison from some decaying animal or vegetable matter . nerves sense this poison and as a consequence vertebrae are drawn out of alignment impinging upon the ganglionic chain of the sympathetic nervous system . page 955 : irritant or acrid poisons are those which produce irritation or inflammation . page 974 : the osteopath and allopath believe poisons to be the cause of fevers . the chiropractor finds bone pressure on nerves the direct cause ; however , these bones may have been drawn out of alignment by poisons . the statements on pages 835 and 974 indicate that poisons , not toxins , cause bones to be drawn out of alignment and impinge upon the spinal nerves . thus , although palmer never specifically said that toxins cause a misalignment type of subluxation , the implication is arguable based on these statements . however , to date , research is lacking that would otherwise support the notion that an individual vertebra can be pulled out of alignment in the fashion posited by palmer . furthermore , a correction of a supposed misalignment subluxation by manipulation has yet to be identified . in fact , the use of premanipulation and postmanipulation radiographs by hart and magnetic resonance images by cramer et al suggest that adjustments do not correct misalignments . this requires doctors of chiropractic ( dcs ) to consider the issue of toxins in a subluxation context that is not related to misalignments . a more functional view is needed , which may involve inflammation and nociceptive processes , rather than pathoanatomical changes.7 , 8 , 9 it is important to note that palmer never described which specific toxins , if any , could promote misalignment subluxations . current discussions about toxins and spinal subluxation also do not implicate specific toxins.2 , 3 in other words , the terms toxins and related toxicity have been consistently described in an imprecise and nebulous fashion in the context of spinal subluxation . nonetheless , many dcs maintain the general perception that an overexposure to yet unnamed toxins can prevent patients from holding adjustments . a similar nebulous scenario applies to colon and liver toxicity , for which nutritional interventions are recommended . no study has yet to define a specific toxin in the colon or liver that then responds to a specifically related detoxification nutritional protocol . this remains the challenge for those within chiropractic who embrace detoxification as a clinical tool . colon cleansing for the purpose of reducing intestinal toxicity and improving systemic health was popularized by a dc , bernard jensen , who wrote a text titled tissue cleansing through bowel management . the 1-week cleansing process involved taking several doses of psyllium husk powder per day , consuming vegetable broths , and supplementing with chlorella . the ingested psyllium is claimed to remove built - up toxic debris , aided by a daily colema , which is a variation of an enema and colonic . after the cleansing period , the recommendation is to avoid processed foods . in other words , the dietary recommendation after the cleanse is to eat more vegetation and avoid refined sugar , flour , and omega-6 fatty acids , which has been termed an anti - inflammatory diet.11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 a pubmed search using the phrase colon cleansing and health leads to 87 published articles , most of which involve cleansing before a colonoscopy . one author criticizes the unfounded claims by those who promote colon cleansing : colon cleansing is a popular therapy among alternative practitioners , but many myths surround it . the scientifically inaccurate way many traditional healers try to explain therapeutic mechanisms is one of the obstacles that inhibits dialog between traditional healers and practitioners of modern medicine . the therapy actually has clinical value , but the explanations used to promote the therapies are the stuff of herbal legends , not scientific fact . colon cleansing is a popular therapy among alternative practitioners , but many myths surround it . the scientifically inaccurate way many traditional healers try to explain therapeutic mechanisms is one of the obstacles that inhibits dialog between traditional healers and practitioners of modern medicine . the therapy actually has clinical value , but the explanations used to promote the therapies are the stuff of herbal legends , not scientific fact . because colon cleansing involves increasing fiber intake as well as eating more vegetables and fruit and avoiding refined sugar , flour , and omega-6 oils , inflammation is reduced . in other words , toxic foods such as refined carbohydrates and oils create inflammation and the diet , the beneficial outcome is inflammation reduction rather than physical or physiological detoxification of the human body . claims that toxic materials can be pulled off the walls of the intestine by fiber , such as powdered psyllium husk , are also inaccurate . the stools of those taking psyllium husk have been studied , and the larger stool mass was determined to contain psyllium fiber , rather than toxins . in other words , the more psyllium one takes , the more psyllium that will be eliminated , leading individuals to misinterpret the eliminated psyllium to be built - up toxins . because we lack data to support the notion that the intestines can be cleansed of built - up debris and toxins , the appropriate choice for promoting intestinal health is to eat fiber - rich vegetation and perhaps supplement with psyllium husk powder to help regulate bowel function . in particular , supplemental psyllium has been reported to be of benefit for patients with constipation , diarrhea , irritable bowel syndrome , inflammatory bowel disease , colon cancer , and type 2 diabetes . the liver has also been targeted as an organ in need of detoxification to help reduce chronic fatigue , food allergies , migraine headaches , fibromyalgia , and generalized arthralgias , which are common presentations in chiropractic practice . practitioners in both the medical and chiropractic professions are guilty of embracing the concept of liver toxicity , despite the lack of scientific evidence . to date , no studies have ever identified specific toxins in the liver that are detoxified by a specific nutritional detoxification program to help such conditions . this approach was popularized by bland et al and appears to have begun with an article published in the journal of applied nutrition . this study argued that a powdered supplement consisting of rice protein , rice syrup solids , and a low - dose multivitamin had the ability to upregulate hepatic detoxification , yet no measures of toxicity were performed . in other words , no specific liver toxin was identified , nor has been identified , that was subsequently detoxified by this nutritional supplement . thus , the appropriateness or clinical relevance of this nutritional approach has yet to be demonstrated in the context of chiropractic practice and health care in general . despite this fact , dcs find this to be a topic of interest and promote this approach for use in clinical practice.26 , 27 , 28 as outlined earlier , the issue of toxicity as a cause of spinal dysfunction , and as an intestinal and liver condition , is of therapeutic interest to dcs . the general topic of body cleansing and detoxification also appears to be of great interest to the general public . a google search performed on june 20 , 2016 , using the phrases detox diet and body detoxification resulted in 13 100 000 and 12 900 000 results , respectively . the challenge thus far , from a scientific and clinical perspective , is that the term detoxification is inappropriately used in a vague and general sense . we supposedly need to detoxify our bodies because air , water , and food contain toxins ; however , no specific toxins have been implicated and measured . in contrast , bacterial endotoxin may be a candidate toxin to consider as a cause of spinal dysfunction , as a therapeutic target to restore intestinal and liver health , and as a promoter of other common conditions encountered in chiropractic practice . the human gastrointestinal tract contains gram - negative bacteria , of which lipopolysaccharide is a structural component of the outer cell membrane . lipopolysaccharide is also known as bacterial endotoxin , which circulates at very low levels in normal disease - free humans ; however , this changes as individuals become unhealthy : although endotoxin ( lipopolysaccharide ( lps ) ) , derived from the cell wall of gram - negative bacteria , circulates at low concentrations in the blood of healthy individuals , the presence of genetic and diet - induced obesity and other metabolic disorders has been associated with a substantial increase in lps concentrations , a condition termed metabolic endotoxemia . although endotoxin ( lipopolysaccharide ( lps ) ) , derived from the cell wall of gram - negative bacteria , circulates at low concentrations in the blood of healthy individuals , the presence of genetic and diet - induced obesity and other metabolic disorders has been associated with a substantial increase in lps concentrations , a condition termed metabolic endotoxemia . the only source of circulating endotoxin can be from the gastrointestinal tract because that is where gram - negative bacteria reside in the human body , which speaks to the importance of maintaining gastrointestinal tract health to promote a homeostatic or symbiotic relationship with our intestinal microbiota . the large intestine appears to be adapted to the presence of high concentrations of bacteria ; compared with the small intestine , it has a relatively small surface area and is not designed for macronutrient and micronutrient absorption , save for short chain fatty acids , vitamin k , sodium , and water . because the small intestine is where the vast majority of nutrients are absorbed , it is supposed to contain far less bacteria.32 , 33 in other words , we are supposed to absorb nutrients and not bacterial components from the small intestine.32 , 33 consider that jejunal cultures may not identify any bacteria in as many as 33% of healthy volunteers . although the jejunum contains bacteria , the concentration should not exceed 10 organisms per milliliter , which is also considered the normal concentration of bacteria in the ileum . in contrast , in the terminal ileum near the ileocecal valve , bacterial concentrations range from 10 to 10 organisms per milliliter , whereas colonic concentrations can reach 10 . consequently , bacterial levels in the small intestine that exceed 10 per milliliter have been referred to as uninvited guests . the diagnosis of small intestinal bacterial overgrowth ( sibo ) is applied to patients when the concentration of bacteria exceeds 10 organisms per milliliter in the jejunum and ileum.33 , 34 not surprisingly , patients with sibo have increased circulating levels of bacterial endotoxin.35 , 36 whether a patient develops sibo or not , research has demonstrated that it is possible to create a state of low - grade metabolic endotoxemia based on poor dietary choices . for example , when normal - weight participants , with an average body mass index of 23 , were fed a typical american breakfast including a cup of tea , 3 slices of toast , and butter , which amounted to 900 calories , lipopolysaccharide levels increased significantly compared with controls , indicating that this common breakfast promotes low - grade endotoxemia . a subsequent study indicated that the overconsumption of calories from refined carbohydrates and fat is also associated with endotoxemia in apparently healthy men . more recently , plasma endotoxin levels increased by 71% when 8 healthy participants were placed on a western dietary pattern characterized by higher intakes of refined grains , sugar , processed meats , and red meat . in contrast , there was a 31% reduction in endotoxin when participants consumed a prudent - style diet characterized by higher intakes of fruits , vegetables , fish , whole grains , and legumes . in these short - term studies , there were no reported symptomatic changes associated with low - grade endotoxemia , which suggests that acute diet - induced endotoxemic events in otherwise healthy individuals go unnoticed . the transition from acute to chronic endotoxemia , and the point at which symptoms may appear , is not clear and obvious , which can be confusing for patients and clinicians and complicates the identification of a cause - effect relationship . in the absence of overt diseases that cause sibo , such as achlorhydria , diabetic autonomic neuropathy , scleroderma , and crohn disease , the most common cause promoter of bacterial overgrowth is a diet that is low in fiber and rich in refined sugar , flour , and oils . the chronic consumption of such a diet will simultaneously reduce gastrointestinal tract motility and feed gastrointestinal tract bacteria , allowing them to overgrow in the small intestine to varying degrees , which can promote chronic low - grade endotoxemia.31 , 41 , 42 whether varying degrees of sibo are related to all conditions that are associated with low - grade endotoxemia is not known . whether or not sibo develops , multiple conditions are known to be associated with elevated levels of circulating endotoxin , such as irritable bowel syndrome and fatty liver disease.32 , 41 , 43 , 44 , 45 this suggests that dcs can still view bowel and liver function in the context of toxicity , with the treatment goal being that of endotoxin reduction . regarding subluxation , no evidence speaks to a relationship with bacterial endotoxemia . however , bacterial endotoxemia is known to be a promoter of obesity , metabolic syndrome , type 2 diabetes , and depression , all of which are associated with an increased risk of back pain , which again permits a dc to embrace the notion that toxins may promote spinal dysfunction . additional common conditions that patients may present with to a chiropractic office and that are known to be promoted by chronic endotoxemia include chronic fatigue , anxiety , impaired cognition , migraine headache , impaired sleep , interstitial cystitis , type 2 diabetes , atherosclerosis , hypertension , general feeling of ill health , and widespread pain that may be diagnosed as fibromyalgia.31 , 32 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 bacterial endotoxemia is also associated with childhood obesity and may be a key contributor to type 2 diabetes and cardiovascular disease later in life . although assessing bacterial endotoxin levels is performed in the research setting , a clinical laboratory test is not available . this should not dissuade clinicians from embracing endotoxin as a candidate toxin in need of detoxification for a few reasons . first , as described earlier , we know that the consumption of refined sugar , flour , and oil leads to postprandial low - grade endotoxemia that has been measured in the clinical research setting.37 , 38 , 39 in other words , a patient who regularly eats these foods is measurably toxic . second , chronic low - grade endotoxemia is correlated to various surrogate markers that can be readily measured and tracked in clinical practice , including elevations in waist circumference , waist - to - hip ratio , total cholesterol , triglycerides , hemoglobin a1c , and serum insulin levels.64 , 65 these objective markers , which are diagnostic for metabolic syndrome , diabetes , and heart disease , can also be viewed as surrogate markers of chronic endotoxemia . third , patients who suffer from postprandial abdominal bloating that occurs within 1 hour of eating could be considered toxic . in the absence of gluten sensitivity , celiac disease , or other gastrointestinal tract pathologic conditions , the most common cause of such bloating is the fermentation of consumed carbohydrates by an overgrown small intestine bacterial population,32 , 49 which leads to endotoxemia.35 , 36 postprandial bloating is an almost universal symptom in patients with irritable bowel syndrome . the prevalence of irritable bowel syndrome is estimated to be between 3% and 28% , suggesting that a substantial number of patients are symptomatically toxic on a regular basis . although no clinical laboratory test is available to measure endotoxin in a practice setting , the condition of endotoxemia is measurable and correlated with various clinical markers . the presence of these clinical markers can be used as evidence for engaging patients in the process of detoxification . in this context toxic because of an excessive intake of refined sugar , flour , and oils , and it is the diet that should be detoxified . sugar and flour serve to promote bacterial growth , which has been referred to as an inflammatory microbiota . thereafter , the consumption of refined carbohydrate and lipid calories from refined oils and animal products will stimulate the release of endotoxin from small intestine gram - negative bacteria , leading to the development of systemic low - grade endotoxemia and related low - grade chronic inflammation . this relationship between diet and endotoxemia indicates that the diet should be viewed as the promoter of toxicity , and the diet should be considered toxic and in need of detoxification . unlike the previously described vague notions of toxicity , viewing the diet as being toxic is very precise and can be made very personal based on the types of refined sugar , flour , and oils preferred by individual patients . detoxified diet would contain little to no proinflammatory sugar and flour , which would eliminate the calories required to create an inflammatory microbiota and reduce systemic endotoxemia . the elimination of lipid calories from the diet is a more complex issue and should focus on avoiding the refined seed oils from corn , sunflower , safflower , cottonseed , peanut , and soy , rather than animal fats . saturated fatty acids , most commonly associated with the consumption of animal fat , are actually involved in the detoxification of circulating endotoxin , which means that dietary saturated fatty acids are actually healthy and anti - inflammatory . this controversial topic will be discussed in more detail in the remainder of this section of the article . consider the fact that the consumption of saturated fatty acids in meat and dairy is encouraged as part of a ketogenic diet , which is anti - inflammatory . studies have demonstrated that ketogenic diets can reverse proinflammatory conditions , such as metabolic syndrome and fatty liver , and reduce blood levels of inflammatory mediators.66 , 67 , 68 , 69 if animal fats and saturated fatty acids , in particular , were proinflammatory and disease promoting , these anti - inflammatory outcomes would not be possible . in fact , research is emerging that may support the use of a ketogenic diet for other proinflammatory conditions , such as obesity , epilepsy , diabetes , cardiovascular disease , cancer , polycystic ovarian syndrome , acne , parkinson disease , alzheimer disease , and brain trauma . additionally , the diets of tropical islanders are not inflammatory when they are devoid of refined sugar , flour , and seed oils but rich in coconut oil.31 , 71 in short , we have been told incorrectly for decades that the saturated fatty acids in both animal products and coconut oil somehow clog blood vessels in a monocausal fashion.72 , 73 , 74 this notion has never been supported by evidence , and recently , even cardiologists are becoming more vocal about this misinformation : recent prospective cohort studies have not supported any significant association between saturated fat intake and cardiovascular risk . instead , saturated fat has been found to be protective.red meat is another major source of saturated fat . consumption of processed meats , but not red meat , has been associated with coronary heart disease and diabetes mellitus , which may be explained by nitrates and sodium as preservatives . recent prospective cohort studies have not supported any significant association between saturated fat intake and cardiovascular risk . instead , saturated fat has been found to be protective.red meat is another major source of saturated fat . consumption of processed meats , but not red meat , has been associated with coronary heart disease and diabetes mellitus , which may be explained by nitrates and sodium as preservatives . from a purely empirical perspective , one could logically conclude that saturated fats could never build up on the walls of arteries , and this is because they are liquid at body temperature and never solid . the only time we witness saturated fatty acids in a solid , or crystallized , form is at room temperature or when refrigerated , with butter being the most obvious example . it is quite disingenuous to suggest that hardened saturated fatty acids at room temperature or in a refrigerator will be similarly hardened in the human body that runs at a temperature of almost 100f . experimental evidence also allows us to conclude that saturated fatty acid and cholesterol can not build up on artery walls . it is common knowledge that high - density lipoprotein ( hdl ) cholesterol is protective against atherosclerosis because of its participation in reverse cholesterol transport . thus , dietary factors that support appropriate hdl levels should be embraced as heart healthy . interestingly , the consumption of dietary saturated fatty acids is known to increase circulating levels of cardioprotective hdl cholesterol,73 , 75 which means that saturated fatty acids are actually heart healthy . more germane to the issue of toxicity is that hdl cholesterol also functions to reduce endotoxemia . in other words , the consumption of saturated fatty acids from animal fat serves to increase circulating levels of hdl that bind to and clear endotoxin from the body,76 , 77 , 78 , 79 , 80 which has been referred to as a humoral detoxification mechanism . this means that dietary saturated fatty acids from animal products have detoxifying properties and also represent a specific example of a named toxin and its detoxification . in summary , a detoxified diet is one that is free of proinflammatory calories from refined sugar , flour , and omega-6 oils . the consumption of meat , cheese , yogurt , and eggs should not be discouraged because they deliver many nutrients , including saturated fatty acids , that aid in the detoxification of circulating bacterial endotoxin . the proinflammatory calories from refined sugar , flour , and oil should be replaced by vegetation , including vegetables , tubers or roots , fruits , nuts , and legumes , which offer anti - inflammatory benefits,11 , 12 , 14 , 15 , 16 , 66 , 67 , 68 , 71 , 81 including the modulation of proinflammatory gastrointestinal tract bacteria and the inhibition of endotoxemia.82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 polyphenolic substances found in vegetation tend not to be absorbed and thus appear to exert their anti - inflammatory actions during their passage through the small and large intestine as they interact with microbial cells.84 , 85 , 86 , 87 , 88 , 89 accordingly , when an anti - inflammatory diet was adopted by patients with the metabolic syndrome , both stool and plasma endotoxin levels were reduced,90 , 91 , 92 indicating that endotoxemia can be managed by appropriate dietary changes . a general limitation is that we currently lack an operational definition of toxicity , and likely will continue to , because there are potentially many toxic substances that affect the human body . for example , in patients with chronic kidney disease , a diet low in plant fiber and symbiotic organisms can alter the normal gastrointestinal microbiome , leading to overgrowth of bacteria that produce renal toxins such as cresyl and indoxyl molecules . endotoxin was chosen as a focus in this article because endotoxemia is a measurable state that has been associated with the expression of many chronic conditions . however , the singular focus in this article on endotoxin is also a limitation . other toxins are potentially viable candidates and should be evaluated by those interested in this field . research studies need to be performed to test if any of the recommendations are viable and can be applied in clinical practice . the issue of toxicity has been a clinical interest within the chiropractic profession since the time of palmer . a common perception that continues today is that toxicity promotes subluxation or leads to a biological state in which adjustments do not hold . it is also believed that toxicity of the intestines and liver can compromise overall health and wellness . unfortunately , to date , none of these perceptions of toxicity is coupled with measurement of an actual toxin or its reduction via an interventional therapy . in contrast , bacterial endotoxemia is a measurable state of toxicity that is associated with many common conditions and is correlated to multiple surrogate clinical markers . endotoxemia also has been correlated directly to the consumption of refined sugar , flour , and oils , which suggests that the focus of detoxification should be the diet itself . until more research is available concept development ( provided idea for the research ) : d.s.design ( planned the methods to generate the results ) : d.s.supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : d.s.data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : d.s.analysis/interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : d.s.literature search ( performed the literature search ) : d.s.writing ( responsible for writing a substantive part of the manuscript ) : d.s.critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : d.s.practical applicationsa diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : d.s . data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : d.s . analysis / interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : d.s . critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : d.s . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia . a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia . a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity .

objectivethe purpose of this commentary is to review the notion of toxicity in the context of chiropractic practice.discussionthe belief that body toxicity is the cause of disease has been promoted for thousands of years . prior to the emergence of the chiropractic profession , the medical profession embraced the notion that the body becomes toxic , requiring detoxification interventions or surgery . the legacy of body toxicity within the chiropractic approach to patient care began with daniel david palmer . today , some sectors within the medical and chiropractic professions continue to embrace the concept of body toxicity and the related need to engage in detoxifying treatments . the most common areas of focus for detoxification are the intestines and liver ; however , the nature of the toxicity in these organs has yet to be defined or measured . in contrast , diet - induced systemic bacterial endotoxemia is a measureable state that is known to be promoted by a diet rich in sugar , flour , and refined oil . this suggests that bacterial endotoxin may be a candidate toxin to consider in the clinical context , as many common conditions , such as obesity , metabolic syndrome , diabetes , interstitial cystitis , depression , and migraine headache , are known to be promoted by endotoxemia.conclusiona diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that lead to systemic , low - grade endotoxemia , which is a common variety of toxicity that is measurable and worthy of research consideration . introducing a diet to reduce endotoxemia , rather than attempting to target a specific organ , appears to be a rational clinical approach for addressing the issue of toxicity .

Introduction Toxins From the Perspective of Early Chiropractic History: Daniel David Palmer Toxicity of the Colon in Relation to Chiropractic Practice Toxicity of the Liver in Relation to Chiropractic Practice Bacterial Endotoxin in Relation to Chiropractic Practice Measuring Bacterial Endotoxin Levels Managing Bacterial Endotoxin by Detoxifying the Diet Limitations Conclusion Funding Sources and Potential Conflicts of Interest Contributorship Information Practical Applications

body toxicity has been viewed as a health risk since the time of ancient egypt , when it was believed that stool putrefaction would lead to systemic disease . the ancient greek humoral theory of disease extended the concept of putrefaction to bile , phlegm , blood , and residues of food . clearly , the notion of body toxicity as a health risk for disease development has been part of patient care for a long time . it therefore might be expected that various sectors of health care , and individuals within health care , might still embrace and promote the issue of body toxicity as a treatment target . the purpose of this commentary is to discuss the issue of toxicity in the context of chiropractic practice , including daniel david palmer s impression of toxicity , a consideration of toxicity regarding the liver and colon , bacterial endotoxemia , and a rational approach to dietary detoxification in the context of addressing bacterial endotoxemia . palmer stated that the determining cause of disease are traumatism , poison , and autosuggestion . in others the place of entrance is not affected , the pathological manifestations being due to nervous disturbance the effect of a poison depends upon the nature of the substance , the amount and the individual.page 124 : poison destroys that which intelligent life has accomplished ; changes physiological to pathological action.page 147 : in the context of abnormal tone in the nervous system , palmer states that this prerequisite to disease is due to pressure , poison or lack of control of the emotions and acts of the patient , a morbid self-consciousness.page 554 : toxicosis is a disease caused by poisons ; a toxic or poisoned state ; the conditions of disease induced by poisons . palmer then gives examples of poisons as being chloroform , chloral , alcohol , digitalis , strychnine , nicotine , picrotoxin , veratrum , and laudanum.page 675 : poisons are not gathered in the tissues or cavities of the body . a chemical may be poisonous or otherwise.page 835 : in the context of developing typhoid fever , palmer states , the predisposing cause is poison from some decaying animal or vegetable matter . in others the place of entrance is not affected , the pathological manifestations being due to nervous disturbance the effect of a poison depends upon the nature of the substance , the amount and the individual . page 147 : in the context of abnormal tone in the nervous system , palmer states that this prerequisite to disease is due to pressure , poison or lack of control of the emotions and acts of the patient , a morbid self - consciousness . page 675 : poisons are not gathered in the tissues or cavities of the body . poison is any animal , vegetable , or mineral substance which when applied externally , or taken into the stomach , or injected into the body by physician or reptile , causes such a change in the animal economy as to produce abnormal functioning , disease or death page 835 : in the context of developing typhoid fever , palmer states , the predisposing cause is poison from some decaying animal or vegetable matter . page 974 : the osteopath and allopath believe poisons to be the cause of fevers . however , to date , research is lacking that would otherwise support the notion that an individual vertebra can be pulled out of alignment in the fashion posited by palmer . this requires doctors of chiropractic ( dcs ) to consider the issue of toxins in a subluxation context that is not related to misalignments . a more functional view is needed , which may involve inflammation and nociceptive processes , rather than pathoanatomical changes.7 , 8 , 9 it is important to note that palmer never described which specific toxins , if any , could promote misalignment subluxations . current discussions about toxins and spinal subluxation also do not implicate specific toxins.2 , 3 in other words , the terms toxins and related toxicity have been consistently described in an imprecise and nebulous fashion in the context of spinal subluxation . no study has yet to define a specific toxin in the colon or liver that then responds to a specifically related detoxification nutritional protocol . colon cleansing for the purpose of reducing intestinal toxicity and improving systemic health was popularized by a dc , bernard jensen , who wrote a text titled tissue cleansing through bowel management . the ingested psyllium is claimed to remove built - up toxic debris , aided by a daily colema , which is a variation of an enema and colonic . in other words , the dietary recommendation after the cleanse is to eat more vegetation and avoid refined sugar , flour , and omega-6 fatty acids , which has been termed an anti - inflammatory diet.11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 a pubmed search using the phrase colon cleansing and health leads to 87 published articles , most of which involve cleansing before a colonoscopy . because colon cleansing involves increasing fiber intake as well as eating more vegetables and fruit and avoiding refined sugar , flour , and omega-6 oils , inflammation is reduced . in other words , toxic foods such as refined carbohydrates and oils create inflammation and the diet , the beneficial outcome is inflammation reduction rather than physical or physiological detoxification of the human body . claims that toxic materials can be pulled off the walls of the intestine by fiber , such as powdered psyllium husk , are also inaccurate . the stools of those taking psyllium husk have been studied , and the larger stool mass was determined to contain psyllium fiber , rather than toxins . because we lack data to support the notion that the intestines can be cleansed of built - up debris and toxins , the appropriate choice for promoting intestinal health is to eat fiber - rich vegetation and perhaps supplement with psyllium husk powder to help regulate bowel function . in particular , supplemental psyllium has been reported to be of benefit for patients with constipation , diarrhea , irritable bowel syndrome , inflammatory bowel disease , colon cancer , and type 2 diabetes . practitioners in both the medical and chiropractic professions are guilty of embracing the concept of liver toxicity , despite the lack of scientific evidence . thus , the appropriateness or clinical relevance of this nutritional approach has yet to be demonstrated in the context of chiropractic practice and health care in general . despite this fact , dcs find this to be a topic of interest and promote this approach for use in clinical practice.26 , 27 , 28 as outlined earlier , the issue of toxicity as a cause of spinal dysfunction , and as an intestinal and liver condition , is of therapeutic interest to dcs . the general topic of body cleansing and detoxification also appears to be of great interest to the general public . we supposedly need to detoxify our bodies because air , water , and food contain toxins ; however , no specific toxins have been implicated and measured . in contrast , bacterial endotoxin may be a candidate toxin to consider as a cause of spinal dysfunction , as a therapeutic target to restore intestinal and liver health , and as a promoter of other common conditions encountered in chiropractic practice . lipopolysaccharide is also known as bacterial endotoxin , which circulates at very low levels in normal disease - free humans ; however , this changes as individuals become unhealthy : although endotoxin ( lipopolysaccharide ( lps ) ) , derived from the cell wall of gram - negative bacteria , circulates at low concentrations in the blood of healthy individuals , the presence of genetic and diet - induced obesity and other metabolic disorders has been associated with a substantial increase in lps concentrations , a condition termed metabolic endotoxemia . although endotoxin ( lipopolysaccharide ( lps ) ) , derived from the cell wall of gram - negative bacteria , circulates at low concentrations in the blood of healthy individuals , the presence of genetic and diet - induced obesity and other metabolic disorders has been associated with a substantial increase in lps concentrations , a condition termed metabolic endotoxemia . the only source of circulating endotoxin can be from the gastrointestinal tract because that is where gram - negative bacteria reside in the human body , which speaks to the importance of maintaining gastrointestinal tract health to promote a homeostatic or symbiotic relationship with our intestinal microbiota . the large intestine appears to be adapted to the presence of high concentrations of bacteria ; compared with the small intestine , it has a relatively small surface area and is not designed for macronutrient and micronutrient absorption , save for short chain fatty acids , vitamin k , sodium , and water . although the jejunum contains bacteria , the concentration should not exceed 10 organisms per milliliter , which is also considered the normal concentration of bacteria in the ileum . in contrast , in the terminal ileum near the ileocecal valve , bacterial concentrations range from 10 to 10 organisms per milliliter , whereas colonic concentrations can reach 10 . the diagnosis of small intestinal bacterial overgrowth ( sibo ) is applied to patients when the concentration of bacteria exceeds 10 organisms per milliliter in the jejunum and ileum.33 , 34 not surprisingly , patients with sibo have increased circulating levels of bacterial endotoxin.35 , 36 whether a patient develops sibo or not , research has demonstrated that it is possible to create a state of low - grade metabolic endotoxemia based on poor dietary choices . for example , when normal - weight participants , with an average body mass index of 23 , were fed a typical american breakfast including a cup of tea , 3 slices of toast , and butter , which amounted to 900 calories , lipopolysaccharide levels increased significantly compared with controls , indicating that this common breakfast promotes low - grade endotoxemia . in contrast , there was a 31% reduction in endotoxin when participants consumed a prudent - style diet characterized by higher intakes of fruits , vegetables , fish , whole grains , and legumes . in these short - term studies , there were no reported symptomatic changes associated with low - grade endotoxemia , which suggests that acute diet - induced endotoxemic events in otherwise healthy individuals go unnoticed . the transition from acute to chronic endotoxemia , and the point at which symptoms may appear , is not clear and obvious , which can be confusing for patients and clinicians and complicates the identification of a cause - effect relationship . in the absence of overt diseases that cause sibo , such as achlorhydria , diabetic autonomic neuropathy , scleroderma , and crohn disease , the most common cause promoter of bacterial overgrowth is a diet that is low in fiber and rich in refined sugar , flour , and oils . the chronic consumption of such a diet will simultaneously reduce gastrointestinal tract motility and feed gastrointestinal tract bacteria , allowing them to overgrow in the small intestine to varying degrees , which can promote chronic low - grade endotoxemia.31 , 41 , 42 whether varying degrees of sibo are related to all conditions that are associated with low - grade endotoxemia is not known . whether or not sibo develops , multiple conditions are known to be associated with elevated levels of circulating endotoxin , such as irritable bowel syndrome and fatty liver disease.32 , 41 , 43 , 44 , 45 this suggests that dcs can still view bowel and liver function in the context of toxicity , with the treatment goal being that of endotoxin reduction . however , bacterial endotoxemia is known to be a promoter of obesity , metabolic syndrome , type 2 diabetes , and depression , all of which are associated with an increased risk of back pain , which again permits a dc to embrace the notion that toxins may promote spinal dysfunction . additional common conditions that patients may present with to a chiropractic office and that are known to be promoted by chronic endotoxemia include chronic fatigue , anxiety , impaired cognition , migraine headache , impaired sleep , interstitial cystitis , type 2 diabetes , atherosclerosis , hypertension , general feeling of ill health , and widespread pain that may be diagnosed as fibromyalgia.31 , 32 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 bacterial endotoxemia is also associated with childhood obesity and may be a key contributor to type 2 diabetes and cardiovascular disease later in life . first , as described earlier , we know that the consumption of refined sugar , flour , and oil leads to postprandial low - grade endotoxemia that has been measured in the clinical research setting.37 , 38 , 39 in other words , a patient who regularly eats these foods is measurably toxic . second , chronic low - grade endotoxemia is correlated to various surrogate markers that can be readily measured and tracked in clinical practice , including elevations in waist circumference , waist - to - hip ratio , total cholesterol , triglycerides , hemoglobin a1c , and serum insulin levels.64 , 65 these objective markers , which are diagnostic for metabolic syndrome , diabetes , and heart disease , can also be viewed as surrogate markers of chronic endotoxemia . in the absence of gluten sensitivity , celiac disease , or other gastrointestinal tract pathologic conditions , the most common cause of such bloating is the fermentation of consumed carbohydrates by an overgrown small intestine bacterial population,32 , 49 which leads to endotoxemia.35 , 36 postprandial bloating is an almost universal symptom in patients with irritable bowel syndrome . although no clinical laboratory test is available to measure endotoxin in a practice setting , the condition of endotoxemia is measurable and correlated with various clinical markers . in this context toxic because of an excessive intake of refined sugar , flour , and oils , and it is the diet that should be detoxified . thereafter , the consumption of refined carbohydrate and lipid calories from refined oils and animal products will stimulate the release of endotoxin from small intestine gram - negative bacteria , leading to the development of systemic low - grade endotoxemia and related low - grade chronic inflammation . this relationship between diet and endotoxemia indicates that the diet should be viewed as the promoter of toxicity , and the diet should be considered toxic and in need of detoxification . unlike the previously described vague notions of toxicity , viewing the diet as being toxic is very precise and can be made very personal based on the types of refined sugar , flour , and oils preferred by individual patients . the elimination of lipid calories from the diet is a more complex issue and should focus on avoiding the refined seed oils from corn , sunflower , safflower , cottonseed , peanut , and soy , rather than animal fats . studies have demonstrated that ketogenic diets can reverse proinflammatory conditions , such as metabolic syndrome and fatty liver , and reduce blood levels of inflammatory mediators.66 , 67 , 68 , 69 if animal fats and saturated fatty acids , in particular , were proinflammatory and disease promoting , these anti - inflammatory outcomes would not be possible . in fact , research is emerging that may support the use of a ketogenic diet for other proinflammatory conditions , such as obesity , epilepsy , diabetes , cardiovascular disease , cancer , polycystic ovarian syndrome , acne , parkinson disease , alzheimer disease , and brain trauma . additionally , the diets of tropical islanders are not inflammatory when they are devoid of refined sugar , flour , and seed oils but rich in coconut oil.31 , 71 in short , we have been told incorrectly for decades that the saturated fatty acids in both animal products and coconut oil somehow clog blood vessels in a monocausal fashion.72 , 73 , 74 this notion has never been supported by evidence , and recently , even cardiologists are becoming more vocal about this misinformation : recent prospective cohort studies have not supported any significant association between saturated fat intake and cardiovascular risk . consumption of processed meats , but not red meat , has been associated with coronary heart disease and diabetes mellitus , which may be explained by nitrates and sodium as preservatives . more germane to the issue of toxicity is that hdl cholesterol also functions to reduce endotoxemia . in summary , a detoxified diet is one that is free of proinflammatory calories from refined sugar , flour , and omega-6 oils . the proinflammatory calories from refined sugar , flour , and oil should be replaced by vegetation , including vegetables , tubers or roots , fruits , nuts , and legumes , which offer anti - inflammatory benefits,11 , 12 , 14 , 15 , 16 , 66 , 67 , 68 , 71 , 81 including the modulation of proinflammatory gastrointestinal tract bacteria and the inhibition of endotoxemia.82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 polyphenolic substances found in vegetation tend not to be absorbed and thus appear to exert their anti - inflammatory actions during their passage through the small and large intestine as they interact with microbial cells.84 , 85 , 86 , 87 , 88 , 89 accordingly , when an anti - inflammatory diet was adopted by patients with the metabolic syndrome , both stool and plasma endotoxin levels were reduced,90 , 91 , 92 indicating that endotoxemia can be managed by appropriate dietary changes . endotoxin was chosen as a focus in this article because endotoxemia is a measurable state that has been associated with the expression of many chronic conditions . the issue of toxicity has been a clinical interest within the chiropractic profession since the time of palmer . it is also believed that toxicity of the intestines and liver can compromise overall health and wellness . in contrast , bacterial endotoxemia is a measurable state of toxicity that is associated with many common conditions and is correlated to multiple surrogate clinical markers . endotoxemia also has been correlated directly to the consumption of refined sugar , flour , and oils , which suggests that the focus of detoxification should be the diet itself . until more research is available concept development ( provided idea for the research ) : d.s.design ( planned the methods to generate the results ) : d.s.supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : d.s.data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : d.s.analysis/interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : d.s.literature search ( performed the literature search ) : d.s.writing ( responsible for writing a substantive part of the manuscript ) : d.s.critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : d.s.practical applicationsa diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia . a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia.a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity . a diet rich in refined sugar , flour , and oils may induce proinflammatory changes within intestinal microbiota that may lead to systemic low - grade endotoxemia . a diet to reduce endotoxemia , rather than attempting to target a specific organ , may be a rational approach for addressing the issue of toxicity .

the olfactory habituation / cross - habituation test ( haxha ) is a noninvasive spontaneous behavioral task that has been used to study the ability to smell and the capacity to discriminate between stimuli ( odors ) in a large variety of animals and humans . the haxha follows the basic principles shown by thompson and spencer in 1966 : when any stimulus is repeatedly evoked , the behavioral response decreases ( habituation ) , not involving sensory adaptation / sensory fatigue or motor fatigue . meanwhile the presentation of a different stimulus leads to a change in the amplitude of the habituated response ( cross - habituation ) [ 1 , 2 ] . the general protocol for olfactory haxha in rodents consists of presenting an odor ( in a paper filter or cotton applicator ) generally in the center of the experimental cage and measuring the time in which an animal is oriented to and within 2 cm of the odorant . to measure the habituation phase , the novel stimulus is presented several times ( trials ) . the cross - habituation phase can be studied by changing the stimulus for a novel unfamiliar odor . this protocol has been adapted to different species , for example , by increasing the number of trials in rats , or by increased intertrial intervals in guinea pigs . the first haxha experiments in rats demonstrated that male and female rodents are able to discriminate between different urine odors independently of hormone status and later this behavioral task was adapted to mice using sex related odors as stimuli . olfactory habituation can be mediated by different circuits of the olfactory system when using paradigms that differ in timescale . short - term habituation following 20 sec odor presentations and short intervals ( 10 sec ) is mediated by neuronal adaptation lasting about 2 minutes in the anterior piriform cortex . long - term habituation [ 810 ] following odor presentations of 50 sec separated by 5 min intertrial intervals persists up to 30 min and is mediated by the olfactory bulb . to study olfactory alterations researchers have also used other behavioral olfactory - guided tasks such as the social transmission of food preference , which combines social interactions and olfaction , the odor - cup sand - digging task , in which the animals have to be trained to dig into one of two cups ( s+ , s ) , the buried food test , which tests the ability of the animals to smell a piece of familiar food such as cereal hidden under the bedding of the cage . in all of these tasks the animals have to be food - regulated in order to obtain responses . for the odor - cued taste avoidance , which combines odor detection and odor discrimination , the mice have to be thirsty to perform the task . the large advantage of the haxha task is that animals do not need to be food- or water - regulated to perform the task . this is critical for aging research where food restriction may interfere with the aging process , for example , by affecting metabolic rate or inducing stress . another advantage is that the haxha test is a spontaneous odor discrimination task which requires no training which may involve cognitive processes unrelated to the ones being under investigation . despite these advantages and ability to target the patency of different neural structures by adjusting the timescale , the standard means to measure the exploration time is by a human experimenter using a stopwatch . this provides the potential for observer bias in cases where subjects can not be tested without prior knowledge of status , as when testing anatomically different phenotypes ( e.g. , fur , body weight ) . further , as individual exploratory bouts can be quite short ( < 1 sec ) the use of a stopwatch limits accuracy . last , a human observer will have limited accuracy in scoring the behavior according to exact criteria such as proximity within 2 cm of the odor source and precise head angle toward it . to avoid these potential confounds altogether we used behavioral video tracking software ( noldus ) , which is able to recognize the nose , the center , and the tail of the mouse at 10 frames / sec . this allowed us to quantify the distance between the nose and the odor stimulus , the head angle to the odorant , and the locomotion of the animal ( position and velocity ) . while it seems to be taken for granted that animals are smelling the target odor while the standard criteria of proximity and orientation are met , odor exploration fundamentally involves sniffing , the rate of which is actively modulated , increasing when rodents explore novel odors . therefore , the assessment of odor - guided ( dis-)habituation should also be guided by sniff rates , which standard haxha tests do not include but which we also measured here using whole - body plethysmography . sniffing is characterized by a rhythmic inhalation and exhalation of air through the nose . this behavior also plays a critical role in shaping how odor information is represented and processed by the nervous system . sniffing behavior in rodents is dynamic , varies with the behavioral context , and is modulated by olfactory and nonolfactory processes . meanwhile , the frequency of sniffing ( sniff rate ) has been used as a parameter to characterize sniffing behaviors in rodents , as they increase from 2 hz to 412 hz when they are investigating novel odors [ 18 , 19 ] . identification of olfactory dysfunction at early stages of sad has been proposed as a promising diagnostic tool , potentially allowing early treatment before the irreversible cognitive deteriorations are established . however , there are many issues in this field to be resolved , including finding optimal behavioral tasks to aid diagnosis . averback described neurodegeneration in the anterior olfactory nucleus in the olfactory bulbs and tracts , caused by cells loss in the presence of amyloid plaques and neurodegenerative tangles . posthumous histological analysis suggested a relationship between the olfactory sensory pathway and the severity of the disease [ 21 , 22 ] . warner et al . first suggested olfactory impairments in patients with ad , employing a standardized smell identification test . however , the lowered ability to distinguish between or recognize different odors is not exclusive for ad , as olfactory dysfunctions have also been detected in parkinson 's disease [ 24 , 25 ] , multiple sclerosis [ 26 , 27 ] , viral infections [ 28 , 29 ] , lesions in the brain , and aging . thus , while there is a need for an olfactory test for ad , it should also be specific . recently , olfactory alterations in transgenic mice models for familial ad were detected with haxha using food and sex - related odors as the stimuli , where olfactory deficits were correlated with the stage of the disease [ 32 , 33 ] . our group studies the olfactory behavior of fus1 ko mice , a novel model of accelerated aging and sporadic alzheimer 's disease ( sad ) , in which deletion of a mitochondrial tumor suppressor protein fus1/tus2 leads to an overproduction of reactive oxygen species ( ros ) inducing oxidative damage to cellular macromolecules [ 34 , 35 ] . our previous studies suggest that the female fus1 ko mice at 10 months old have alterations in their habituation to nonsocial odors and deteriorations in their cross - habituation for social odors ( ms submitted for publication ) . here , we propose a novel approach to accurately assess haxha using the noldus system to track the behavior of every animal combined with whole - body plethysmography to noninvasively evaluate their sniffing . we evaluated how the classical haxha odor exploration criteria relate to sniffing in both wild type and fus1 ko mice . we further sought to find improved nonolfactory parameters , to be used in cases when sniffing can not be measured . we confirm that standard criteria accurately capture sniffing - mediated olfactory exploration and suggest that velocity combined with a relaxed distance criterion outperforms odor exploration quantification . in this study we compared the behavior of young fus1 ko/129sv and wt/129sv mice of different genders generated by dr . the groups in the study were 4 - 5-month - old female fus1 ko ( fus ko ; n = 13 ) and wt mice ( n = 7 ) . the vivarium had a 12 h/12 h inverted light cycle with lights off at 10:30 am . all animals were housed individually in polycarbonate cages ( 12 12 25 cm ) with controlled humidity ( 40% ) and temperature ( 22c ) and were provided with nestlets . a cotton - tipped wood applicator ( puritan ref 806-wc ) was presented mounted in a removable holder on the bottom of the cage , located 1 cm above the cage floor . we saturated the cotton applicator with one of 4 odorants : mineral oil ( mo ) , amyl acetate ( aa ) 1% ( in mo ) , phenyl ethanol ( pe ) 1% ( in mo ) , and social odorant ( s , obtained by swabbing the cage of a female mouse ) . a total of 12 trials were performed per mouse , where each odorant was presented three times in succession per daily session to yield the following order : mo13 , aa13 , pe13 , and s13 . the total duration of a single session was 35 min per mouse , each trial consisting of 2 min per odorant exposure and an intertrial interval of 1 min between stimuli . odor exploration was defined as being oriented toward the applicator tip while the nose was within 2 cm of it . this test evaluates if mice are able to spontaneously recognize a novel odorant stimulus by spending more time smelling the applicator ( cross - habituation phase ) , as opposed to the time that the mice spend on each repeated stimulus ( habituation phase ) . in order to reduce timing errors and experimenter bias in measuring the habituation / cross - habituation task and to know if these behavioral responses were specific to the olfactory abilities of the animals , we combined a system to automatically record behavioral responses using the noldus behavioral tracking system ( ethovision xt , version 10.1 , noldus information technology b.v . , wageningen , netherlands ) with sniffing analysis obtained by whole - body plethysmography . we used an air - sealed experimental semitransparent white acrylic box ( 26 38 16 cm ) with a usb camera ( logitech hd pro c920 , 1920 1080 pixels ) mounted at the ceiling of the box , aimed downward . noldus software analyzed the camera input to identify and score the behavior of the animal . in noldus , we marked a circular area ( od = 4 cm , a = 12.6 cm ) in the middle of the cage floor where a cotton swab was placed . we saturated the cotton applicator with one of the following experimental odors : mo , aa , pe , and s. to measure sniffing noninvasively , a pressure sensor ( buxco , trd5700 ) communicated with the experimental box . the transducer signals were amplified 10x ( a - m systems , differential ac amplifier model 1700 ) , band - pass filtered between 0.1 and 40 hz and 60 hz notch filtered ( a - m systems ) , and subsequently filtered between 0.1 and 40 hz with an 8th order linkwitz - riley filter using a minidsp 2 4 processor ( minidsp , hong kong ) . the data obtained from the sensor was stored using a neuroplex system ( redshirtimaging , decatur , ga , usa ) , synchronized to the start of each trial using the noldus mini - usb i / o box . during 2 minutes of each trial the behavior of a mouse was recorded , and when the rodent 's nose was oriented to the cotton within a 2 cm distance a signal was generated via the noldus mini - usb i / o box , identifying the period that the mouse was exploring the stimulus , and sent to the neuroplex data - acquisition system . noldus also scored the number of times the mouse oriented to the stimulus and the distance and speed traveled by the rodent during every trial . the data was analyzed using noldus for behavioral responses and using matlab for sniff rates for the period that the animal was exploring versus not exploring the stimulus . matlab ( r2016a , the mathworks , ma ) was used to analyze all raw sniffing data and the noldus signal from neuroplex and video data from noldus exported in excel format at 100 ms bins . only the trial types pe3 , s1 , s2 , and s3 were used , as s1 was by far the most explored stimulus . matlab output was organized in excel ( 2016 for mac , microsoft ) for calculating means and sem ( sd/n ) and graphing . for each trial ( 120 s duration ) sniffs were identified by filtering the neuroplex data ( 200 samples / s ) with a 4th - order butterworth bandpass ( 320 hz ) filter and finding peaks in z - scored data exceeding 0.4 s.d . average sniff rates and amplitudes during noldus identified exploration on / off times were calculated . neuroplex variable acquisition delays common to its bnc - only mode were taken into account . for deeper analyses the following procedure was used to integrate the neuroplex - based sniffing signal ( 200 s / s ) with the neuroplex video tracking output ( 10 s / s ) . mouse nose and core location ( x , y ) and velocity data with rare missing values were completed by using last - known locations and velocities , except for start values which were set to 1 ( location ) or 0 ( velocity ) . instantaneous sniff rates , velocity ( cm / s ) , distance ( cm ) to odor ( at x = 0 , y = 0 ) , and head angle to odor were assigned to each 100 ms bin and subsequently convolved with a 30-bin gaussian low - pass filter and end - corrected . first - order regressions were calculated between sniff rates and distance , velocity , and the angle to odorized cotton tip ( matlab regression function ) . multiple regression ( matlab fit function ) was performed of 1st and 2nd order ( poly11 and poly22 ) onto sniff rate ( including nonzero constants ) . this was also performed for each trial type ( pe3 , s1 , s2 , and s3 ) and all trials combined per group ( wild type ( wt ) or fus ko ) by combining data bins of all trials into population vectors . stepwise multiple regression was computed using population vectors for regressing angle , distance , and velocity ( matlab stepwiselm ) onto sniff rate on this . population vectors were also used to parametrically explore how combinations of exploration criteria affected exploration on / off time and sniff rates . the olfactory haxha task is used to evaluate the patency of the olfactory system by asking how much time an animal spontaneously explores new or previously presented odor objects . it is hence assumed that the exploration time is equivalent to the time smelling to date sniffing has not been used as a key marker of this behavior . using our noldus video tracking system combined with whole - body plethysmography we were able to address the question of whether the criteria used thus far to measure exploration , namely , distance and head angle to the object , are indeed related to enhanced sniff rates . figure 1 shows this to be the case : sniff rates are enhanced during periods that the wt mice were within 2 cm of the cotton tip and with their head aimed to it within 20 ( onfreq ) , as compared to other periods ( offfreq ) , for each trial type ( pe3 , s1 , s2 , and s3 ) . for s1 trials this increased from 6.5 hz to 9.5 hz . pe3 shows low exploration time ( ontime < 1 sec ) indicative of habituation , followed by a cross - habituation of ~14 sec exploration to the first social odor presentation ( s1 ) . this was followed by moderate , if apparently , inconsistent habituation during trials s2 and s3 ( ~8 sec ) . in contrast to the sniff rate being increased during proximity and orientation to the object , there was no evidence that sniff amplitudes were modulated by trial type . figure 2 shows that sniff rates followed the same pattern as wt mice , with s1 sniff rates at 10.1 hz during proximity and orientation to the odorant , versus 6.0 hz otherwise . ( note that the s1 exploration time was corrected from a rare ( all data checked ) noldus output error ( 22.34 sec , mouse fus627 , set to 0 as the mouse was well over 2 cm away from the odorant ) ) . we conclude that the proximity criterion of < 2 cm and < 20 to the odorant is indicative of olfactory exploration during habituation and cross - habituation for female mice of the 129 wt strain as well as for their fus ko chronic oxidative stress counterparts . figure 3 illustrates this finding for two wt mice during s1 trials by showing their path ( large solid circle : start ; small solid circle : end ) in the cage ( one dot per 100 ms bin , 1201 dots total ) , the head angle to the cotton tip ( smaller size indicating smaller angle ) , and sniff rate ( color coded ) . it is evident that sniff rates tend to be high and head angles low when they are near the centrally located odorant ( dotted bulls - eye ) , though not exclusively so . in the hypothetical absence of a relation between aforementioned criteria and sniff rates it would be untenable that such criteria measured olfactory exploration . in light of our findings that these criteria are predictive of sniff rate , the subtler question arises of whether the classical proximity and orientation criteria are optimal , either in isolation or combined , as a proxy for sniffing - mediated odorant exploration when sniff rate is not or can not be measured ( as is the general approach ) . figure 4 shows the mean ( sem , across 7 wt mice ) linear regression coefficient between sniff rate and proximity ( distance ) , orientation ( angle ) , and the speed of wt mice 's trajectory ( velocity ) across time bins . as expected from the prior results , distance is mostly negatively correlated , in particular during the cross - habituation s1 trials ( r = 0.49 ) when sniff rate is modulated most strongly . however , the correlation between angle and sniff rate is quite inconsistent across trial types . interestingly , the velocity of the mice was strongly and consistently positively correlated with sniff rate ( r = 0.470.59 ; figure 4 , right ) . for fus ko mice , shown in figure 5 , a similar set of relationships was evident , but now both angle and distance negatively correlated during s1 trials ( r = 0.38 ) . in a complimentary approach time bins were categorized to belong to one of four sniff rate quartiles ( analyzed per trial ) and associated distance , angle , and velocity were assessed . figure 6(a ) shows the expected mean sniff rate ( across bins , followed by across animals ) increase from the 025th percentile of sniff rate ( sniff_25 ) , 2550th percentile ( sniff_2550 ) , and 5075th percentile ( sniff_5075 ) to the 75100th percentile ( sniff_75 ) . whereas the lowest quartile shows similar sniff rates across trial types , at higher quartiles s1 sniff rates are highest . exploration of the proximity during time bins associated with each sniff rate quartile shows that distance is smallest during s1 trials especially at the top quartile ( figure 6(b ) ) . this indicates that small distance to the odorant during cross - habituation has a high specificity as a marker of olfactory exploration , a further validation of the proximity criterion . although angle showed similar tendencies as distance , it lacked the specificity thereof ( figure 6(c ) ) : orientation to the odor source occurred in overlapping degrees across sniff rate quartiles , notably s2 at lowest three quartiles . consistent with the strong and trial - type invariant positive correlation between velocity and sniff rate discussed before ( figures 4 and 5 , right ) , we found that at higher sniff rate quartiles mice consistently showed higher velocities , albeit with different functions between trial types ( figure 6(d ) ) . the same analysis for fus ko mice ( not shown ) confirmed these wt findings , although with somewhat lower specificity of distance and higher specificity of angle for s1 trials than wt . acceleration was also explored and found to be inconsistent across quartiles , trial types , and subject groups ( not shown ) . we further explored at what fraction of time when sniffing at each sniff rate quartile the wt mice would satisfy the categorical proximity ( < 2 cm ) , orientation ( < 20 ) , or their combined ( < 2 cm & < 20 ) criterion ( figure 7 ) . it can be seen that during faster sniffing at the top quartile mice spent ~30% of their time in close proximity to the odorant ( dist_on_75 ) during s1 trials but much less time during other trials or at lower quartiles ( < 13% , figure 7 , left ) . during s1 trials mice spent 46% of time oriented to the odorant when sniffing fast ( angle_on_75 ) and less than 21% at lower sniff rates . however , during s2 trials they were similarly oriented to the odor source even at the lowest quartile . fus ko data shows similar patterns , though with somewhat lower specificity for proximity and higher for orientation than wt shown here . thus far we have shown that complimentary analytical approaches confirm that proximity and ( to a lesser degree ) orientation to the odorant relate to enhanced sniffing , especially during s1 cross - habituation trials , lending credit to this long - used means of measuring odor object exploration duration ( in the absence of a direct sniff rate measure ) . we also found that velocity may be an additionally useful factor to consider in quantifying odor exploration in the haxha test , due to its strong and consistent relation to sniff rate . we therefore used multiple regression to see how well combinations of proximity , orientation , and velocity can predict sniff frequency parametrically ( figure 8) . population vectors were used to allow all time bins from all mice to be included in a single regression ( each vector spanning 7 mice 1201 bins = 8407 time bins for s1 trials ; 84074 trials = 33,628 bins for all trials ) . while we found that distance and angle combined could explain 35% of wt s1 sniff rate variance ( adjusted r , 2nd - order regression , dist - angle 2nd ) , distance and velocity explained a rather high 60% ( dist - veloc 2nd ) of wt s1 trial sniff rate variance , and > 40% of sniff rate variance across all wt and fus trials ( wt all , fus ko all ) . distance and angle only explained up to 19% of sniff rate variance across all trials in both groups . figure 9 shows the respective sniff rate state - spaces for wt s1 trials and the regression equations ( ( a ) angle and distance ; ( b ) velocity and distance ; top : 1st - order , bottom , 2nd - order ) . a stepwise multiple regression ( p value to enter : 0.05 ) included all 3 factors and explained roughly as much as the 2-way 2nd - order velocity - distance regression ( 3954% , figure 8 , right - most bars ) . these data suggest that the combined criterion of proximity and velocity may be more useful than proximity and orientation in estimating sniff - mediated odor exploration . in an effort to establish new criteria based on the above findings we used the s1 trial population vectors to see which exploration times ( figure 10 ) and exploration sniff rates ( figure 11 ) would result from using different criterion thresholds and their combinations . optimal criteria should yield the highest exploration time without substantially lowering the sniff rate during it . figure 10 ( top ) shows the fraction of time the mice would be considered to be exploring with distance criteria of less than 1 , 2 , 4 , or 8 cm ( left , d1d8 ) , angle of less than 5 , 10 , 20 , or 40 ( middle , a5a40 ) , or velocity of more than 4 , 2 , 1 , or 0.5 cm / s ( right , v4v0.5 ) . the distance threshold < 2 cm ( d2 ) was marked , including a horizontal line for reference , as it is the standard criterion and yields exploration for 11% of time ( 13.2 sec for a 120 sec trial , like the trial - based noldus result of 14.1 sec in figure 1 , right ) . figure 10 ( bottom ) shows the fraction of time explored for their combinations at 3 distance thresholds . it can , for example , be seen that combining d2 with a20 does not substantially alter exploration time ( 11% ) , suggesting that these thresholds largely overlap over time , calling into question the usefulness of adding orientation to the d2 criterion . figure 11 ( top ) shows that no single threshold can substantially improve on d2 , yielding 9.6 hz sniffing during the 11% exploration time ( close to 9.5 hz onfreq in figure 1 ; 6.5 hz is shown as y - axis bar cut - off as this is the offfreq baseline sniff rate , i.e. , when not exploring ) . whereas the strictest angle thresholds yield peak rates of ~8.5 hz , sniff rates decrease somewhat with stricter velocity thresholds ( from 9.0 to 8.5 hz ) . sniff rate would increase slightly by tightening the distance threshold from < 2 to < 1 cm from odor source , but exploration time would drop dramatically from 11% to 5% ( figure 10 , top : d2 versus d1 ) . nearly all the combined criteria ( figure 11 , bottom ) show sniff rates similar or somewhat higher than when applying the d2 criterion ( orange line ) . at d2 the various angle thresholds yield similar rates of 9.69.9 hz ( figure 11 , bottom left ) . relaxing the distance threshold to < 4 cm requires an angle threshold < 10 to retain such rates but reduces exploration time from 11% to 7% ( figure 10 , bottom left ) . combining proximity with velocity ( figure 11 , middle ) shows that it can yield highest exploration sniff rates ( d1 and v2 ; 11.3 hz ) but identifies only 0.02% as exploration . in contrast , relaxed distance threshold < 4 cm and velocity > 0.5 cm / s ( d4 and v0.5 , marked ) yielded the same 9.6 hz as the standard but increased exploration time from 11% to 15% , thereby being suggestive of a criterion better able to identify sniff - mediated odor exploration . adding orientation to this combination we next tested this new criterion of distance threshold < 4 cm and velocity > 0.5 cm / s ( d4-v0.5 ) to individual trials of wt ( figure 12 , middle ) and fus ko mice ( figure 13 , middle ) . the left graph in these figures ( onfreq , offfreq , and ontime ) is identical to that of figures 1 and 2 using the noldus d < 2 cm and a < 20 criterion ( using unsmoothed data ) and is shown for reference . the neighboring graphs ( marked _ b ) show these results upon removal of trials of mice yielding < 1 sec exploration time , which helped robustness in particular for the d4-v0.5 criterion by raising s2 and s3 sniff rates during exploration and reducing their sem ( sniff_on_di4xve05_b ) for both wt and fus ko mice . the d4-v0.5 criterion increased wt pe nonexploratory sniff rates slightly but consistently ( sniff_off_di4xve05_b ) over the standard d2-a20 criterion ( offfreq_b , p < 0.001 , paired 2-sided t - test , figure 12 ) and also consistently , if slightly , increased fus ko pe3 , s2 , and s3 nonexploratory sniff rates ( p < 0.001 , p < 0.05 , and p < 0.001 , resp . , paired 2-sided t - test , figure 13 ) . the d4-v0.5 criterion increased wt s1 trial exploration time ( explore_on_di4xve05_b ) over the standard d2-a20 criterion ( ontime_b ) by 39% from 14.1 sec to 18.2 sec ( orange line ) , without decreasing sniff rate ( 9.5 hz , figure 12 ) . meanwhile , s2 and s3 exploration times of wt mice decreased together with a larger reduction in sem . this high sem using d2-a20 was mostly due to a single outlying mouse ( wt517 ) yielding 33 and 56 sec of exploration time for s2 and s3 , respectively ( data was verified for correct assessment ) . the new criterion is apparently more robust in avoiding such pitfalls , which can allow for a statistically more powerful ( discriminating ) assessment of habituation and cross - habituation . indeed , whereas habituation was weak for s2 ( p = 0.04 , paired 1-sided t - test ) and was not significant for s3 using the d2-a20 criterion , it was highly significant for s2 and s3 ( p < 0.001 ) using the d4-v0.5 criterion . other outcomes were roughly similar between the two criteria and for fus ko mice ( figure 13 ) statistical conclusions based on exploration time did not differ between the two criteria . the difference in time bins meeting these two criteria was also assessed ( % overlap ) and was ~69% for s1s3 ( crit_diffpct_div4ve05 , figure 12 ) . the difference between the noldus unsmoothed d2-a20 exploration time ( usb - based acquired ) output and the post hoc smoothed d2-a20 exploration time output was < 3% ( crit_diffpct ) . the fus ko criterion comparison yielded similar results as for wt ( figure 13 ) . s1 exploration time increased by 15% ( 6.5 sec to 7.5 sec ) , s2 by 44% ( 4.5 sec to 6.5 sec ) , and s3 by 0.5 sec ( 0.9 to 1.4 sec ) , without a concomitant increase in variance or reduction in associated sniff rate . exploratory time bins also differed to a degree similar to wt ( figure 13 , right ) . as we propose that exploration of an odor object should ultimately be guided by both proximity and the direct means of exploring it via sniffing , we lastly also show the scores using the criterion of distance < 2 cm and sniff rate > 8.2 hz ( figure 14 ) . this sniff rate threshold was chosen so as to be highly unusual when not exploring . it was calculated from the mean + 1.96 sd rate ( 97.5th percentile ) using di < 4 cm and ve > 0.5 cm / s ( sniff_off_di4xve05_b , figures 12 and 13 ) and was the same for both groups of mice . the results were very similar to the results using the di < 4 cm and ve > 0.5 cm / s criterion . the present work explores the criteria used to score odor exploration time during the haxha task , using sniff rate as the ultimate guide . thorough analysis was afforded through the combined use of video tracking ( noldus ) and whole - body plethysmography ( buxco ) . although it could be recommended to measure sniff rate during the haxha task to aid determination of exploration time in general , the sniff measure may typically not be available for various reasons . we hence sought to corroborate the validity of the criteria used thus far in the literature and explored if improvements were possible in absence of sniffing data in a total of 19 mice across 4 trial types . we found that the use of < 2 cm proximity to the odorant alone , or in combination with the somewhat redundant head orientation criterion of < 20 , provided a remarkably accurate estimate of the odor exploration time . sniff rates were clearly elevated during such exploration times ( figures 1 and 2 ) , and distance and angle were correlated to sniff rate ( figures 4 and 5 ) , depended on trial type and sniff rate ( figures 6 and 7 ) and together explained 35% of sniff rate during s1 trials ( figures 8 and 9 ) . the exploration sniff rate using the proximity criterion alone or the combined proximity - orientation criterion also could not be significantly improved upon without a drastic loss of identified exploration time ( figures 10 and 11 ) . we conclude that the commonly used proximity criterion ( < 2 cm ) alone or combined with head angle ( < 20 ) is a very effective tool to estimate odor exploration in the haxha test . we also explored the use of other behavioral variables to estimate odor exploration time . whereas the acceleration of mice was found to be an inconsistent predictor ( not shown ) , velocity was remarkably consistent ( figure 4 ) across trial types and subjects . velocity was strikingly related to sniff rate , albeit with different functions across trial types ( figure 6 ) , suggesting that sniffing and moving are somehow coupled and in a context - dependent way . proximity and velocity combined explained 60% of sniff rate during s1 trials , double that of the standard criterion ( figures 8 and 9 ) . although using distance and velocity as criterion was unable to appreciably increase sniff rate during exploration time over the standard without massively reducing the exploration time , using velocity ( > 0.5 cm ) combined with a relaxed distance criterion ( < 4 cm ) retained the high sniff rate while increasing s1 exploration time ( figures 10 and 11 ) . when tested on a trial - trial basis the new criterion typically increased exploration time by 1544% , and was more robust by avoiding outliers leading to substantially different and better statistical conclusions in case of wt mice ( figures 12 and 13 ) . for 129 strain mice we can hence recommend the criterion of proximity < 2 cm and velocity > 0.5 cm / s over the standard criterion of proximity < 2 cm and head angle < it is clear that mice olfactorily explore the entire haxha environment as indicated by increased sniff rate when traveling through the box ( figure 3 ) and sniff fast only 30% of the time near the odorant even during s1 trials ( figure 7 ) . furthermore , while velocity appeared strongly related to sniff rate in general , at very high velocities ( > 4 cm / s ) it was only apparent when very near the odor source ( figure 11 , d1v2 ) . when further away from the source higher velocities appeared to coincide with reduced sniff rates ( e.g. , see widely spaced circles in figure 3 ) evidenced by the saddle form of the velocity - distance regression in figure 9 . the general relationship between velocity and sniffing was somewhat surprising to us as we expected the mice to also show stop and sniff behavior which we did not find evidence for given the effectiveness of the velocity threshold criterion . it hence appears that 129 mice modulate their body position at least somewhat while sniffing the cotton tip . it should be pointed out that , unlike common setups where the tip is mounted on the cage lid sufficiently elevated from the floor to induce rearing and concomitant low velocity once reared , our cotton tip was mounted 1 cm from the floor so as not to require rearing . while not being the focus of this paper , we found that fus ko mice showed reduced habituation and cross - habituation compared to wt mice ( di4-ve05 ) . both groups showed significant cross - habituation ( pe3 versus s1 ) , but wt mice explored the s1 odorant for longer duration ( 18.2 3.9 sec , figure 12 , center ) than fus ko mice ( 7.5 2.1 sec , figure 13 , center ) . this difference was quite significant using the new di4-ve05 exploration criterion ( p = 0.019 , 1-sided unpaired t - test ) and using the di2-an20 criterion ( p = 0.046 , 1-sided unpaired t - test ) . wt mice significantly cross - habituated during the s2 trial ( 5.2 2.6 sec ) , whereas the fus ko mice did not ( 6.5 2.8 sec ) . the complete haxha sessions consisted of 3 presentations each of mineral oil ( mo , diluent control ) , followed by amyl acetate ( aa ) , pe , and finally s. we intentionally omitted stimulus presentations prior to pe3 from this paper for sake of clarity of an already complex data set . the chosen trials were deemed sufficient to demonstrate large differences in exploration time accompanied by similarly large differences in sniff rates . for this entire series ( mo , aa , pe , and s ) repeated measures anova showed a significant effect of trial on exploration time for wt mice ( p < 0.01 , f2.3,30.0 = 5.8 ) and for fus ko mice ( p < 0.001 , f3.0,62.0 = 11.7 ) when using the standard noldus criteria . wt mice only showed significant cross - habituation between pe3 and s1 and no significant habituation . fus mice showed significant cross - habituation ( increase ) between aa3 and pe1 ( p < 0.05 ) and pe3 and s1 ( p < 0.001 ) and significant habituation ( decrease from first to third trial ) for mo and pe ( p < 0.05 ) and s ( p < 0.001 ; all bonferroni - corrected post hoc t - test on exploration time based on standard criteria ) . these results support the notion that the fus mice displayed haxha , whereas this is less evident for the wt mice ( due to absent habituation ) in which case differences in interest in pe and s may be responsible for the different exploration times . as proof of principle we also showed that the criterion based on both sniff rate and proximity is sensitive and robust ( figure 14 ) . it yielded remarkably similar results to the other indirect approaches to assess olfactory guided exploration , again attesting to their effectiveness . the most notable difference was the higher fus ko exploration time from 6.5 1.8 ( di2an20 ) and 7.5 2.1 ( di4ve05 ) to 9.8 2.9 sec , ( di4sniff8.2 ) which subsequently was marginally significantly lower than that of wt ( 17.9 4.5 sec , p = 0.076 , 1-sided unpaired t - test ) . other sniff thresholds may show somewhat different results , but preliminary exploration suggests that the outcome is rather robust to sniff rate threshold ( not shown ) . in conclusion , we confirm that standard haxha exploration criteria are fairly accurate at assessing sniff - modulated odorant exploration . we suggest that using velocity or sniffing itself rather than head orientation , combined with proximity , as criterion provides more accurate and more robust results . we further suggest that combined use of video tracking and sniff measurements is optimally suited to perform haxha experiments .

the habituation / cross - habituation test ( haxha ) is a spontaneous odor discrimination task that has been used for many decades to evaluate olfactory function in animals . animals are presented repeatedly with the same odorant after which a new odorant is introduced . the time the animal explores the odor object is measured . an animal is considered to cross - habituate during the novel stimulus trial when the exploration time is higher than the prior trial and indicates the degree of olfactory patency . on the other hand , habituation across the repeated trials involves decreased exploration time and is related to memory patency , especially at long intervals . classically exploration is timed using a stopwatch when the animal is within 2 cm of the object and aimed toward it . these criteria are intuitive , but it is unclear how they relate to olfactory exploration , that is , sniffing . we used video tracking combined with plethysmography to improve accuracy , avoid observer bias , and propose more robust criteria for exploratory scoring when sniff measures are not available . we also demonstrate that sniff rate combined with proximity is the most direct measure of odorant exploration and provide a robust and sensitive criterion .

1. Introduction 2. Experimental Procedures 3. Results 4. Discussion

the olfactory habituation / cross - habituation test ( haxha ) is a noninvasive spontaneous behavioral task that has been used to study the ability to smell and the capacity to discriminate between stimuli ( odors ) in a large variety of animals and humans . meanwhile the presentation of a different stimulus leads to a change in the amplitude of the habituated response ( cross - habituation ) [ 1 , 2 ] . the general protocol for olfactory haxha in rodents consists of presenting an odor ( in a paper filter or cotton applicator ) generally in the center of the experimental cage and measuring the time in which an animal is oriented to and within 2 cm of the odorant . to measure the habituation phase , the novel stimulus is presented several times ( trials ) . the cross - habituation phase can be studied by changing the stimulus for a novel unfamiliar odor . for the odor - cued taste avoidance , which combines odor detection and odor discrimination , the mice have to be thirsty to perform the task . another advantage is that the haxha test is a spontaneous odor discrimination task which requires no training which may involve cognitive processes unrelated to the ones being under investigation . despite these advantages and ability to target the patency of different neural structures by adjusting the timescale , the standard means to measure the exploration time is by a human experimenter using a stopwatch . last , a human observer will have limited accuracy in scoring the behavior according to exact criteria such as proximity within 2 cm of the odor source and precise head angle toward it . to avoid these potential confounds altogether we used behavioral video tracking software ( noldus ) , which is able to recognize the nose , the center , and the tail of the mouse at 10 frames / sec . this allowed us to quantify the distance between the nose and the odor stimulus , the head angle to the odorant , and the locomotion of the animal ( position and velocity ) . sniffing behavior in rodents is dynamic , varies with the behavioral context , and is modulated by olfactory and nonolfactory processes . meanwhile , the frequency of sniffing ( sniff rate ) has been used as a parameter to characterize sniffing behaviors in rodents , as they increase from 2 hz to 412 hz when they are investigating novel odors [ 18 , 19 ] . recently , olfactory alterations in transgenic mice models for familial ad were detected with haxha using food and sex - related odors as the stimuli , where olfactory deficits were correlated with the stage of the disease [ 32 , 33 ] . our previous studies suggest that the female fus1 ko mice at 10 months old have alterations in their habituation to nonsocial odors and deteriorations in their cross - habituation for social odors ( ms submitted for publication ) . here , we propose a novel approach to accurately assess haxha using the noldus system to track the behavior of every animal combined with whole - body plethysmography to noninvasively evaluate their sniffing . we confirm that standard criteria accurately capture sniffing - mediated olfactory exploration and suggest that velocity combined with a relaxed distance criterion outperforms odor exploration quantification . a cotton - tipped wood applicator ( puritan ref 806-wc ) was presented mounted in a removable holder on the bottom of the cage , located 1 cm above the cage floor . odor exploration was defined as being oriented toward the applicator tip while the nose was within 2 cm of it . this test evaluates if mice are able to spontaneously recognize a novel odorant stimulus by spending more time smelling the applicator ( cross - habituation phase ) , as opposed to the time that the mice spend on each repeated stimulus ( habituation phase ) . in order to reduce timing errors and experimenter bias in measuring the habituation / cross - habituation task and to know if these behavioral responses were specific to the olfactory abilities of the animals , we combined a system to automatically record behavioral responses using the noldus behavioral tracking system ( ethovision xt , version 10.1 , noldus information technology b.v . noldus software analyzed the camera input to identify and score the behavior of the animal . we saturated the cotton applicator with one of the following experimental odors : mo , aa , pe , and s. to measure sniffing noninvasively , a pressure sensor ( buxco , trd5700 ) communicated with the experimental box . the transducer signals were amplified 10x ( a - m systems , differential ac amplifier model 1700 ) , band - pass filtered between 0.1 and 40 hz and 60 hz notch filtered ( a - m systems ) , and subsequently filtered between 0.1 and 40 hz with an 8th order linkwitz - riley filter using a minidsp 2 4 processor ( minidsp , hong kong ) . during 2 minutes of each trial the behavior of a mouse was recorded , and when the rodent 's nose was oriented to the cotton within a 2 cm distance a signal was generated via the noldus mini - usb i / o box , identifying the period that the mouse was exploring the stimulus , and sent to the neuroplex data - acquisition system . only the trial types pe3 , s1 , s2 , and s3 were used , as s1 was by far the most explored stimulus . for deeper analyses the following procedure was used to integrate the neuroplex - based sniffing signal ( 200 s / s ) with the neuroplex video tracking output ( 10 s / s ) . stepwise multiple regression was computed using population vectors for regressing angle , distance , and velocity ( matlab stepwiselm ) onto sniff rate on this . the olfactory haxha task is used to evaluate the patency of the olfactory system by asking how much time an animal spontaneously explores new or previously presented odor objects . it is hence assumed that the exploration time is equivalent to the time smelling to date sniffing has not been used as a key marker of this behavior . using our noldus video tracking system combined with whole - body plethysmography we were able to address the question of whether the criteria used thus far to measure exploration , namely , distance and head angle to the object , are indeed related to enhanced sniff rates . figure 1 shows this to be the case : sniff rates are enhanced during periods that the wt mice were within 2 cm of the cotton tip and with their head aimed to it within 20 ( onfreq ) , as compared to other periods ( offfreq ) , for each trial type ( pe3 , s1 , s2 , and s3 ) . pe3 shows low exploration time ( ontime < 1 sec ) indicative of habituation , followed by a cross - habituation of ~14 sec exploration to the first social odor presentation ( s1 ) . in contrast to the sniff rate being increased during proximity and orientation to the object , there was no evidence that sniff amplitudes were modulated by trial type . figure 2 shows that sniff rates followed the same pattern as wt mice , with s1 sniff rates at 10.1 hz during proximity and orientation to the odorant , versus 6.0 hz otherwise . we conclude that the proximity criterion of < 2 cm and < 20 to the odorant is indicative of olfactory exploration during habituation and cross - habituation for female mice of the 129 wt strain as well as for their fus ko chronic oxidative stress counterparts . figure 3 illustrates this finding for two wt mice during s1 trials by showing their path ( large solid circle : start ; small solid circle : end ) in the cage ( one dot per 100 ms bin , 1201 dots total ) , the head angle to the cotton tip ( smaller size indicating smaller angle ) , and sniff rate ( color coded ) . in light of our findings that these criteria are predictive of sniff rate , the subtler question arises of whether the classical proximity and orientation criteria are optimal , either in isolation or combined , as a proxy for sniffing - mediated odorant exploration when sniff rate is not or can not be measured ( as is the general approach ) . as expected from the prior results , distance is mostly negatively correlated , in particular during the cross - habituation s1 trials ( r = 0.49 ) when sniff rate is modulated most strongly . in a complimentary approach time bins were categorized to belong to one of four sniff rate quartiles ( analyzed per trial ) and associated distance , angle , and velocity were assessed . exploration of the proximity during time bins associated with each sniff rate quartile shows that distance is smallest during s1 trials especially at the top quartile ( figure 6(b ) ) . this indicates that small distance to the odorant during cross - habituation has a high specificity as a marker of olfactory exploration , a further validation of the proximity criterion . although angle showed similar tendencies as distance , it lacked the specificity thereof ( figure 6(c ) ) : orientation to the odor source occurred in overlapping degrees across sniff rate quartiles , notably s2 at lowest three quartiles . we further explored at what fraction of time when sniffing at each sniff rate quartile the wt mice would satisfy the categorical proximity ( < 2 cm ) , orientation ( < 20 ) , or their combined ( < 2 cm & < 20 ) criterion ( figure 7 ) . thus far we have shown that complimentary analytical approaches confirm that proximity and ( to a lesser degree ) orientation to the odorant relate to enhanced sniffing , especially during s1 cross - habituation trials , lending credit to this long - used means of measuring odor object exploration duration ( in the absence of a direct sniff rate measure ) . we also found that velocity may be an additionally useful factor to consider in quantifying odor exploration in the haxha test , due to its strong and consistent relation to sniff rate . while we found that distance and angle combined could explain 35% of wt s1 sniff rate variance ( adjusted r , 2nd - order regression , dist - angle 2nd ) , distance and velocity explained a rather high 60% ( dist - veloc 2nd ) of wt s1 trial sniff rate variance , and > 40% of sniff rate variance across all wt and fus trials ( wt all , fus ko all ) . in an effort to establish new criteria based on the above findings we used the s1 trial population vectors to see which exploration times ( figure 10 ) and exploration sniff rates ( figure 11 ) would result from using different criterion thresholds and their combinations . optimal criteria should yield the highest exploration time without substantially lowering the sniff rate during it . figure 10 ( top ) shows the fraction of time the mice would be considered to be exploring with distance criteria of less than 1 , 2 , 4 , or 8 cm ( left , d1d8 ) , angle of less than 5 , 10 , 20 , or 40 ( middle , a5a40 ) , or velocity of more than 4 , 2 , 1 , or 0.5 cm / s ( right , v4v0.5 ) . the distance threshold < 2 cm ( d2 ) was marked , including a horizontal line for reference , as it is the standard criterion and yields exploration for 11% of time ( 13.2 sec for a 120 sec trial , like the trial - based noldus result of 14.1 sec in figure 1 , right ) . figure 11 ( top ) shows that no single threshold can substantially improve on d2 , yielding 9.6 hz sniffing during the 11% exploration time ( close to 9.5 hz onfreq in figure 1 ; 6.5 hz is shown as y - axis bar cut - off as this is the offfreq baseline sniff rate , i.e. sniff rate would increase slightly by tightening the distance threshold from < 2 to < 1 cm from odor source , but exploration time would drop dramatically from 11% to 5% ( figure 10 , top : d2 versus d1 ) . in contrast , relaxed distance threshold < 4 cm and velocity > 0.5 cm / s ( d4 and v0.5 , marked ) yielded the same 9.6 hz as the standard but increased exploration time from 11% to 15% , thereby being suggestive of a criterion better able to identify sniff - mediated odor exploration . the left graph in these figures ( onfreq , offfreq , and ontime ) is identical to that of figures 1 and 2 using the noldus d < 2 cm and a < 20 criterion ( using unsmoothed data ) and is shown for reference . the neighboring graphs ( marked _ b ) show these results upon removal of trials of mice yielding < 1 sec exploration time , which helped robustness in particular for the d4-v0.5 criterion by raising s2 and s3 sniff rates during exploration and reducing their sem ( sniff_on_di4xve05_b ) for both wt and fus ko mice . the d4-v0.5 criterion increased wt s1 trial exploration time ( explore_on_di4xve05_b ) over the standard d2-a20 criterion ( ontime_b ) by 39% from 14.1 sec to 18.2 sec ( orange line ) , without decreasing sniff rate ( 9.5 hz , figure 12 ) . the new criterion is apparently more robust in avoiding such pitfalls , which can allow for a statistically more powerful ( discriminating ) assessment of habituation and cross - habituation . s1 exploration time increased by 15% ( 6.5 sec to 7.5 sec ) , s2 by 44% ( 4.5 sec to 6.5 sec ) , and s3 by 0.5 sec ( 0.9 to 1.4 sec ) , without a concomitant increase in variance or reduction in associated sniff rate . as we propose that exploration of an odor object should ultimately be guided by both proximity and the direct means of exploring it via sniffing , we lastly also show the scores using the criterion of distance < 2 cm and sniff rate > 8.2 hz ( figure 14 ) . the present work explores the criteria used to score odor exploration time during the haxha task , using sniff rate as the ultimate guide . although it could be recommended to measure sniff rate during the haxha task to aid determination of exploration time in general , the sniff measure may typically not be available for various reasons . we found that the use of < 2 cm proximity to the odorant alone , or in combination with the somewhat redundant head orientation criterion of < 20 , provided a remarkably accurate estimate of the odor exploration time . sniff rates were clearly elevated during such exploration times ( figures 1 and 2 ) , and distance and angle were correlated to sniff rate ( figures 4 and 5 ) , depended on trial type and sniff rate ( figures 6 and 7 ) and together explained 35% of sniff rate during s1 trials ( figures 8 and 9 ) . the exploration sniff rate using the proximity criterion alone or the combined proximity - orientation criterion also could not be significantly improved upon without a drastic loss of identified exploration time ( figures 10 and 11 ) . we conclude that the commonly used proximity criterion ( < 2 cm ) alone or combined with head angle ( < 20 ) is a very effective tool to estimate odor exploration in the haxha test . we also explored the use of other behavioral variables to estimate odor exploration time . velocity was strikingly related to sniff rate , albeit with different functions across trial types ( figure 6 ) , suggesting that sniffing and moving are somehow coupled and in a context - dependent way . proximity and velocity combined explained 60% of sniff rate during s1 trials , double that of the standard criterion ( figures 8 and 9 ) . although using distance and velocity as criterion was unable to appreciably increase sniff rate during exploration time over the standard without massively reducing the exploration time , using velocity ( > 0.5 cm ) combined with a relaxed distance criterion ( < 4 cm ) retained the high sniff rate while increasing s1 exploration time ( figures 10 and 11 ) . when tested on a trial - trial basis the new criterion typically increased exploration time by 1544% , and was more robust by avoiding outliers leading to substantially different and better statistical conclusions in case of wt mice ( figures 12 and 13 ) . for 129 strain mice we can hence recommend the criterion of proximity < 2 cm and velocity > 0.5 cm / s over the standard criterion of proximity < 2 cm and head angle < it is clear that mice olfactorily explore the entire haxha environment as indicated by increased sniff rate when traveling through the box ( figure 3 ) and sniff fast only 30% of the time near the odorant even during s1 trials ( figure 7 ) . furthermore , while velocity appeared strongly related to sniff rate in general , at very high velocities ( > 4 cm / s ) it was only apparent when very near the odor source ( figure 11 , d1v2 ) . while not being the focus of this paper , we found that fus ko mice showed reduced habituation and cross - habituation compared to wt mice ( di4-ve05 ) . both groups showed significant cross - habituation ( pe3 versus s1 ) , but wt mice explored the s1 odorant for longer duration ( 18.2 3.9 sec , figure 12 , center ) than fus ko mice ( 7.5 2.1 sec , figure 13 , center ) . wt mice significantly cross - habituated during the s2 trial ( 5.2 2.6 sec ) , whereas the fus ko mice did not ( 6.5 2.8 sec ) . for this entire series ( mo , aa , pe , and s ) repeated measures anova showed a significant effect of trial on exploration time for wt mice ( p < 0.01 , f2.3,30.0 = 5.8 ) and for fus ko mice ( p < 0.001 , f3.0,62.0 = 11.7 ) when using the standard noldus criteria . wt mice only showed significant cross - habituation between pe3 and s1 and no significant habituation . fus mice showed significant cross - habituation ( increase ) between aa3 and pe1 ( p < 0.05 ) and pe3 and s1 ( p < 0.001 ) and significant habituation ( decrease from first to third trial ) for mo and pe ( p < 0.05 ) and s ( p < 0.001 ; all bonferroni - corrected post hoc t - test on exploration time based on standard criteria ) . as proof of principle we also showed that the criterion based on both sniff rate and proximity is sensitive and robust ( figure 14 ) . it yielded remarkably similar results to the other indirect approaches to assess olfactory guided exploration , again attesting to their effectiveness . the most notable difference was the higher fus ko exploration time from 6.5 1.8 ( di2an20 ) and 7.5 2.1 ( di4ve05 ) to 9.8 2.9 sec , ( di4sniff8.2 ) which subsequently was marginally significantly lower than that of wt ( 17.9 4.5 sec , p = 0.076 , 1-sided unpaired t - test ) . other sniff thresholds may show somewhat different results , but preliminary exploration suggests that the outcome is rather robust to sniff rate threshold ( not shown ) . in conclusion , we confirm that standard haxha exploration criteria are fairly accurate at assessing sniff - modulated odorant exploration . we suggest that using velocity or sniffing itself rather than head orientation , combined with proximity , as criterion provides more accurate and more robust results .

heart failure ( hf ) is a complex clinical syndrome and global public health problem affecting an estimated 26 million people worldwide.1 despite increased utilization of pharmacological2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 and device therapy options10 , 11 , 12 , 13 that improve clinical outcomes in randomized controlled trials , morbidity and mortality in hf remain a major burden to patients , their caregivers , and national healthcare systems . patients with hf frequently experience worsening symptoms related to accumulation of excess intravascular volume and congestion , requiring hospitalization to provide intravenous medical support to restore normal volume.14 , 15 , 16 , 17 , 18 , 19 heart failure is cited as the most frequent cause of hospitalization in the us medicare population , resulting in > 1 million admissions per year ( accounting for 12% of all hospitalizations).20 , 21 the economic impact of hf in the usa is profound , with the total costs of hf estimated to increase from us$31 billion in 2012 to us$70 billion in 2030 secondary to an ageing population.22 costeffective hf management strategies are required to address this growing problem . elevated cardiac filling pressures are associated with higher rates of rehospitalization and mortality in patients with hf.23 , 24 independent of lvef , rises in cardiac filling pressures can often be detected several weeks prior to patients experiencing symptoms of hf decompensation that require hospitalization.25 , 26 remote monitoring of intracardiac and pulmonary artery pressures ( paps ) in patients with hf using implantable haemodynamic monitoring devices can provide physicians with access to actionable pathophysiological information and help improve the hf management decisionmaking process necessary to prevent hf hospitalizations.27 , 28 , 29 , 30 a novel wireless pap measurement system ( cardiomems hf system , st . atlanta , ga , usa ) was evaluated in the cardiomems heart sensor allows monitoring of pressure to improve outcomes in new york heart association ( nyha ) functional class iii heart failure patients ( champion ) trial.30 , 31 the champion trial was a prospective , multicentre , randomized , singleblind clinical study in 550 patients that tested the incremental impact of papguided hf management on clinical outcomes compared with hf management based on current american college of cardiology foundation / american heart association practice guidelines only . the papguided hf management group in the champion trial experienced a significant reduction in hf hospitalization rates , a greater reduction in paps , fewer patients hospitalized for hf , and more days alive and outside of the hospital for hf , and exhibited an improvement in quality of life when compared with guidelinedirected standard of care hf management only ( control group).30 these longterm benefits were seen in patients with hf and preserved ef,32 secondary pulmonary hypertension,33 and comorbid chronic obstructive cad.34 hospitalization reductions were seen after an average of 18 months of randomized followup , with additional longterm benefits noted in the 13 months of openaccess , which immediately followed the end of the randomized followup in the trial.35 based on these data , the cardiomems hf system was approved by the us food and drug administration for hf management in nyha class iii hf patients with a hf hospitalization within the last 12 months . this study is a comprehensive analysis of the costeffectiveness of this treatment strategy in the context of the us medical system . a markov model was utilized to approximate the course of management observed in the champion trial for the treatment and control groups using monte carlo simulation . the objective was to estimate the costs and costeffectiveness over a time horizon extended beyond the study followup period . healthcare utilization event rates , survival , and quality of life were based on study data . we took the perspective of the payer , and focused on the medicare and private insurance patient populations as they represent the vast majority of patients eligible for the cardiomems hf system in the usa , and cost data were available for these two patient populations to conduct our analyses . model endpoints included cost of treatment and preferenceweighted survival , which were then used to calculate incremental cost per life year gained and cost per qualityadjusted life year ( qaly ) gained for the treatment compared with control . stable hf , hospitalized for hf , hospitalized for other cause , and death ( figure 1 ) . each simulated patient transitioned through the states in cycles of 1 month , incurring costs and accumulating effects associated with each state . the total time horizon of the simulation for the base case was 5 years ( or 60 cycles ) . costs and effects were discounted at 3% per year.36 a total of 100 000 patients were simulated in each group . markov model used to approximate the course of management observed in the champion trial for the treatment and control groups . stable hf state was characterized as a patient that received typical care for hf including physician visits , prescription drugs , longterm care , and outpatient hospital visits . a patient in the hospitalized for hf state was characterized as a patient undergoing inpatient hospitalization related to a primary diagnosis of hf . since the average length of stay for a hf hospitalization in the usa is 5.2 days for all patients according to the healthcare cost and utilization project ( hcup ) national statistics ( 2012),37 we assumed that in this state , a patient incurred the same expense as in the stable hf state in addition to the cost of hf hospitalization . in the model simulation , stable hf state , and the treatment group started the simulation with an implantrelated hospitalization . a patient in the hospitalized for other cause state was characterized as a patient undergoing inpatient hospitalization for any reason other than a primary diagnosis of hf . since the average length of stay for a hospitalization for any cause in the usa is 4.5 days for all patients according to the hcup national statistics ( 2012),37 we assumed that in this state , a patient incurred the same expense as in the stable hf state in addition to the cost of othercause hospitalization . as part of the prospective champion trial design , all patients initially underwent implantation of the pap sensor prior to randomization . all patients then remained in their randomized study group until the last patient to be enrolled completed at least 6 months of study followup ( randomized access period ) . at the conclusion of the randomized access period , all active patients transitioned to a followup period where study physicians then had access to pap information for all patients ( open access period ) . for this costeffectiveness analysis ( cea ) , estimates of the transition probabilities among states , mortality , and eq5d preference weight utilities in each state came from the complete randomized access period of the champion trial ( table 1 ) . all hospitalizations were adjudicated by a clinical event classification ( cec ) committee as part of the champion trial and classified in one of two ways : inpatient hospitalization associated with hf or inpatient hospitalization not associated with hf . longterm clinical outcomes from champion trial randomized access period35 nnt , number needed to treat ; rrr , relative risk reduction . hazard ratio ( hr ) , 95% confidence interval ( ci ) , and pvalue from the andersen gill model . hr and 95% ci from the cox proportional hazards model , pvalue from logrank test . healthrelated quality of life ( hrqol ) was assessed via the eq5d3 l38 questionnaire at baseline , 6 months , and 12 months in the champion trial . for the model , we utilized us populationbased eq5d3 l preference weights for this economic analysis.39 qalys were accumulated based on the assumption that the preference weight was constant between measurement intervals . health insurance is purchased in the private marketplace or provided by the government to certain groups ( e.g. medicare insurance to the elderly and disabled population ) . because of this , we used a payer mix based on the age distribution in the champion study cohort . patients less than 65 years old at implant were assumed to be paid through private insurance , and those 65 years or older at implant were assumed to be paid by medicare . in the usa , the implant of the cardiomems hf system is associated with the msdrg ( medicare severity diagnosis related groups ) payment for 264 accompanied by the icd9cm ( international classification of diseases , ninth revision , clinical modification ) procedure code of 38.26 : insertion of an implantable pressure sensor without a lead for intracardiac or great vessel haemodynamic monitoring . in the base case , the cost of system implantation in the treatment group was us$17 75040 and we made the assumption that all implants occurred on a unique scheduled day for each patient , and did not occur during a preexisting hf hospitalization . payer costs for hospitalizations postimplant ( which are reimbursements ) were determined from the truven health marketscan april 2008 to march 2013 commercial claims and encounters and medicare supplemental and coordination of benefits database . this marketscan database represents the deidentified health services of employees , dependents , and retirees in the usa with primary or medicare supplemental coverage through privately insured feeforservice , pointofservice , or capitated health plans . all enrolment records and inpatient , outpatient , ancillary , and drug claims were tabulated . we identified a hf cohort from this claims database by ascertaining patients with an inpatient hospitalization with primary diagnosis of hf ( icd9cm diagnosis code of 428.x ) . the hf cohort consisted of 200 471 patients that had medicare advantage insurance and private insurance . the medicare patients consisted of 50% male patients with an average age of 80 8 years . the private insurance patients consisted of 60% male patients with an average age of 57 9 years . the distribution of geographic locations for all patients were : northeast ( 20% ) , north central ( 33% ) , south ( 31% ) , and west ( 14% ) . in this hf cohort , we identified payments for three types of healthcare utilization : average cost of inpatient hospitalization with primary diagnosis code of hf , average cost of inpatient hospitalization not related to hf , and annual costs for outpatient healthcare utilization of any type for these patients ( table 2 ) . these payments corresponded to the states of hospitalized for hf , hospitalized for other cause , and the payments included facility cost and professional fees associated with the event , adjusted to 2014 us dollars based on the consumer price index ( cpi ) inflation from the bureau of labor statistics . stable hf included the majority of expenses a nonhospitalized hf patient incurs receiving medical care ; this included physician visits , prescription drugs , longterm care , and outpatient hospital visits . in addition to this , a patient implanted with the cardiomems hf system was assumed to incur a monthly cost of us$45 associated with the professional and technical components of reimbursement for remote physiological monitoring . since remote physiological monitoring is conducted for various reasons for patients that have not been implanted with the cardiomems hf system , we assumed that 25% of the standard of care patients also incur this monthly cost . the champion trial reported eight device and systemrelated complications ( dsrcs ) during 575 implant attempts.3 all of these events occurred within the first 30 days of implant . using the details from hospital admission and discharge records for each one of the eight dsrcs , we ascertained an msdrg for each such hospitalization , and then determined an average cost of dsrcs based on the 2014 reimbursement for the msdrgs . no further device or systemrelated complications or sensor failures were reported in an average of 31 months following implant.36 the primary efficacy endpoint of the champion trial was the rate of hfrelated hospitalizations . thus , our primary effectiveness endpoint was the incremental costeffectiveness ratio ( icer ) comparing the costs and qalys of hf hospitalization outcomes in the pap treatment and control groups . oneway sensitivity analyses were performed to assess the impact of varying selected model parameters while holding other variables fixed at their base case values . these parameters included hf hospitalization rates , mortality rates , cost of hf hospitalization , monitor implant cost , and payer mix . the goal was to understand how robust the base case results were to uncertainty about the values used . in addition , sensitivity analyses were conducted on the costs included in the analysis : hf hospitalizations only , allcause hospitalizations , and all chronic hf management costs including allcause hospitalizations . two probabilistic sensitivity analyses ( psas ) were performed with 100 000 resamples of the model parameters to examine the impact of their combined uncertainty . tabulation of distributions used for the parameters in the probabilistic sensitivity analyses are provided in the supplementary material online , table s1 . an assumption in this model is that the outpatient costs increase due to an improved survival benefit ( not due to other changes ) . each simulated patient transitioned through the states in cycles of 1 month , incurring costs and accumulating effects associated with each state . the total time horizon of the simulation for the base case was 5 years ( or 60 cycles ) . costs and effects were discounted at 3% per year.36 a total of 100 000 patients were simulated in each group . markov model used to approximate the course of management observed in the champion trial for the treatment and control groups . stable hf state was characterized as a patient that received typical care for hf including physician visits , prescription drugs , longterm care , and outpatient hospital visits . a patient in the hospitalized for hf state was characterized as a patient undergoing inpatient hospitalization related to a primary diagnosis of hf . since the average length of stay for a hf hospitalization in the usa is 5.2 days for all patients according to the healthcare cost and utilization project ( hcup ) national statistics ( 2012),37 we assumed that in this state , a patient incurred the same expense as in the stable hf state in addition to the cost of hf hospitalization . in the model simulation , stable hf state , and the treatment group started the simulation with an implantrelated hospitalization . a patient in the hospitalized for other cause state was characterized as a patient undergoing inpatient hospitalization for any reason other than a primary diagnosis of hf . since the average length of stay for a hospitalization for any cause in the usa is 4.5 days for all patients according to the hcup national statistics ( 2012),37 we assumed that in this state , a patient incurred the same expense as in the stable hf state in addition to the cost of othercause hospitalization . as part of the prospective champion trial design , all patients initially underwent implantation of the pap sensor prior to randomization . all patients then remained in their randomized study group until the last patient to be enrolled completed at least 6 months of study followup ( randomized access period ) . at the conclusion of the randomized access period , all active patients transitioned to a followup period where study physicians then had access to pap information for all patients ( open access period ) . for this costeffectiveness analysis ( cea ) , estimates of the transition probabilities among states , mortality , and eq5d preference weight utilities in each state came from the complete randomized access period of the champion trial ( table 1 ) . all hospitalizations were adjudicated by a clinical event classification ( cec ) committee as part of the champion trial and classified in one of two ways : inpatient hospitalization associated with hf or inpatient hospitalization not associated with hf . longterm clinical outcomes from champion trial randomized access period35 nnt , number needed to treat ; rrr , relative risk reduction . hazard ratio ( hr ) , 95% confidence interval ( ci ) , and pvalue from the andersen gill model . hr and 95% ci from the cox proportional hazards model , pvalue from logrank test . healthrelated quality of life ( hrqol ) was assessed via the eq5d3 l38 questionnaire at baseline , 6 months , and 12 months in the champion trial . for the model , we utilized us populationbased eq5d3 l preference weights for this economic analysis.39 qalys were accumulated based on the assumption that the preference weight was constant between measurement intervals . health insurance is purchased in the private marketplace or provided by the government to certain groups ( e.g. medicare insurance to the elderly and disabled population ) . because of this , we used a payer mix based on the age distribution in the champion study cohort . patients less than 65 years old at implant were assumed to be paid through private insurance , and those 65 years or older at implant were assumed to be paid by medicare . in the usa , the implant of the cardiomems hf system is associated with the msdrg ( medicare severity diagnosis related groups ) payment for 264 accompanied by the icd9cm ( international classification of diseases , ninth revision , clinical modification ) procedure code of 38.26 : insertion of an implantable pressure sensor without a lead for intracardiac or great vessel haemodynamic monitoring . in the base case , the cost of system implantation in the treatment group was us$17 75040 and we made the assumption that all implants occurred on a unique scheduled day for each patient , and did not occur during a preexisting hf hospitalization . payer costs for hospitalizations postimplant ( which are reimbursements ) were determined from the truven health marketscan april 2008 to march 2013 commercial claims and encounters and medicare supplemental and coordination of benefits database . this marketscan database represents the deidentified health services of employees , dependents , and retirees in the usa with primary or medicare supplemental coverage through privately insured feeforservice , pointofservice , or capitated health plans . all enrolment records and inpatient , outpatient , ancillary , and drug claims were tabulated . we identified a hf cohort from this claims database by ascertaining patients with an inpatient hospitalization with primary diagnosis of hf ( icd9cm diagnosis code of 428.x ) . the hf cohort consisted of 200 471 patients that had medicare advantage insurance and private insurance . the medicare patients consisted of 50% male patients with an average age of 80 8 years . the private insurance patients consisted of 60% male patients with an average age of 57 9 years . the distribution of geographic locations for all patients were : northeast ( 20% ) , north central ( 33% ) , south ( 31% ) , and west ( 14% ) . in this hf cohort , we identified payments for three types of healthcare utilization : average cost of inpatient hospitalization with primary diagnosis code of hf , average cost of inpatient hospitalization not related to hf , and annual costs for outpatient healthcare utilization of any type for these patients ( table 2 ) . the payments included facility cost and professional fees associated with the event , adjusted to 2014 us dollars based on the consumer price index ( cpi ) inflation from the bureau of labor statistics . stable hf included the majority of expenses a nonhospitalized hf patient incurs receiving medical care ; this included physician visits , prescription drugs , longterm care , and outpatient hospital visits . in addition to this , a patient implanted with the cardiomems hf system was assumed to incur a monthly cost of us$45 associated with the professional and technical components of reimbursement for remote physiological monitoring . since remote physiological monitoring is conducted for various reasons for patients that have not been implanted with the cardiomems hf system , we assumed that 25% of the standard of care patients also incur this monthly cost . the champion trial reported eight device and systemrelated complications ( dsrcs ) during 575 implant attempts.3 all of these events occurred within the first 30 days of implant . using the details from hospital admission and discharge records for each one of the eight dsrcs , we ascertained an msdrg for each such hospitalization , and then determined an average cost of dsrcs based on the 2014 reimbursement for the msdrgs . no further device or systemrelated complications or sensor failures thus , our primary effectiveness endpoint was the incremental costeffectiveness ratio ( icer ) comparing the costs and qalys of hf hospitalization outcomes in the pap treatment and control groups . oneway sensitivity analyses were performed to assess the impact of varying selected model parameters while holding other variables fixed at their base case values . these parameters included hf hospitalization rates , mortality rates , cost of hf hospitalization , monitor implant cost , and payer mix . the goal was to understand how robust the base case results were to uncertainty about the values used . in addition , sensitivity analyses were conducted on the costs included in the analysis : hf hospitalizations only , allcause hospitalizations , and all chronic hf management costs including allcause hospitalizations . two probabilistic sensitivity analyses ( psas ) were performed with 100 000 resamples of the model parameters to examine the impact of their combined uncertainty . tabulation of distributions used for the parameters in the probabilistic sensitivity analyses are provided in the supplementary material online , table s1 . an assumption in this model is that the outpatient costs increase due to an improved survival benefit ( not due to other changes ) . the purpose of pap monitoring via cardiomems was to reduce hf hospitalizations , hence the primary cea focused on comparing hf hospitalization outcomes . the costs associated with hf hospitalizations , device and systemrelated complications , and remote physiological monitoring were accumulated . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$56 974 ; patients in the control group had qalys of 2.16 with a total cost of us$52 149 . costeffectiveness analysis basecase costs and survival over a 5year time horizon cea , costeffectiveness analysis ; hf , heart failure ; qaly , quality adjusted life year . heart failure management is expensive , and , historically , studies related to crt management of hf patients have compared allcause hospitalization outcomes . although the cardiomems hf system did not have a significant impact on nonhfrelated hospitalizations , we compared all hospitalizations for the two groups to enable a comparison with historical studies . the costs associated with allcause hospitalizations , device and systemrelated complications , and remote physiological monitoring were accumulated . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . the total cost of patient management is the sum of various components for both hf and nonhf management care . the costs associated with hospitalizations for any reason , device and systemrelated complications , and remote physiological monitoring were accumulated . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . the icer was sensitive to the time horizon ( figure 2 ) . compared with the base case icer of us$12 262/qaly at 5 years , the icers at 4 years and 7 years were us$34 909/qaly and us$5412/qaly , respectively . the icers decreased over time ; this is a common observation when a highcost procedure occurs at the beginning of the time horizon , indicating that the therapy is costeffective primarily resulting from averted hospitalizations . the icers were also assessed when hf hospitalization rates , hf hospitalization costs , and mortality rates were varied . the icers varied between us$10 960/qaly and us$14 311/qaly when hf hospitalization rate varied between the 95% confidence interval of the hospitalization rates from the champion trial . the sensitivity around hf hospitalization cost showed the icers to vary between us$6172/qaly and us$30 696/qaly . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the icers varied between us$8456/qaly and us$16 854/qaly when the mortality rates varied between the 95% confidence bounds of the champion trial mortality rates . the icer was sensitive to the implant cost , and for an implant cost range of us$15 556 to us$26 704 , the icer ranged from us$6686/qaly and us$35 018/qaly . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . according to the hcup nis ( nationwide inpatient sample ) data set from 2012,37 amongst all inpatient hospitalizations associated with a primary or secondary diagnosis of hf identified via icd9cm code 428.x , there were 5 021 800 discharges for hf with the payer as medicare , and 674 680 discharges for hf in patients that had private insurance . using these two payer categories ( medicare and private payer ) , 88% of hf hospitalizations occurred in medicare beneficiaries . using this 88% ( medicare ) and 12% ( private insurance ) distribution to form the assumption of patient mix , the icer was us$20 734/qaly . for the primary cea ( using hf hospitalization costs only ) , the psa showed that at the willingnesstopay threshold of us$25 000 , > 85% of the simulations were costeffective ; > 99% were costeffective at the us$50 000 threshold . further , for the cea using comprehensive patient management costs , the psa showed that at the willingnesstopay threshold of us$50 000 , 87% of the simulations were costeffective ; > 99% were so at the us$100 000 threshold . the purpose of pap monitoring via cardiomems was to reduce hf hospitalizations , hence the primary cea focused on comparing hf hospitalization outcomes . the costs associated with hf hospitalizations , device and systemrelated complications , and remote physiological monitoring were accumulated . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$56 974 ; patients in the control group had qalys of 2.16 with a total cost of us$52 149 . costeffectiveness analysis basecase costs and survival over a 5year time horizon cea , costeffectiveness analysis ; hf , heart failure ; qaly , quality adjusted life year . heart failure management is expensive , and , historically , studies related to crt management of hf patients have compared allcause hospitalization outcomes . although the cardiomems hf system did not have a significant impact on nonhfrelated hospitalizations , we compared all hospitalizations for the two groups to enable a comparison with historical studies . the costs associated with allcause hospitalizations , device and systemrelated complications , and remote physiological monitoring were accumulated . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . the total cost of patient management is the sum of various components for both hf and nonhf management care . the costs associated with hospitalizations for any reason , device and systemrelated complications , and remote physiological monitoring were accumulated . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . the icer was sensitive to the time horizon ( figure 2 ) . compared with the base case icer of us$12 262/qaly at 5 years , the icers at 4 years and 7 years were us$34 909/qaly and us$5412/qaly , respectively . the icers decreased over time ; this is a common observation when a highcost procedure occurs at the beginning of the time horizon , indicating that the therapy is costeffective primarily resulting from averted hospitalizations . the icers were also assessed when hf hospitalization rates , hf hospitalization costs , and mortality rates were varied . the icers varied between us$10 960/qaly and us$14 311/qaly when hf hospitalization rate varied between the 95% confidence interval of the hospitalization rates from the champion trial . the sensitivity around hf hospitalization cost showed the icers to vary between us$6172/qaly and us$30 696/qaly . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the icers varied between us$8456/qaly and us$16 854/qaly when the mortality rates varied between the 95% confidence bounds of the champion trial mortality rates . the icer was sensitive to the implant cost , and for an implant cost range of us$15 556 to us$26 704 , the icer ranged from us$6686/qaly and us$35 018/qaly . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . according to the hcup nis ( nationwide inpatient sample ) data set from 2012,37 amongst all inpatient hospitalizations associated with a primary or secondary diagnosis of hf identified via icd9cm code 428.x , there were 5 021 800 discharges for hf with the payer as medicare , and 674 680 discharges for hf in patients that had private insurance . using these two payer categories ( medicare and private payer ) , 88% of hf hospitalizations occurred in medicare beneficiaries . using this 88% ( medicare ) and 12% ( private insurance ) distribution to form the assumption of patient mix , the icer was us$20 734/qaly . for the primary cea ( using hf hospitalization costs only ) , the psa showed that at the willingnesstopay threshold of us$25 000 , > 85% of the simulations were costeffective ; > 99% were costeffective at the us$50 000 threshold . further , for the cea using comprehensive patient management costs , the psa showed that at the willingnesstopay threshold of us$50 000 , 87% of the simulations were costeffective ; > 99% were so at the us$100 000 threshold . heart failure management is expensive , and , historically , studies related to crt management of hf patients have compared allcause hospitalization outcomes . although the cardiomems hf system did not have a significant impact on nonhfrelated hospitalizations , we compared all hospitalizations for the two groups to enable a comparison with historical studies . the costs associated with allcause hospitalizations , device and systemrelated complications , and remote physiological monitoring were accumulated . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . the total cost of patient management is the sum of various components for both hf and nonhf management care . the costs associated with hospitalizations for any reason , device and systemrelated complications , and remote physiological monitoring were accumulated . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . the icer was sensitive to the time horizon ( figure 2 ) . compared with the base case icer of us$12 262/qaly at 5 years , the icers at 4 years and 7 years were us$34 909/qaly and us$5412/qaly , respectively . the icers decreased over time ; this is a common observation when a highcost procedure occurs at the beginning of the time horizon , indicating that the therapy is costeffective primarily resulting from averted hospitalizations . the icers were also assessed when hf hospitalization rates , hf hospitalization costs , and mortality rates were varied . the icers varied between us$10 960/qaly and us$14 311/qaly when hf hospitalization rate varied between the 95% confidence interval of the hospitalization rates from the champion trial . the sensitivity around hf hospitalization cost showed the icers to vary between us$6172/qaly and us$30 696/qaly . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the icers varied between us$8456/qaly and us$16 854/qaly when the mortality rates varied between the 95% confidence bounds of the champion trial mortality rates . the icer was sensitive to the implant cost , and for an implant cost range of us$15 556 to us$26 704 , the icer ranged from us$6686/qaly and us$35 018/qaly . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . according to the hcup nis ( nationwide inpatient sample ) data set from 2012,37 amongst all inpatient hospitalizations associated with a primary or secondary diagnosis of hf identified via icd9cm code 428.x , there were 5 021 800 discharges for hf with the payer as medicare , and 674 680 discharges for hf in patients that had private insurance . using these two payer categories ( medicare and private payer ) , 88% of hf hospitalizations occurred in medicare beneficiaries . using this 88% ( medicare ) and 12% ( private insurance ) distribution to form the assumption of patient mix , the icer was us$20 734/qaly . for the primary cea ( using hf hospitalization costs only ) , the psa showed that at the willingnesstopay threshold of us$25 000 , > 85% of the simulations were costeffective ; > 99% were costeffective at the us$50 000 threshold . further , for the cea using comprehensive patient management costs , the psa showed that at the willingnesstopay threshold of us$50 000 , 87% of the simulations were costeffective ; > 99% were so at the us$100 000 threshold . this study demonstrated that haemodynamicguided hf management is a costeffective strategy to improve outcomes in outpatient management of patients with chronic hf . costeffectiveness calculations determined that this strategy was well below the most commonly used threshold of us$50 000 per qaly . this threshold has become an accepted benchmark for costeffectiveness in the usa and is often attributed to the us decision to mandate medicare coverage for patients with endstage renal disease in the 1970s.41 some economists as well as the world health organization ( who ) have argued , on the basis of plausible assumptions about people 's values and attitudes toward risk , for a threshold of 23 times the per capita annual income , which would imply a us threshold of us$110 000 to us$160 000 per qaly.42 this , indeed , would be a contemporary estimate of the costs of treating endstage renal disease with haemodialysis . various factors result in a therapy being costeffective , and in the case of papguided hf care , the costeffectiveness is attributable to the reduction in hf hospitalization rates , reduction in mortality , and improvement in quality of life . for the base case , as well as allcost sensitivity analyses , the uncertainty around the implant reimbursement was handled in our model by a sensitivity analysis around the implant cost range of us$15 556 and us$26 704 . for this wide range , management of hf with pharmacological therapies and implantable device therapy has been studied extensively to prove their clinical effectiveness.43 the icers for medical device therapy for hf patients are between us$10 900 and us$303 000/qaly , depending on the intervention considered . costeffectiveness of pharmacological therapies such as ace inhibitors , arbs , and betablockers has been assessed in the usa and europe , and they have been shown to be costeffective , with icers over a lifetime lower than us$17 900/qaly . implantable cardioverter defibrillators ( icds ) , crtp pacemakers , and crtd defibrillations have been evaluated in the hf population for which these devices are known to have clinical benefit . costeffectiveness studies of icds are sensitive to the patient population selected in each individual trial , and icers vary from us$34 000 to > us$70 000 per qaly gained over a lifetime.43 icers across countries are variable due to variations in healthcare practices and prices . as an illustration , feldman et al . showed that the icer for crtp vs. optimal medical therapy is us$22 900/qaly in the usa ; an icer of us$10 900us$29 700 in the uk ; an icer of us$37 200/qaly in spain , and an icer of us$14 600/qaly in belgium.44 the companion trial , for example , conducted a comparison of crtp vs. optimal medical therapy and crtd vs. optimal medical therapy . implant costs for a crtd were assumed to be us$29 500 and for a crtp were us$20 500 . inclusion criteria included nyha functional class iii or iv plus a hf treatment in the preceding 12 months over and above other criteria . the icers for crtp and crtd vs. optimal medical therapy were us$19 600 and us$43 000 , respectively , over a 7year time horizon.44 using the cpi for medical care inflation of an average of 4.03% , the icer for crtp vs. optimal medical therapy and crtd vs. optimal medical therapy would be us$27 513/qaly and us$60 360/qaly , respectively . modelling the champion trial costeffectiveness over a 7year time horizon and using allcause hospitalization costs , which is similar to the methods used in companion , compares favourably with this at us$15 231/qaly ( us$ 2014 ) . 45 that concluded that the cardiomems device is costeffective with an icer of us$82 301 in patients with reduced ef and us$47 768 in those with preserved ef . our analysis produced different estimates of the icers for several reasons : first , sandhu et al . secondly , the parameter estimates in the sandhu model differed from those in our model . for example , sandhu and colleagues assumed the cost of a hf hospitalization in the usa to be us$12 832 . our analysis used realworld claims data from 200 471 hf patients , which demonstrated an average payer cost of us$16 770 for medicare and us$30 100 for private insurance patients . thirdly , sandhu and colleagues mapped mlhfq ( minnesota living with heart failure questionnaire ) scores into the eq5d scores based on an existing algorithm , which is an acceptable method , but inferior to direct measurement of utilities . our analysis used the eq5d utilities which were measured at baseline and several times during followup in the champion trial . finally , sandhu et al . used mortality rates based on the relative risk of death associated with hospitalization ; our analysis used mortality rates observed in the champion trial . methodological differences in the model perspective , time horizon , and parameters resulted in different estimates of the icer . however , both our analysis and the report from sandhu et al . conclude that using the cardiomems hf system to manage hf patients is costeffective in the us setting . in spite of introduction of effective hf medical and device therapies , average annual medical expenditures per medicare hf beneficiary are estimated to be us$33 247,46 with total annual medicare costs estimated at us$40 billion . reductions in hospitalizations seen in the champion trial are very encouraging and suggest that haemodynamic monitoring of hf patients will provide a muchneeded tool to assist outpatient management of highrisk patients . detailed information about guidelinedirected medical therapy use at baseline and changes during the 6 months of haemodynamicguided care in the champion trial was recently published by costanzo et al . 47 both the control and treatment groups started the trial with high prevalence of guidelinedirected medical therapies at baseline at target doses . both groups were receiving significantly higher doses of loop diuretics at the end of the 6month efficacy endpoint ; however , more increases and decreases in diuretics were seen in the treatment group compared with the controls . additionally , treatment group patients had significant increases in neurohormonal intervention and vasodilator therapies , which were not seen in the control group . the trial demonstrated that active personalization of hf management , guided by frequent haemodynamic assessment , was associated with less need for hospitalization and improvement in delivery of diseasemodifying medications . within the us healthcare system , management of hf using a pap sensor the centers for medicare and medicaid services ( cms ) has instituted financial penalties for hospitals with a higher than expected hf readmission rate for medicare patients . the potential impact of the cardiomems hf system on hf 30day readmissions was not studied in this model . adoption of this treatment strategy at hospitals struggling with hf hospitalization and 30day readmissions could potentially help address an unmet need within the us healthcare system , and may be addressed in separate analyses . heart failure is a global burden ; while it is reasonable to expect that the clinical outcomes in europe would be similar to those found in the current usbased study , a direct cost comparison using simple currency translation is not an accurate method to assess the economic impact in europe . hence , we present here a cea study from the us payer perspective and expect that this will pave the way for future studies in individual countries wherein the unique measures that are relevant to each healthcare system and cost structure for each country are appropriately dealt with . the largest costs in our primary cea are the implant hospitalization cost and the hf hospitalization cost . this study includes a sensitivity analysis using the cost of hf hospitalization at us$8358us$25 155 . the costs for hf hospitalizations in many european countries are quite variable and sometimes much lower ; for example at 2515 in the uk and 2400 in germany . for the lower of the two , assuming the same cost of implant as the us model , the icer would be us$45 002/qaly . the icers , when considered for hf management or comprehensive management , were well below the conventional us acceptability threshold of us$50 000 . heart failure remains an increasing global problem.22 , 48 coupled with the ageing population and thus increasing numbers of hf patients , the pressure on healthcare payers to reduce hospitalizations will continue unabated . strategies such as cardiomems , which decrease the rate of hospitalization , are likely to be only more costeffective in future .

abstractaimshaemodynamicguided heart failure ( hf ) management effectively reduces decompensation events and need for hospitalizations . the economic benefit of clinical improvement requires further study.methods and resultsan estimate of the costeffectiveness of haemodynamicguided hf management was made based on observations published in the randomized , prospective singleblinded champion trial . a comprehensive analysis was performed including healthcare utilization event rates , survival , and quality of life demonstrated in the randomized portion of the trial ( 18 months ) . markov modelling with monte carlo simulation was used to approximate comprehensive costs and qualityadjusted life years ( qalys ) from a payer perspective . unit costs were estimated using the truven health marketscan database from april 2008 to march 2013 . over a 5year horizon , patients in the treatment group had average qalys of 2.56 with a total cost of us$56 974 ; patients in the control group had qalys of 2.16 with a total cost of us$52 149 . the incremental costeffectiveness ratio ( icer ) was us$12 262 per qaly . using comprehensive cost modelling , including all anticipated costs of hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits over 5 years , the treatment group had a total cost of us$212 004 and the control group had a total cost of us$200 360 . the icer was us$29 593 per qaly.conclusionsstandard economic modelling suggests that pulmonary artery pressureguided management of hf using the cardiomems hf system is costeffective from the uspayer perspective . this analysis provides the background for further modelling in specific country healthcare systems and cost structures .

Introduction Methods Model structure Cost of healthcare utilization Costeffectiveness outcome measures Results Primary costeffectiveness analysis: comparison of heart failure hospitalization outcomes Sensitivity analyses of cost sources Costeffectiveness analysis: comparing allcause hospitalization outcomes Costeffectiveness analysis: comparing comprehensive management Oneway sensitivity analyses Probabilistic sensitivity analyses Discussion Conclusions Supporting information

heart failure ( hf ) is a complex clinical syndrome and global public health problem affecting an estimated 26 million people worldwide.1 despite increased utilization of pharmacological2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 and device therapy options10 , 11 , 12 , 13 that improve clinical outcomes in randomized controlled trials , morbidity and mortality in hf remain a major burden to patients , their caregivers , and national healthcare systems . patients with hf frequently experience worsening symptoms related to accumulation of excess intravascular volume and congestion , requiring hospitalization to provide intravenous medical support to restore normal volume.14 , 15 , 16 , 17 , 18 , 19 heart failure is cited as the most frequent cause of hospitalization in the us medicare population , resulting in > 1 million admissions per year ( accounting for 12% of all hospitalizations).20 , 21 the economic impact of hf in the usa is profound , with the total costs of hf estimated to increase from us$31 billion in 2012 to us$70 billion in 2030 secondary to an ageing population.22 costeffective hf management strategies are required to address this growing problem . elevated cardiac filling pressures are associated with higher rates of rehospitalization and mortality in patients with hf.23 , 24 independent of lvef , rises in cardiac filling pressures can often be detected several weeks prior to patients experiencing symptoms of hf decompensation that require hospitalization.25 , 26 remote monitoring of intracardiac and pulmonary artery pressures ( paps ) in patients with hf using implantable haemodynamic monitoring devices can provide physicians with access to actionable pathophysiological information and help improve the hf management decisionmaking process necessary to prevent hf hospitalizations.27 , 28 , 29 , 30 a novel wireless pap measurement system ( cardiomems hf system , st . atlanta , ga , usa ) was evaluated in the cardiomems heart sensor allows monitoring of pressure to improve outcomes in new york heart association ( nyha ) functional class iii heart failure patients ( champion ) trial.30 , 31 the champion trial was a prospective , multicentre , randomized , singleblind clinical study in 550 patients that tested the incremental impact of papguided hf management on clinical outcomes compared with hf management based on current american college of cardiology foundation / american heart association practice guidelines only . the papguided hf management group in the champion trial experienced a significant reduction in hf hospitalization rates , a greater reduction in paps , fewer patients hospitalized for hf , and more days alive and outside of the hospital for hf , and exhibited an improvement in quality of life when compared with guidelinedirected standard of care hf management only ( control group).30 these longterm benefits were seen in patients with hf and preserved ef,32 secondary pulmonary hypertension,33 and comorbid chronic obstructive cad.34 hospitalization reductions were seen after an average of 18 months of randomized followup , with additional longterm benefits noted in the 13 months of openaccess , which immediately followed the end of the randomized followup in the trial.35 based on these data , the cardiomems hf system was approved by the us food and drug administration for hf management in nyha class iii hf patients with a hf hospitalization within the last 12 months . this study is a comprehensive analysis of the costeffectiveness of this treatment strategy in the context of the us medical system . a markov model was utilized to approximate the course of management observed in the champion trial for the treatment and control groups using monte carlo simulation . healthcare utilization event rates , survival , and quality of life were based on study data . we took the perspective of the payer , and focused on the medicare and private insurance patient populations as they represent the vast majority of patients eligible for the cardiomems hf system in the usa , and cost data were available for these two patient populations to conduct our analyses . model endpoints included cost of treatment and preferenceweighted survival , which were then used to calculate incremental cost per life year gained and cost per qualityadjusted life year ( qaly ) gained for the treatment compared with control . markov model used to approximate the course of management observed in the champion trial for the treatment and control groups . stable hf state was characterized as a patient that received typical care for hf including physician visits , prescription drugs , longterm care , and outpatient hospital visits . in the model simulation , stable hf state , and the treatment group started the simulation with an implantrelated hospitalization . for this costeffectiveness analysis ( cea ) , estimates of the transition probabilities among states , mortality , and eq5d preference weight utilities in each state came from the complete randomized access period of the champion trial ( table 1 ) . healthrelated quality of life ( hrqol ) was assessed via the eq5d3 l38 questionnaire at baseline , 6 months , and 12 months in the champion trial . in the usa , the implant of the cardiomems hf system is associated with the msdrg ( medicare severity diagnosis related groups ) payment for 264 accompanied by the icd9cm ( international classification of diseases , ninth revision , clinical modification ) procedure code of 38.26 : insertion of an implantable pressure sensor without a lead for intracardiac or great vessel haemodynamic monitoring . in the base case , the cost of system implantation in the treatment group was us$17 75040 and we made the assumption that all implants occurred on a unique scheduled day for each patient , and did not occur during a preexisting hf hospitalization . payer costs for hospitalizations postimplant ( which are reimbursements ) were determined from the truven health marketscan april 2008 to march 2013 commercial claims and encounters and medicare supplemental and coordination of benefits database . in this hf cohort , we identified payments for three types of healthcare utilization : average cost of inpatient hospitalization with primary diagnosis code of hf , average cost of inpatient hospitalization not related to hf , and annual costs for outpatient healthcare utilization of any type for these patients ( table 2 ) . these payments corresponded to the states of hospitalized for hf , hospitalized for other cause , and the payments included facility cost and professional fees associated with the event , adjusted to 2014 us dollars based on the consumer price index ( cpi ) inflation from the bureau of labor statistics . stable hf included the majority of expenses a nonhospitalized hf patient incurs receiving medical care ; this included physician visits , prescription drugs , longterm care , and outpatient hospital visits . in addition to this , a patient implanted with the cardiomems hf system was assumed to incur a monthly cost of us$45 associated with the professional and technical components of reimbursement for remote physiological monitoring . since remote physiological monitoring is conducted for various reasons for patients that have not been implanted with the cardiomems hf system , we assumed that 25% of the standard of care patients also incur this monthly cost . using the details from hospital admission and discharge records for each one of the eight dsrcs , we ascertained an msdrg for each such hospitalization , and then determined an average cost of dsrcs based on the 2014 reimbursement for the msdrgs . thus , our primary effectiveness endpoint was the incremental costeffectiveness ratio ( icer ) comparing the costs and qalys of hf hospitalization outcomes in the pap treatment and control groups . these parameters included hf hospitalization rates , mortality rates , cost of hf hospitalization , monitor implant cost , and payer mix . in addition , sensitivity analyses were conducted on the costs included in the analysis : hf hospitalizations only , allcause hospitalizations , and all chronic hf management costs including allcause hospitalizations . markov model used to approximate the course of management observed in the champion trial for the treatment and control groups . stable hf state was characterized as a patient that received typical care for hf including physician visits , prescription drugs , longterm care , and outpatient hospital visits . in the model simulation , stable hf state , and the treatment group started the simulation with an implantrelated hospitalization . for this costeffectiveness analysis ( cea ) , estimates of the transition probabilities among states , mortality , and eq5d preference weight utilities in each state came from the complete randomized access period of the champion trial ( table 1 ) . healthrelated quality of life ( hrqol ) was assessed via the eq5d3 l38 questionnaire at baseline , 6 months , and 12 months in the champion trial . in the usa , the implant of the cardiomems hf system is associated with the msdrg ( medicare severity diagnosis related groups ) payment for 264 accompanied by the icd9cm ( international classification of diseases , ninth revision , clinical modification ) procedure code of 38.26 : insertion of an implantable pressure sensor without a lead for intracardiac or great vessel haemodynamic monitoring . in the base case , the cost of system implantation in the treatment group was us$17 75040 and we made the assumption that all implants occurred on a unique scheduled day for each patient , and did not occur during a preexisting hf hospitalization . payer costs for hospitalizations postimplant ( which are reimbursements ) were determined from the truven health marketscan april 2008 to march 2013 commercial claims and encounters and medicare supplemental and coordination of benefits database . in this hf cohort , we identified payments for three types of healthcare utilization : average cost of inpatient hospitalization with primary diagnosis code of hf , average cost of inpatient hospitalization not related to hf , and annual costs for outpatient healthcare utilization of any type for these patients ( table 2 ) . stable hf included the majority of expenses a nonhospitalized hf patient incurs receiving medical care ; this included physician visits , prescription drugs , longterm care , and outpatient hospital visits . in addition to this , a patient implanted with the cardiomems hf system was assumed to incur a monthly cost of us$45 associated with the professional and technical components of reimbursement for remote physiological monitoring . since remote physiological monitoring is conducted for various reasons for patients that have not been implanted with the cardiomems hf system , we assumed that 25% of the standard of care patients also incur this monthly cost . using the details from hospital admission and discharge records for each one of the eight dsrcs , we ascertained an msdrg for each such hospitalization , and then determined an average cost of dsrcs based on the 2014 reimbursement for the msdrgs . no further device or systemrelated complications or sensor failures thus , our primary effectiveness endpoint was the incremental costeffectiveness ratio ( icer ) comparing the costs and qalys of hf hospitalization outcomes in the pap treatment and control groups . these parameters included hf hospitalization rates , mortality rates , cost of hf hospitalization , monitor implant cost , and payer mix . in addition , sensitivity analyses were conducted on the costs included in the analysis : hf hospitalizations only , allcause hospitalizations , and all chronic hf management costs including allcause hospitalizations . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$56 974 ; patients in the control group had qalys of 2.16 with a total cost of us$52 149 . costeffectiveness analysis basecase costs and survival over a 5year time horizon cea , costeffectiveness analysis ; hf , heart failure ; qaly , quality adjusted life year . although the cardiomems hf system did not have a significant impact on nonhfrelated hospitalizations , we compared all hospitalizations for the two groups to enable a comparison with historical studies . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . the total cost of patient management is the sum of various components for both hf and nonhf management care . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the icer was sensitive to the implant cost , and for an implant cost range of us$15 556 to us$26 704 , the icer ranged from us$6686/qaly and us$35 018/qaly . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$56 974 ; patients in the control group had qalys of 2.16 with a total cost of us$52 149 . costeffectiveness analysis basecase costs and survival over a 5year time horizon cea , costeffectiveness analysis ; hf , heart failure ; qaly , quality adjusted life year . heart failure management is expensive , and , historically , studies related to crt management of hf patients have compared allcause hospitalization outcomes . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . heart failure management is expensive , and , historically , studies related to crt management of hf patients have compared allcause hospitalization outcomes . although the cardiomems hf system did not have a significant impact on nonhfrelated hospitalizations , we compared all hospitalizations for the two groups to enable a comparison with historical studies . over a 5year time horizon , patients in the treatment group had average qalys of 2.56 ( discounted at 3% ) with a total cost of us$140 966 ; patients in the control group had qalys of 2.16 with a total cost of us$133 681 . the total cost of patient management is the sum of various components for both hf and nonhf management care . to these were added the costs of patient management , which include hf and nonhf hospitalizations , physician visits , prescription drugs , longterm care , and outpatient hospital visits . over the 5year time period , patients in the treatment group had a total cost of us$212 004 and patients in the control group had a total cost of us$200 360 . when the cost of hf hospitalization was 50% higher than the base case , the icer at a 5year time horizon of us$6172 resulted from the treatment arm accumulating lower costs compared with the control arm . the icer was sensitive to the implant cost , and for an implant cost range of us$15 556 to us$26 704 , the icer ranged from us$6686/qaly and us$35 018/qaly . the implant costs low and high ranges were determined using the national drg payment for msdrg code 264 with and without the new technology addon payment ( ntap ) in the case of the inpatient hospitalizations , and the apc ( ambulatory payment classification ) code 0319 with and without the passthrough payment . this threshold has become an accepted benchmark for costeffectiveness in the usa and is often attributed to the us decision to mandate medicare coverage for patients with endstage renal disease in the 1970s.41 some economists as well as the world health organization ( who ) have argued , on the basis of plausible assumptions about people 's values and attitudes toward risk , for a threshold of 23 times the per capita annual income , which would imply a us threshold of us$110 000 to us$160 000 per qaly.42 this , indeed , would be a contemporary estimate of the costs of treating endstage renal disease with haemodialysis . various factors result in a therapy being costeffective , and in the case of papguided hf care , the costeffectiveness is attributable to the reduction in hf hospitalization rates , reduction in mortality , and improvement in quality of life . costeffectiveness of pharmacological therapies such as ace inhibitors , arbs , and betablockers has been assessed in the usa and europe , and they have been shown to be costeffective , with icers over a lifetime lower than us$17 900/qaly . the icers for crtp and crtd vs. optimal medical therapy were us$19 600 and us$43 000 , respectively , over a 7year time horizon.44 using the cpi for medical care inflation of an average of 4.03% , the icer for crtp vs. optimal medical therapy and crtd vs. optimal medical therapy would be us$27 513/qaly and us$60 360/qaly , respectively . methodological differences in the model perspective , time horizon , and parameters resulted in different estimates of the icer . conclude that using the cardiomems hf system to manage hf patients is costeffective in the us setting . both groups were receiving significantly higher doses of loop diuretics at the end of the 6month efficacy endpoint ; however , more increases and decreases in diuretics were seen in the treatment group compared with the controls . additionally , treatment group patients had significant increases in neurohormonal intervention and vasodilator therapies , which were not seen in the control group . the trial demonstrated that active personalization of hf management , guided by frequent haemodynamic assessment , was associated with less need for hospitalization and improvement in delivery of diseasemodifying medications . within the us healthcare system , management of hf using a pap sensor the centers for medicare and medicaid services ( cms ) has instituted financial penalties for hospitals with a higher than expected hf readmission rate for medicare patients . the potential impact of the cardiomems hf system on hf 30day readmissions was not studied in this model . for the lower of the two , assuming the same cost of implant as the us model , the icer would be us$45 002/qaly .

while genetic risk is certainly a reasonable clue to follow to search for the developmental biomarkers of schizophrenia , there are inherent difficulties in this strategy . although the heritability for schizophrenia is estimated to be as high as 70% , the illness clearly does not have a pattern of inheritance in any population or even in single families that is consistent with the effect of a single gene . thus , like many common illnesses such as diabetes and hypertension , it is more likely that multiple genes are involved . multigenic illnesses were once considered unanalyzable by genetic linkage techniques , but the use of large sample sizes , dense chromosomal maps , and improved statistical methodology has led to the detection of a number of genetic loci . for some of these loci , promising candidate genes have been identified and , for some of these genes , polymorphisms have been discovered that are associated with schizophrenia and would seem to alter gene function to produce a neurobiological effect . most of the genes identified have some role in the development or function of neurotransmission . consideration of the finding of multigenic inheritance illuminates the problem of detecting biomarkers for schizophrenia . for example , if two genes on different chromosomes are hypothesized to be responsible for all cases of schizophrenia , then , for 1% of the population to have schizophrenia , the frequency of the allele associated with schizophrenia must be approximately 5% per chromosome for each gene . an individual would then have 5% chance of inheriting a disease allele for the first gene from mother and 5% from father , for a total of approximately 10% . the combined probability of inheriting disease alleles in both genes would be 10% times 10% , or 1% total risk . if a parent had schizophrenia , so that he or she carried disease alleles for both genes , the probability of transmitting both would be 50% for each disease allele , since one of two chromosomes , one carrying the disease - associated allele and one not , is transmitted to an off - spring through the sperm or egg . the probability of transmitting disease alleles for both genes would be 50% times 50% , or 25% . since the observed transmission of risk from parent to child is only about 10% , however , the two - gene model comes closer than a one - gene model , for which parent child transmission would be 50% . additional complexities could include the involvement of more than two genes or environmental factors to produce the illness , which could range from perinatal developmental injury to psychosocial stressors . the implication of this theoretical exercise for the early assessment of risk factors is that disease allele frequencies of 5% per chromosome ( termed the allele frequency ) or about 10% per individual , considering the possibility of the occurrence of a disease allele on either of the two chromosomes , mean that a biomarker associated with a specific gene would occur in about 10% of the population . thus , even if we perfectly understood both the molecular biology and the associated neurobiological deficits for one of the genes that convey risk for schizophrenia , and if that gene were involved in all cases of schizophrenia , then we would still detect its presence in 10% of the population , which would include the 1% who would actually develop schizophrenia . treatment of 10% of adolescents with neuroleptic drugs , to delay the onset of psychotic symptoms that might occur in only 1 out of 10 of the identified individuals would probably be viewed as medically unsound from the perspective of risk and benefit . of course , if we could identify all the genes and all their interactions with each other and with environmental factors , then we might be able to restrict the identification to a subset of individuals who were more likely to develop schizophrenia , but such a complete understanding has not yet been achieved . the actual findings with genetic linkages and candidate genes associated with risk for schizophrenia suggest that the theoretical two - gene exercise underestimates the prevalence of genetic risk in the population . for example , we have conducted genetic linkage analysis for schizophrenia in the national institute of mental health ( nimh ) genetics initiative families . the first 88 families were selected to have two ill individuals , generally a pair of siblings , who met the then current diagnostic criteria ( diagnostic and statistical manual of mental disorders , third edition , revised [ dsm - iii - r ] ) for schizophrenia or schizoaffective disorder , with at least one individual meeting criteria for schizophrenia . the families were collected by a consortium of investigators at harvard university , washington university , and columbia university and the genotyping was performed by millennium pharmaceuticals . several genetic analyses of the sample have been published , which point to loci on a number of chromosomes . a maximum likelihood statistical analysis was used to determine the association between genetic markers on each of the chromosomes and the inheritance pattern of the illness in each family . the analysis determined the common logarithm of the ratio of the odds that there is a true linkage between chromosomal markers and the illness , as opposed to a chance association , termed the lod score . scores above 3.3 are considered indicative of linkage , through lower lod scores also considered worthy of further investigation . chromosomes 15 and 10 contain two of the more significance loci ( table i ) . significance of a linkage finding is based on several factors , the most relevant of which is the pathophysiological significance of the underlying putative genetic variant that causes the illness . genetic linkage does not directly identify the variant ; rather , it finds genetic markers that are close to or linked to an undiscovered genetic variant that actually contributes to risk for the illness . thus , other factors can equally influence the lod score , such as genetic homogeneity of the population , so that the number of other possible genetic causes of schizophrenia in the population is limited . accordingly , although findings with chromosomes 15 and 10 have been replicated in other populations , there are considerable differences in genetic findings for schizophrenia across different populations and studies . thus , the selection of chromosomal loci in this example is only one of several strategies that could be used . the more powerful genetic analytic strategies use likelihood analyses that assume a model of inheritance , which can be either dominant or recessive . additional parameters that are specified are the allele frequency of each putative allele and the penetrances , which are the probability that each putative genotype will produce illness . thus , if a and a are two alleles of a putative gene associated with risk for schizophrenia , and the a variant is associated with risk and the a variant is not , there would be four possible genotypes if we list the paternally inherited allele , followed by the maternally inherited allele : aa , aa , aa , and aa . the frequency of each genotype is the product of the two allele frequencies . for a dominant genetic model with complete penetrance , the penetrance is 1.0 for any genotype containing a. for a gene with only two alleles , the two allele frequencies add to 1.0 , and the sum of the products of the penetrances of each genotype and the frequency of each genotype is the frequency of the illness in the general population . the frequency of the illness in the general population ( 0.01 ) is a piece of real data that anchors the model in reality thus , in the example in table ii , the sum of the gene frequencies , which are the products of the two allele frequencies , is 1 , accounting for all genotypes . the penetrances of the three genotypes that contain a , either as the homozygote aa or the heterozygotes aa and aa , are 1.0 , because this is a completely dominant illness . the penetrance of aa , a genotype that does not convey risk , is 0.00103 , which essentially accounts for the possibility of other genetic and nongenetic causes of illness that do not involve this gene . the sum of the product of the gene frequencies is 0.01 , which is the frequency of schizophrenia in the population . these parameters were used in the initial analyses for a canadian linkage study that identified a locus on chromosome 1 with the highest statistical confidence level yet reported for a genetic linkage finding in schizophrenia . the assumptions of the model , in addition to dominant transmission , is that the gene that conveys risk for schizophrenia is highly penetrant so that the genotypes containing a always produce illness and there are very few cases ( 1/10 ) that do not have this particular genetic cause . the model posits that schizophrenia is caused by a gene that has an extremely deleterious effect on brain function , resulting in a very severe illness , chronic schizophrenia . a similar model has been used to discover other major genetic influences in schizophrenia . in the canadian study , it turned out that a similar recessive model better explained the data . returning to the nimh families , the finding of multiple genetic signal calls this simple model into question . considering only the two most positive linkages , chromosomes 15 and 10 one possibility is that there are two types of schizophrenia , a 15 type and a 10 type , where the data are best explained as two independent findings , with some families having one type and some having another , a heterogeneous model . a second possibility is that both genetic factors are active in the same families and that schizophrenia results from their additive effect . this model implies that neither gene by itself produces an effect that is completely devastating to normal brain function , but when deficits from the two genes occur together , a threshold is crossed , and a brain dysfunction occurs that results in psychosis . the genetic model becomes more complex , but the products of genotypes and penetrances must still add to 0.01 , the population frequency of schizophrenia ( table iii ) . for the additive model , if we allow the frequency of the putative disease variants in each of the two genes to vary we see that the maximum likelihood occurs with much higher allele frequencies than the 0.0045 frequency that we initially assumed ( figure 1 ) . at this high frequency , penetrance is also reduced ( table iii ) , indicating that individuals who carry the genotypes associated with illness now have only a 25% probability of having the illness . a comparison of this additive model with the heterogeneous model , by subtracting their respective maximum likelihood , reveals that the additive model is significantly more likely . thus , genetic linkage data in the nimh population supports the hypothesis that schizophrenia is caused by multiple genes acting together and the variants associated with the illness are common in the population . reproduced from reference 2 : freedman r , leonard s , olincy a , et al . for many of the linkage signals , candidate genes are now being identified . at chromosomal region 15q14 , the most extensively studied candidate gene is chrna7 , the gene for the 7-nicotinic acetylcholine receptor . this gene was first identified by neurobiological research into a pathophysiological abnormality in schizophrenia , the failure to inhibit the p50 auditory evoked response to the second of paired stimuli . this inhibitory deficit , one of many such physiological sensory gating deficits characterized in schizophrenia , is related to patients inability to maintain sustained attention , one of the notable neuropsychological deficits of schizophrenia . they describe being overwhelmed or flooded by sensory stimuli in their environment that most normal subjects can ignore . deficits in prepulse inhibition of startle , poor performance in the continuous performance test , and various smooth pursuit eye tracking abnormalities have all been characterized as failures in inhibitory and sensory gating function in schizophrenia . deficits in inhibitory neurons , including failures of migration , diminished expression of inhibitory neurotransmitters , and loss of the neurons themselves , have all been found in postmortem studies of brain tissue for persons who had schizophrenia . deficits in expression of 7-nicotinic receptors , which are highly expressed on many interneurons , are consistent with the other deficits found in these interneurons . animal models with genetically diminished expression of the 7-nicotinic receptor have deficits in inhibition of auditory evoked responses that resemble the deficit in schizophrenia . these models suggest that nicotinic receptor activation of interneurons in the hippocampus is a critical mechanism in sensory inhibition . thus , deficits in inhibition in general and diminished nicotinic receptor activation of inhibitory interneurons in particular appear to be two of the neurobiological features of schizophrenia ( figure 2 ) . genome - wide linkage studies initially related the loss of p50 inhibition in schizophrenia to the chromosomal region 15q14 locus of chrna7 . mutating screening in the chrna7 gene resulted in the discovery of single nucleotide polymorphisms in the promoter and other abnormalities that have been associated with schizophrenia itself and also with the failure to inhibit p50 responses in schizophrenia ( figure 3 ) . while the significance of all of these polymorphisms is still being sorted out , the allele frequency of these polymorphisms is relatively high , as was predicted from the linkage data . taking all these genes into account , it would seem that the majority of the population has at least one of the genetic risk factors for schizophrenia . reproduced from reference 9 : leonard s , gault j , hopkins j , et al . association of promoter variants in the 7-nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia . thus , the somewhat unexpected result of genetic research to date is that genetic risk is much more wide - spread than initially posited . identification of individuals at risk by genetic means alone is not likely to select individuals who have a high probability of actually developing schizophrenia . first , for chrna7 and for many of the other genes being discovered for schizophrenia , the neurobiological phenotypes are being elucidated . most of the genes are associated with dysfunction in the mechanisms of neurotransmission . for chrna7 , there is an apparent link to an inhibitory dysfunction that can be measured physiologically , in both animal models and humans . to the extent that this dysfunction and similar dysfunctions can be traced through development , a biological developmental course of schizophrenia can be ascertained , so that the window for possible prevention can be determined . while this time course could have been established without the genetic information , the genetic associations and linkages relate the physiological dysfunction , eg , diminished sensory gating , to a well - identified biological element , eg , diminished activation of 7-nicotinic receptors . the dominant model with complete penetrance posited a model of pathophysiology in which a single genetic deficit produces a near catastrophic effect on brain function that results in schizophrenia . the more complex model posited here suggests that schizophrenia may be the coincidence in a single individual of multiple deficits , none of which in themselves are particularly problematic . small changes in the pathophysiological effect of any one of these deficits could have significant effects on the development of illness . in adolescence , persons at risk for schizophrenia can be identified in two waysby the individual having a parent who has schizophrenia , particularly in a family that already has other cases of schizophrenia , or by the individual displaying prodromal features of schizophrenia . palau is a small island nation in micronesia in which many individuals with schizophrenia have been found in large , multiply affected families . adolescents in these families adolescents identified by self - report and follow - up interview as displaying prodromal symptoms also have an elevated prevalence of diminished p50 inhibition . thus , diminished p50 inhibition is present in adolescents at risk for schizophrenia before the onset of psychotic symptoms ( figure 4 ) . attempts to measure p50 in younger children have shown that normal adult levels of p50 inhibition are present in only about half of preadolescent children . in these younger children , diminished levels of inhibition do not correlate with any obvious psychopathology or familial risk . rather , the children with diminished p50 inhibition tend to display increased levels of activity that are well within the normal range for children of their age . adrenergic activity , for example , diminishes inhibitory interneuron response and is correlated with diminished p50 inhibition in normals . while it is not known whether normal children with diminished p50 inhibition have increased adrenergic activity it is known that children are much better able than adults to tolerate psychotomimetic drugs , such as amphetamine and ketamine , both of which increase catecholaminergic activity . whether the apparent maturation of p50 inhibition at the end of adolescence and the loss of relative protection against psychotomimetic effects of drugs and the peak incidence of development of schizophrenia during this transition to adulthood have a common neurobiological basis is a question that has yet to be addressed . experimental elimination of adrenergic activity by pharmacological means in animals results in the normalization of the inhibition of auditory responses to repeated stimuli . similar experiments in normal children would be informative about the neurobiology of p50 inhibition , but exposure of these children to medication is problematic . however , loss of adrenergic activity is one of the physiological concomitants of rapid eye movement ( rem ) sleep . therefore , a natural state exists in which p50 inhibition can be measured without the interference of adrenergic activity . in adults , recordings during rem show the same differences in p50 inhibition between normals and schizophrenics that have been observed during the waking state . for schoolaged children , the rem state would also be achieved after sleep deprivation or by all - night recording , but for newborns , rem is a frequently achieved state during the day . therefore , we have recorded p50 inhibition during the first 3 months of life . in initial experiments , this method is used to avoid the confound of premature birth in the calculation of perinatal development . by the first 3 months of life , most infants have developed near - adult levels of p50 inhibition ( figure 5 ) . recording of infants at risk for schizophrenia is a logical next step in determining whether or not the neurobiological processes that result in abnormal p50 inhibition in schizophrenia are present as early as the neonatal period . reproduced from reference 12 : kisley ma , olincy a , robbins e , et al . sensory gating impairment associated with schizophrenia the task consists of following a slowly moving target , which the subject must follow with his or her eyes , while eye movement is monitored using infrared reflectometry . normal persons are able to move their eyes precisely , so that the image of the target always remains in the small foveal region of the retina . persons with schizophrenia and some of their relatives have diminished performance of the task . one of the elements of this abnormality is the inability to inhibit saccadic eye movements , so that their eyes jump ahead of the target and then wait for the target to catch up ( figure 6 ) . functional magnetic resonance imaging during the task reveals increased hemodynamic activity in the hippocampus ( figure 7 ) . this increased activity is consistent with the putative decreased hippocampal inhibition that has also been proposed as a mechanism for the diminished inhibitory gating of the p50 response . reproduced from reference 14 : tregellas jr , tanabe jl , miller de , ross rg , olincy a , freedman r. neurobiology of smooth pursuit eye movement deficits in schizophrenia : an fmri study . am j psychiatry . 2004;161:315 - 321 . about half of children with a parent who has schizophrenia also have abnormal smooth pursuit eye movements with the intrusion of saccades , which can be detected as early as age 6 . this abnormality is thus the earliest studied physiological dysfunction associated with the eventual appearance of schizophrenia ( figure 8) . the abnormality is associated with neuropsychological evidence of attention dysfunction , one of the cognitive abnormalities found in children at risk for schizophrenia . thus , the use of physiological dysfunctions associated with the genetic risk for schizophrenia to identify putative windows during which preventive efforts might be possible points to the expression of these dysfunctions before the onset of schizophrenia . dysfunction is certainly present in adolescents at risk and in school - aged children , but the development of inhibitory function in the early perinatal period suggests that it is reasonable to look even earlier . reproduced from reference 16 : ross rg . early expression of a pathophysiological feature of schizophrenia : saccadic intrusions into smooth - pursuit eye movements in school - age children vulnerable to schizophrenia . the identification of a window for a developmental effect is a major clue to the mechanism of developmental abnormality in schizophrenia , but it does not immediately identify possible mechanisms . an advantage of a genetically associated pathophysiological feature is that the cellular mechanism can be immediately deduced from the gene 's product . most of this work is necessarily performed in animal models , because neurobiological investigation at the cellular level can not generally be performed in human beings . as in the previous section , the example will come from our work on chrna7 and inhibitory brain mechanisms , but similar examples are possible with many of the other genes currently being investigated for schizophrenia . 7-nicotinic receptors are formed early in development , when neurons first differentiate from the neuroepithelium . during adult life in rodents , , there is prominent expression in the hippocampus as well , but also in the cingulate cortex and in the nucleus reticularis thalami , which is a thin sheet of inhibitory neurons that surrounds the thalamus . in rodents and in humans , hippocampal pyramidal neurons have diminished response to repeated stimuli , making the hippocampus is a putative source of the diminished p50 response to repeated stimuli that can be modeled in rodents . 7-nicotinic receptors are found both presynaptically and postsynaptically throughout the hippocampus , the area studied most thoroughly at the ultrastructural level using electron microscopy 7-nicotinic receptors are found within glutamate synapses , where they anchor to common postsynaptic densities . however , the most prominent expression is postsynaptic on interneurons throughout the hippocampus ( figure 9 ) . during perinatal development there is also prominent expression on pyramidal neurons in the ca1 region , but this expression soon disappears early in postnatal development . indeed , the perinatal expression of 7-nicotinic receptors is greater than at any other time during the life cycle ( figure 10 ) . the dba/2 inbred mouse strain has lower levels of 7-receptors in the hippocampus and diminished inhibition of the hippocampal evoked response to the repeated auditory stimuli . although p50 has several sources in human brain , it can be recorded from the hippocampus . the dba/2 mouse also has polymorphisms in the chrna7 gene , outside the amino acid coding region , which correlate with diminished expression of the gene . thus , the dba/2 mouse mimics pathophysiological features found in many persons with schizophrenia , ie , diminished expression of 7-nicotinic receptors and polymorphisms in chrna7 , as well as diminished inhibition of the p50-type response to repeated stimuli . in dba/2 mice , the expression of 7-nicotinic receptors is diminished in the ca3 region of the hippocampus , but is relatively abundant in some areas of ca1 . these differences are maintained when the region of mouse chromosome 7 that contains chrna7 is selectively bred onto the genetic backgrounds of other inbred strains of mice . during development , dba/2 mice lag behind comparison strains in the development of an adult pattern of 7-nicotinic receptor expression ( figure 11 ) . reproduced from reference 23 : adams ce , stitzel ja , collins ac , freedman r. 7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons . comparison of 7-nicotinic acetylcholine receptor development in the hippocampal formation of c3h and dba/2 mice . copyright 2003 , elsevier . like n - methyl - d - aspartate ( nmda ) type glutamate receptors , activation of 7-receptors admits calcium ions , as well as other cations , into neurons and thereby activates nitric oxide synthetase , to produce the second messenger nitric oxide . presumably this second messenger system is part of the mechanism of the development of neuronal circuitry that underlies reciprocal excitation and inhibition in the hippocampus . early in development , cholinergic receptors can depolarize neurons before they receive glutamatergic synapses , which eventually will become the primary depolarizing or excitatory receptors of the central nervous system . the depolarization produced by 7-receptors may thus be critical to early circuit formation at about the time of birth , in both rodents and primates . indeed , dba/2 mice , in addition to their deficiencies in 7-nicotinic receptors , have alterations in the expression of glutamate receptors , eg , decreased ampa ( -amino3-hydroxy-5-methyl-4-isoxazole proprionic acid ) and kainate receptors and increased nmda receptors , a pathological change that has also been found in the hippocampus of schizophrenics . thus , developmental abnormalities in 7-nicotinic receptors may have effects beyond cholinergic neurotransmission . these nicotinic developmental abnormalities may affect the development of the major glutamatergic pathways that support most information - processing mechanisms in the brain . several of the genes associated with schizophrenia are involved in the formation of glutamatergic synapses as well , suggesting that the development of these synapses may be a final common pathway for several genetic risk factors . prom the adult perspective , chrna7 deficiencies appear as elementary problems in inhibition , which have neurocognitive significance for the individual . however , from the neurodevelopmental perspective , chrna7 deficiencies could have a profound effect on the development of the excitatory and inhibitory synapses . thus , remediation of chrna7 deficiencies in adult life by pharmacological means might not sufficiently reverse their developmental insult . the significance of developmental pathophysiology for the prevention of schizophrenia is the possibility that an intervention can be performed at some time in the life cycle before the onset of schizophrenia , possibly in the perinatal period . whether a strategy to activate nicotinic cholinergic receptors can be formulated and whether it would be effective in all cases or only in individuals who have polymorphisms in chrna7 receptors is unknown . biomarkers such as diminished p50 inhibition or saccadic intrusions into smooth pursuit eye movements could be helpful to establish the timing and effectiveness of the intervention . more generally , the identification of the time course and cellular mechanism of developmental abnormalities associated with schizophrenia thus has the potential to identify possible strategies for reversal of these developmental abnormalities . pharmacological strategies to activate deficient receptors or to replace deficient growth factors are within the range of current biological possibilities . as genetic information about schizophrenia and other mental disorders increases , the possibilities for such interventions are likely to increase . in some cases , the genetic information may indicate unique mechanisms , but in other cases the mechanisms may converge to common targets , such as the development of excitatory and inhibitory synapses . the use of biomarkers for studies of the development of schizophrenia can thus have implications for treatment discovery even in the absence of the marker 's sensitivity and specificity at the levels necessary for diagnostic purposes . more extensive animal model studies are necessary to design such interventions , but mouse strains with similar genetic and neurobiological features would seem to be useful for such work . eventually , safe treatments that can be applied population wide may result from the consideration of developmental processes that produce biomarkers related to schizophrenia .

biological traits that are predictive of the later development of psychosis have not yet been identified . the complex , multidetermined nature of schizophrenia and other psychoses makes it unlikely that any single biomarker will be both sensitive and specific enough to unambiguously identify individuals who will later become psychotic . however , current genetic research has begun to identify genes associated with schizophrenia , some of which have phenotypes that appear early in life . while these phenotypes have low predictive power for identifying individuals who will become psychotic , they do serve as biomarkers for pathophysiological processes that can become the targets of prevention strategies . examples are given from work on the role of the 7-nicotinic receptor and its gene chrna7 on chromosome 15 in the neurobiology and genetic transmission of schizophrenia .

Genetic risk schizophrenia The developmental time course of physiological dysfunction in schizophrenia The developmental effects of gene dysfunction Significance for possible interventions

while genetic risk is certainly a reasonable clue to follow to search for the developmental biomarkers of schizophrenia , there are inherent difficulties in this strategy . for some of these loci , promising candidate genes have been identified and , for some of these genes , polymorphisms have been discovered that are associated with schizophrenia and would seem to alter gene function to produce a neurobiological effect . most of the genes identified have some role in the development or function of neurotransmission . consideration of the finding of multigenic inheritance illuminates the problem of detecting biomarkers for schizophrenia . for example , if two genes on different chromosomes are hypothesized to be responsible for all cases of schizophrenia , then , for 1% of the population to have schizophrenia , the frequency of the allele associated with schizophrenia must be approximately 5% per chromosome for each gene . if a parent had schizophrenia , so that he or she carried disease alleles for both genes , the probability of transmitting both would be 50% for each disease allele , since one of two chromosomes , one carrying the disease - associated allele and one not , is transmitted to an off - spring through the sperm or egg . the probability of transmitting disease alleles for both genes would be 50% times 50% , or 25% . since the observed transmission of risk from parent to child is only about 10% , however , the two - gene model comes closer than a one - gene model , for which parent child transmission would be 50% . the implication of this theoretical exercise for the early assessment of risk factors is that disease allele frequencies of 5% per chromosome ( termed the allele frequency ) or about 10% per individual , considering the possibility of the occurrence of a disease allele on either of the two chromosomes , mean that a biomarker associated with a specific gene would occur in about 10% of the population . thus , even if we perfectly understood both the molecular biology and the associated neurobiological deficits for one of the genes that convey risk for schizophrenia , and if that gene were involved in all cases of schizophrenia , then we would still detect its presence in 10% of the population , which would include the 1% who would actually develop schizophrenia . treatment of 10% of adolescents with neuroleptic drugs , to delay the onset of psychotic symptoms that might occur in only 1 out of 10 of the identified individuals would probably be viewed as medically unsound from the perspective of risk and benefit . of course , if we could identify all the genes and all their interactions with each other and with environmental factors , then we might be able to restrict the identification to a subset of individuals who were more likely to develop schizophrenia , but such a complete understanding has not yet been achieved . the actual findings with genetic linkages and candidate genes associated with risk for schizophrenia suggest that the theoretical two - gene exercise underestimates the prevalence of genetic risk in the population . for example , we have conducted genetic linkage analysis for schizophrenia in the national institute of mental health ( nimh ) genetics initiative families . several genetic analyses of the sample have been published , which point to loci on a number of chromosomes . a maximum likelihood statistical analysis was used to determine the association between genetic markers on each of the chromosomes and the inheritance pattern of the illness in each family . the analysis determined the common logarithm of the ratio of the odds that there is a true linkage between chromosomal markers and the illness , as opposed to a chance association , termed the lod score . chromosomes 15 and 10 contain two of the more significance loci ( table i ) . significance of a linkage finding is based on several factors , the most relevant of which is the pathophysiological significance of the underlying putative genetic variant that causes the illness . genetic linkage does not directly identify the variant ; rather , it finds genetic markers that are close to or linked to an undiscovered genetic variant that actually contributes to risk for the illness . thus , other factors can equally influence the lod score , such as genetic homogeneity of the population , so that the number of other possible genetic causes of schizophrenia in the population is limited . the more powerful genetic analytic strategies use likelihood analyses that assume a model of inheritance , which can be either dominant or recessive . additional parameters that are specified are the allele frequency of each putative allele and the penetrances , which are the probability that each putative genotype will produce illness . thus , if a and a are two alleles of a putative gene associated with risk for schizophrenia , and the a variant is associated with risk and the a variant is not , there would be four possible genotypes if we list the paternally inherited allele , followed by the maternally inherited allele : aa , aa , aa , and aa . the frequency of each genotype is the product of the two allele frequencies . for a dominant genetic model with complete penetrance , the penetrance is 1.0 for any genotype containing a. for a gene with only two alleles , the two allele frequencies add to 1.0 , and the sum of the products of the penetrances of each genotype and the frequency of each genotype is the frequency of the illness in the general population . the frequency of the illness in the general population ( 0.01 ) is a piece of real data that anchors the model in reality thus , in the example in table ii , the sum of the gene frequencies , which are the products of the two allele frequencies , is 1 , accounting for all genotypes . the penetrances of the three genotypes that contain a , either as the homozygote aa or the heterozygotes aa and aa , are 1.0 , because this is a completely dominant illness . the sum of the product of the gene frequencies is 0.01 , which is the frequency of schizophrenia in the population . these parameters were used in the initial analyses for a canadian linkage study that identified a locus on chromosome 1 with the highest statistical confidence level yet reported for a genetic linkage finding in schizophrenia . the assumptions of the model , in addition to dominant transmission , is that the gene that conveys risk for schizophrenia is highly penetrant so that the genotypes containing a always produce illness and there are very few cases ( 1/10 ) that do not have this particular genetic cause . in the canadian study , it turned out that a similar recessive model better explained the data . considering only the two most positive linkages , chromosomes 15 and 10 one possibility is that there are two types of schizophrenia , a 15 type and a 10 type , where the data are best explained as two independent findings , with some families having one type and some having another , a heterogeneous model . a second possibility is that both genetic factors are active in the same families and that schizophrenia results from their additive effect . the genetic model becomes more complex , but the products of genotypes and penetrances must still add to 0.01 , the population frequency of schizophrenia ( table iii ) . for the additive model , if we allow the frequency of the putative disease variants in each of the two genes to vary we see that the maximum likelihood occurs with much higher allele frequencies than the 0.0045 frequency that we initially assumed ( figure 1 ) . at this high frequency , penetrance is also reduced ( table iii ) , indicating that individuals who carry the genotypes associated with illness now have only a 25% probability of having the illness . a comparison of this additive model with the heterogeneous model , by subtracting their respective maximum likelihood , reveals that the additive model is significantly more likely . thus , genetic linkage data in the nimh population supports the hypothesis that schizophrenia is caused by multiple genes acting together and the variants associated with the illness are common in the population . for many of the linkage signals , candidate genes are now being identified . at chromosomal region 15q14 , the most extensively studied candidate gene is chrna7 , the gene for the 7-nicotinic acetylcholine receptor . this gene was first identified by neurobiological research into a pathophysiological abnormality in schizophrenia , the failure to inhibit the p50 auditory evoked response to the second of paired stimuli . this inhibitory deficit , one of many such physiological sensory gating deficits characterized in schizophrenia , is related to patients inability to maintain sustained attention , one of the notable neuropsychological deficits of schizophrenia . deficits in prepulse inhibition of startle , poor performance in the continuous performance test , and various smooth pursuit eye tracking abnormalities have all been characterized as failures in inhibitory and sensory gating function in schizophrenia . deficits in inhibitory neurons , including failures of migration , diminished expression of inhibitory neurotransmitters , and loss of the neurons themselves , have all been found in postmortem studies of brain tissue for persons who had schizophrenia . animal models with genetically diminished expression of the 7-nicotinic receptor have deficits in inhibition of auditory evoked responses that resemble the deficit in schizophrenia . these models suggest that nicotinic receptor activation of interneurons in the hippocampus is a critical mechanism in sensory inhibition . thus , deficits in inhibition in general and diminished nicotinic receptor activation of inhibitory interneurons in particular appear to be two of the neurobiological features of schizophrenia ( figure 2 ) . genome - wide linkage studies initially related the loss of p50 inhibition in schizophrenia to the chromosomal region 15q14 locus of chrna7 . mutating screening in the chrna7 gene resulted in the discovery of single nucleotide polymorphisms in the promoter and other abnormalities that have been associated with schizophrenia itself and also with the failure to inhibit p50 responses in schizophrenia ( figure 3 ) . taking all these genes into account , it would seem that the majority of the population has at least one of the genetic risk factors for schizophrenia . association of promoter variants in the 7-nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia . thus , the somewhat unexpected result of genetic research to date is that genetic risk is much more wide - spread than initially posited . identification of individuals at risk by genetic means alone is not likely to select individuals who have a high probability of actually developing schizophrenia . first , for chrna7 and for many of the other genes being discovered for schizophrenia , the neurobiological phenotypes are being elucidated . most of the genes are associated with dysfunction in the mechanisms of neurotransmission . for chrna7 , there is an apparent link to an inhibitory dysfunction that can be measured physiologically , in both animal models and humans . to the extent that this dysfunction and similar dysfunctions can be traced through development , a biological developmental course of schizophrenia can be ascertained , so that the window for possible prevention can be determined . the more complex model posited here suggests that schizophrenia may be the coincidence in a single individual of multiple deficits , none of which in themselves are particularly problematic . small changes in the pathophysiological effect of any one of these deficits could have significant effects on the development of illness . in adolescence , persons at risk for schizophrenia can be identified in two waysby the individual having a parent who has schizophrenia , particularly in a family that already has other cases of schizophrenia , or by the individual displaying prodromal features of schizophrenia . palau is a small island nation in micronesia in which many individuals with schizophrenia have been found in large , multiply affected families . thus , diminished p50 inhibition is present in adolescents at risk for schizophrenia before the onset of psychotic symptoms ( figure 4 ) . rather , the children with diminished p50 inhibition tend to display increased levels of activity that are well within the normal range for children of their age . while it is not known whether normal children with diminished p50 inhibition have increased adrenergic activity it is known that children are much better able than adults to tolerate psychotomimetic drugs , such as amphetamine and ketamine , both of which increase catecholaminergic activity . whether the apparent maturation of p50 inhibition at the end of adolescence and the loss of relative protection against psychotomimetic effects of drugs and the peak incidence of development of schizophrenia during this transition to adulthood have a common neurobiological basis is a question that has yet to be addressed . experimental elimination of adrenergic activity by pharmacological means in animals results in the normalization of the inhibition of auditory responses to repeated stimuli . similar experiments in normal children would be informative about the neurobiology of p50 inhibition , but exposure of these children to medication is problematic . however , loss of adrenergic activity is one of the physiological concomitants of rapid eye movement ( rem ) sleep . therefore , we have recorded p50 inhibition during the first 3 months of life . in initial experiments , this method is used to avoid the confound of premature birth in the calculation of perinatal development . recording of infants at risk for schizophrenia is a logical next step in determining whether or not the neurobiological processes that result in abnormal p50 inhibition in schizophrenia are present as early as the neonatal period . sensory gating impairment associated with schizophrenia the task consists of following a slowly moving target , which the subject must follow with his or her eyes , while eye movement is monitored using infrared reflectometry . normal persons are able to move their eyes precisely , so that the image of the target always remains in the small foveal region of the retina . persons with schizophrenia and some of their relatives have diminished performance of the task . one of the elements of this abnormality is the inability to inhibit saccadic eye movements , so that their eyes jump ahead of the target and then wait for the target to catch up ( figure 6 ) . functional magnetic resonance imaging during the task reveals increased hemodynamic activity in the hippocampus ( figure 7 ) . this increased activity is consistent with the putative decreased hippocampal inhibition that has also been proposed as a mechanism for the diminished inhibitory gating of the p50 response . this abnormality is thus the earliest studied physiological dysfunction associated with the eventual appearance of schizophrenia ( figure 8) . the abnormality is associated with neuropsychological evidence of attention dysfunction , one of the cognitive abnormalities found in children at risk for schizophrenia . thus , the use of physiological dysfunctions associated with the genetic risk for schizophrenia to identify putative windows during which preventive efforts might be possible points to the expression of these dysfunctions before the onset of schizophrenia . dysfunction is certainly present in adolescents at risk and in school - aged children , but the development of inhibitory function in the early perinatal period suggests that it is reasonable to look even earlier . early expression of a pathophysiological feature of schizophrenia : saccadic intrusions into smooth - pursuit eye movements in school - age children vulnerable to schizophrenia . the identification of a window for a developmental effect is a major clue to the mechanism of developmental abnormality in schizophrenia , but it does not immediately identify possible mechanisms . most of this work is necessarily performed in animal models , because neurobiological investigation at the cellular level can not generally be performed in human beings . as in the previous section , the example will come from our work on chrna7 and inhibitory brain mechanisms , but similar examples are possible with many of the other genes currently being investigated for schizophrenia . 7-nicotinic receptors are formed early in development , when neurons first differentiate from the neuroepithelium . during adult life in rodents , , there is prominent expression in the hippocampus as well , but also in the cingulate cortex and in the nucleus reticularis thalami , which is a thin sheet of inhibitory neurons that surrounds the thalamus . in rodents and in humans , hippocampal pyramidal neurons have diminished response to repeated stimuli , making the hippocampus is a putative source of the diminished p50 response to repeated stimuli that can be modeled in rodents . however , the most prominent expression is postsynaptic on interneurons throughout the hippocampus ( figure 9 ) . during perinatal development there is also prominent expression on pyramidal neurons in the ca1 region , but this expression soon disappears early in postnatal development . the dba/2 inbred mouse strain has lower levels of 7-receptors in the hippocampus and diminished inhibition of the hippocampal evoked response to the repeated auditory stimuli . the dba/2 mouse also has polymorphisms in the chrna7 gene , outside the amino acid coding region , which correlate with diminished expression of the gene . thus , the dba/2 mouse mimics pathophysiological features found in many persons with schizophrenia , ie , diminished expression of 7-nicotinic receptors and polymorphisms in chrna7 , as well as diminished inhibition of the p50-type response to repeated stimuli . in dba/2 mice , the expression of 7-nicotinic receptors is diminished in the ca3 region of the hippocampus , but is relatively abundant in some areas of ca1 . during development , dba/2 mice lag behind comparison strains in the development of an adult pattern of 7-nicotinic receptor expression ( figure 11 ) . reproduced from reference 23 : adams ce , stitzel ja , collins ac , freedman r. 7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons . comparison of 7-nicotinic acetylcholine receptor development in the hippocampal formation of c3h and dba/2 mice . presumably this second messenger system is part of the mechanism of the development of neuronal circuitry that underlies reciprocal excitation and inhibition in the hippocampus . early in development , cholinergic receptors can depolarize neurons before they receive glutamatergic synapses , which eventually will become the primary depolarizing or excitatory receptors of the central nervous system . indeed , dba/2 mice , in addition to their deficiencies in 7-nicotinic receptors , have alterations in the expression of glutamate receptors , eg , decreased ampa ( -amino3-hydroxy-5-methyl-4-isoxazole proprionic acid ) and kainate receptors and increased nmda receptors , a pathological change that has also been found in the hippocampus of schizophrenics . these nicotinic developmental abnormalities may affect the development of the major glutamatergic pathways that support most information - processing mechanisms in the brain . several of the genes associated with schizophrenia are involved in the formation of glutamatergic synapses as well , suggesting that the development of these synapses may be a final common pathway for several genetic risk factors . prom the adult perspective , chrna7 deficiencies appear as elementary problems in inhibition , which have neurocognitive significance for the individual . however , from the neurodevelopmental perspective , chrna7 deficiencies could have a profound effect on the development of the excitatory and inhibitory synapses . the significance of developmental pathophysiology for the prevention of schizophrenia is the possibility that an intervention can be performed at some time in the life cycle before the onset of schizophrenia , possibly in the perinatal period . whether a strategy to activate nicotinic cholinergic receptors can be formulated and whether it would be effective in all cases or only in individuals who have polymorphisms in chrna7 receptors is unknown . biomarkers such as diminished p50 inhibition or saccadic intrusions into smooth pursuit eye movements could be helpful to establish the timing and effectiveness of the intervention . more generally , the identification of the time course and cellular mechanism of developmental abnormalities associated with schizophrenia thus has the potential to identify possible strategies for reversal of these developmental abnormalities . as genetic information about schizophrenia and other mental disorders increases , the possibilities for such interventions are likely to increase . in some cases , the genetic information may indicate unique mechanisms , but in other cases the mechanisms may converge to common targets , such as the development of excitatory and inhibitory synapses . the use of biomarkers for studies of the development of schizophrenia can thus have implications for treatment discovery even in the absence of the marker 's sensitivity and specificity at the levels necessary for diagnostic purposes . eventually , safe treatments that can be applied population wide may result from the consideration of developmental processes that produce biomarkers related to schizophrenia .

a clinical need exists to optimally identify those who will sustain fragility fractures . simply utilizing bone mineral density ( bmd ) measurement alone is not adequate , as the majority of people who sustain osteoporosis - related fractures do not have osteoporosis by bmd t - score [ 14 ] . as such , major efforts to improve fracture risk estimation continue , e.g. , development of calculators such as frax , garvan and qfracture that include clinical risk factors [ 511 ] . however , using existing calculators some people at low estimated risk sustain fractures and not all at high risk do fracture . this increase is much greater than the corresponding bmd decline [ 14 , 15 ] . as such , age encompasses factors that increase fracture risk , including , among others , declining functional status and increased falls risk . sarcopenia , the age - related decline in muscle mass and function , is associated with increased risk for falls , frailty and fractures [ 1624 ] and may well explain some / much of the increased risk currently attributed to age . consistent with this , in the dubbo study age was not associated with fracture risk after correction for muscle strength and body sway [ 25 , 26 ] . it is possible that individuals with both osteoporosis and sarcopenia ( sarco - osteoporosis ) are at greatest fracture risk [ 2730 ] . indeed , a recent report of over 300 women with hip fracture found 45 % to have both osteoporosis and sarcopenia . therefore , evaluation of sarco - osteoporosis as a fracture risk factor , and assessment of approaches to improve this diagnosis , is appropriate . current consensus sarcopenia definitions include appendicular lean mass ( alm ) adjusted for height ( alm / ht ) [ 20 , 22 , 32 ] . however , height loss is common with advancing age and confounds body mass index ( bmi ) and body composition assessment in older adults [ 33 , 34 ] . as many older adults are shorter than their tallest height , their alm / ht value is higher than if no height loss had occurred thereby making them less likely to be classified as sarcopenic . moreover , differing amounts of height loss between individuals will introduce unappreciated variability into any clinical cut point alm / ht ratio selected to include in the definition of sarcopenia . recognizing this potential confounder , some prior sarcopenia studies have adjusted for height loss [ 34 , 35 ] . however , to our knowledge , the effect of using tallest reported rather than current height on sarcopenia prevalence has not been reported . however , spine bmd as measured by dual - energy x - ray absorptiometry ( dxa ) is commonly elevated in older adults by degenerative changes [ 3638 ] . proximal femur measurements , while less frequently affected , are not immune to the impact of degenerative changes . in contrast , bmd measurement at the one - third radius is unaffected by degenerative changes . prior work finds radius bmd measurement to be important in older adults , and documents the ability of this site to predict fracture risk . thus , it is possible that routine performance of one - third radius bmd measurement in older adults might improve fracture risk prediction . the overarching purpose of this work is to suggest potential approaches that are easily applicable that may enhance fracture prediction and to begin their evaluation . to this end , we hypothesized that use of historical tallest height in the alm / ht ratio and inclusion of radius bmd t - score might enhance identification of sarcopenia and osteoporosis , respectively , thereby facilitating sarco - osteoporosis recognition and potentially fracture risk estimation in older adults . to test this hypothesis , the aims of this study in a group of adults age 60 + years were to : ( 1 ) evaluate the effect of inclusion of one - third radius bmd measurement on t - score distribution and use of historical height vs. current height on the alm / ht ratio and ( 2 ) define the prevalence of sarcopenia , osteoporosis and sarco - osteoporosis with and without using one - third radius bmd and historical height . here we report the effect of these approaches on prevalence of osteoporosis , sarcopenia and sarco - osteoporosis in a convenience sample of 304 community dwelling adults age 60 years and over . data from three studies performed at the university of wisconsin osteoporosis clinical research program were included in this analysis . these studies include the midus ( mid - life in the united states ) ii study , precision , a dxa precision research study comparing bmd and body composition between two ge healthcare lunar densitometers , ( idxa and prodigy ) and jump , a study examining jump mechanography as a tool to assess function in older adults . participants from the midus biomarker project study ( n = 44 ) , precision ( n = 164 ) and jump ( n = 96 ) comprise a sample of older community dwelling adults , primarily from the madison , wi area . all studies were reviewed and approved by the university of wisconsin health sciences irb and all participants signed an informed consent document prior to conduct of any study procedure . a ge healthcare lunar ( madison , wi ) prodigy or idxa densitometer was used for all bmd and body composition measurements . excellent bmd and body composition assessment agreement is observed between the two densitometers used for this study . routine densitometry quality assurance procedures were followed ; no instrument drift or shift was detected during these studies . scans of the lumbar spine , proximal femur , non - dominant radius and total body were performed in routine clinical manner by international society for clinical densitometry ( iscd ) certified technologists . the lowest bmd t - score of the lumbar spine , femoral neck , total proximal femur or one - third radius was chosen to define osteoporosis using the world health organization ( who ) t - score based classification system . the lowest t - score at any of the above sites was used to classify each individual s skeletal status as recommended by the iscd . alm includes only lean mass , excluding fat and bone mass , of the arms and legs as depicted in fig . 1 . height for this ratio was based either on stadiometer - measured height performed on the day of dxa scanning or by participant self - reported tallest historical height . commonly utilized definitions of osteoporosis and sarcopenia were applied , i.e. , a t - score 2.5 at the lumbar spine or hip and alm / current height < 5.45 kg / m ( female ) and < 7.26 kg / m ( male ) . for the sensitive definitions , a one - third radius t - score ( derived using the ge normative gender matched database ) of 2.5 was considered osteoporosis and the alm / ht ratio utilized historical tallest height and the sarcopenia cut points noted above of 5.45 and 7.26 kg / m were applied.fig . examples of the bone ( left ) and soft tissue ( right ) dxa image of an individual . appendicular lean mass ( alm ) is defined as the lean mass , excluding bone and fat , in the arms ( light gray region ) and legs ( dark gray region ) appendicular lean mass regions of interest . examples of the bone ( left ) and soft tissue ( right ) dxa image of an individual . appendicular lean mass ( alm ) is defined as the lean mass , excluding bone and fat , in the arms ( light gray region ) and legs ( dark gray region ) the potential importance of using a dxa site not affected by degenerative change was explored by defining the prevalence of obvious degenerative disease . to this end , all dxa spine images were reviewed by one experienced densitometrist ( nb ) . degenerative changes were considered to be present if obvious osteophytes , vertebral fractures , or focal degenerative changes involving less than an entire vertebral body , or a 1 t - score discrepancy between vertebral bodies was observed . demographic comparisons and evaluation of t - score comparability were performed using t - tests with statview ( cary , nc ) software . pearson and fisher chi - square statistics ( analyze - it ; leeds uk ) were used to compare the prevalence of osteoporosis , sarcopenia and sarco - osteoporosis using standard or sensitive definitions in the three age groups ( 6069 , 7079 and 80 + years ) . data from three studies performed at the university of wisconsin osteoporosis clinical research program were included in this analysis . these studies include the midus ( mid - life in the united states ) ii study , precision , a dxa precision research study comparing bmd and body composition between two ge healthcare lunar densitometers , ( idxa and prodigy ) and jump , a study examining jump mechanography as a tool to assess function in older adults . participants from the midus biomarker project study ( n = 44 ) , precision ( n = 164 ) and jump ( n = 96 ) comprise a sample of older community dwelling adults , primarily from the madison , wi area . all studies were reviewed and approved by the university of wisconsin health sciences irb and all participants signed an informed consent document prior to conduct of any study procedure . a ge healthcare lunar ( madison , wi ) prodigy or idxa densitometer was used for all bmd and body composition measurements . excellent bmd and body composition assessment agreement is observed between the two densitometers used for this study . routine densitometry quality assurance procedures were followed ; no instrument drift or shift was detected during these studies . scans of the lumbar spine , proximal femur , non - dominant radius and total body were performed in routine clinical manner by international society for clinical densitometry ( iscd ) certified technologists . the lowest bmd t - score of the lumbar spine , femoral neck , total proximal femur or one - third radius was chosen to define osteoporosis using the world health organization ( who ) t - score based classification system . the lowest t - score at any of the above sites was used to classify each individual s skeletal status as recommended by the iscd . alm includes only lean mass , excluding fat and bone mass , of the arms and legs as depicted in fig . 1 . height for this ratio was based either on stadiometer - measured height performed on the day of dxa scanning or by participant self - reported tallest historical height . commonly utilized definitions of osteoporosis and sarcopenia were applied , i.e. , a t - score 2.5 at the lumbar spine or hip and alm / current height < 5.45 kg / m ( female ) and < 7.26 kg / m ( male ) . for the sensitive definitions , a one - third radius t - score ( derived using the ge normative gender matched database ) of 2.5 was considered osteoporosis and the alm / ht ratio utilized historical tallest height and the sarcopenia cut points noted above of 5.45 and 7.26 examples of the bone ( left ) and soft tissue ( right ) dxa image of an individual . appendicular lean mass ( alm ) is defined as the lean mass , excluding bone and fat , in the arms ( light gray region ) and legs ( dark gray region ) appendicular lean mass regions of interest . examples of the bone ( left ) and soft tissue ( right ) dxa image of an individual . appendicular lean mass ( alm ) is defined as the lean mass , excluding bone and fat , in the arms ( light gray region ) and legs ( dark gray region ) the potential importance of using a dxa site not affected by degenerative change was explored by defining the prevalence of obvious degenerative disease . to this end , all dxa spine images were reviewed by one experienced densitometrist ( nb ) . degenerative changes were considered to be present if obvious osteophytes , vertebral fractures , or focal degenerative changes involving less than an entire vertebral body , or a 1 t - score discrepancy between vertebral bodies was observed . demographic comparisons and evaluation of t - score comparability were performed using t - tests with statview ( cary , nc ) software . pearson and fisher chi - square statistics ( analyze - it ; leeds uk ) were used to compare the prevalence of osteoporosis , sarcopenia and sarco - osteoporosis using standard or sensitive definitions in the three age groups ( 6069 , 7079 and 80 + years ) . a total of 304 individuals ( 158 females , 146 males ) age 60 and above were included in this cross - sectional analysis . their age and bmi ( mean , sd and range ) was 75.5 ( 7.5 ) years ( 6095 ) and 27.1 ( 4.9 ) kg / m ( 15.151.6 ) , respectively . the male cohort was slightly ( p < 0.05 ) older . as could be expected , the males were taller and heavier ( p < 0.001 ) than the females ; however , mean bmi did not differ . bmd t - score was lower ( p < 0.05 ) at all sites in women . similarly , alm / ht was lower ( p < 0.001 ) in women . participants were stratified by decade of age : 6069 ( n = 89 ) ; 7079 ( n = 119 ) ; and 80 + ( n = 96).table 1study participant demographic dataage ( years)bmi ( kg / m)height loss ( cm)l1l4 t - scorefemoral neck t - scoretotal femur t - scoreone - third radius t - scorealm / height ( kg / m)female n = 15874.5 * ( 7.0)27.1 ( 5.5)3.7 * ( 2.9)0.7 ( 1.7)1.5 * ( 0.9)1.0 * ( 1.1)1.9 ( 1.4)6.5 ( 1.0)male n = 14676.5 ( 7.9)27.2 ( 4.0)4.5 ( 3.1)+0.9 ( 2.1)1.1 ( 1.0)0.7 ( 1.1)0.5 ( 1.1)8.1 ( 1.0)data are presented as mean ( sd)different than males : * p < 0.05 ; p < 0.001 study participant demographic data data are presented as mean ( sd ) different than males : * p < 0.05 ; p < 0.001 obvious degenerative changes ( examples in fig . 2 ) were apparent on the dxa image in virtually all ( 93 % ) of these volunteers . only 14 of 158 ( 9 % ) of the women and eight of 146 ( 5 % ) of these men had no apparent degenerative changes . as a result , lumbar spine t - scores deviated from normal to a lesser degree than in the radius in both men and women ( p < 0.0001 ; fig . 3 ) . of those 14 individuals without obvious degenerative changes on their dxa image , 59 % were less than age 70.fig . examples of what were considered as obvious degenerative changes on the dxa images are presented here . the image on the left is a 70-year - old man in whom focal abnormalities at l2 and l3 are apparent . the individual vertebral body t - scores are consistent ; from l1 to l4 these values are + 1.1 , + 4.5 , + 4.2 and + 1.7 ; thus , a > 1 t - score difference is present between adjacent vertebrae . the 86-year - old female on the right has apparent degenerative changes at l2 , l3 and l4 with obvious osteophytes at l2 and l3 . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectivelyfig . 3comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . the broad range of lumbar spine t - scores in males is striking with values above + 5 in some men . this was not the case in the one - third radius site examples of spinal degenerative changes . examples of what were considered as obvious degenerative changes on the dxa images are presented here . the image on the left is a 70-year - old man in whom focal abnormalities at l2 and l3 are apparent . the individual vertebral body t - scores are consistent ; from l1 to l4 these values are + 1.1 , + 4.5 , + 4.2 and + 1.7 ; thus , a > 1 t - score difference is present between adjacent vertebrae . the 86-year - old female on the right has apparent degenerative changes at l2 , l3 and l4 with obvious osteophytes at l2 and l3 . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectively comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . the broad range of lumbar spine t - scores in males is striking with values above + 5 in some men . this was not the case in the one - third radius site self - reported tallest height was greater than current stadiometer - measured height in 97 % ( 296/304 ) of study participants . males had a larger mean reported height loss than females ( 4.5 vs. 3.7 cm ; p < 0.05 ; table 1 ; fig . 4a ) . use of reported tallest height in the alm / ht ratio reduced this value by a mean of 4.9 % in men and 4.4 % in women ( fig . the distribution curves ( using a normal continuous fit function ) for the amount of reported historical height loss ( a ) shows a mean of 4.5 cm in men and 3.7 cm in women . the variability of height loss is striking with some individuals reporting more than 10 cm of height loss . the negative values ( suggesting height gain ) are evidence of limitations of historical tallest height . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) historical height change and effect on alm / ht ratio . the distribution curves ( using a normal continuous fit function ) for the amount of reported historical height loss ( a ) shows a mean of 4.5 cm in men and 3.7 cm in women . the variability of height loss is striking with some individuals reporting more than 10 cm of height loss . the negative values ( suggesting height gain ) are evidence of limitations of historical tallest height . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) the prevalence of sarcopenia increased with age ( p < 0.01 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarcopenia than the standard criteria ( p < 0.0001 ) . overall , 15 % of this cohort was classified as sarcopenic with standard criteria whereas the sensitive criteria identified 26 % .fig . 5prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . sarcopenia was more common in individuals age 80 + than 7079 and 6069 years ( p < 0.01 ) regardless of criteria used ( a ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . similarly , osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.001 ) regardless of criteria used ( b ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . sarcopenia was more common in individuals age 80 + than 7079 and 6069 years ( p < 0.01 ) regardless of criteria used ( a ) . 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . similarly , osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.001 ) regardless of criteria used ( b ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria osteoporosis prevalence increased with age ( p < 0.001 ) with both the standard and sensitive criteria ( fig . the sensitive criteria identified more individuals with osteoporosis than the standard criteria ( p < 0.0001 ) . overall , 17 % of this cohort was classified as osteoporotic with standard criteria whereas the sensitive criteria identified 29 % . sarco - osteoporosis prevalence increased with age ( p < 0.05 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarco - osteoporosis than the standard criteria ( p < 0.0001 ) . the prevalence of osteoporosis and sarcopenia were similar ; however , the prevalence of sarco - osteoporosis was less than either ( p < 0.0001 ) . specifically , 5 % of the entire cohort was classified as having sarco - osteoporosis with standard criteria whereas the sensitive criteria identified 11 % . a total of 304 individuals ( 158 females , 146 males ) age 60 and above were included in this cross - sectional analysis . their age and bmi ( mean , sd and range ) was 75.5 ( 7.5 ) years ( 6095 ) and 27.1 ( 4.9 ) kg / m ( 15.151.6 ) , respectively . the male cohort was slightly ( p < 0.05 ) older . as could be expected , the males were taller and heavier ( p < 0.001 ) than the females ; however , mean bmi did not differ . bmd t - score was lower ( p < 0.05 ) at all sites in women . similarly , alm / ht was lower ( p < 0.001 ) in women . participants were stratified by decade of age : 6069 ( n = 89 ) ; 7079 ( n = 119 ) ; and 80 + ( n = 96).table 1study participant demographic dataage ( years)bmi ( kg / m)height loss ( cm)l1l4 t - scorefemoral neck t - scoretotal femur t - scoreone - third radius t - scorealm / height ( kg / m)female n = 15874.5 * ( 7.0)27.1 ( 5.5)3.7 * ( 2.9)0.7 ( 1.7)1.5 * ( 0.9)1.0 * ( 1.1)1.9 ( 1.4)6.5 ( 1.0)male n = 14676.5 ( 7.9)27.2 ( 4.0)4.5 ( 3.1)+0.9 ( 2.1)1.1 ( 1.0)0.7 ( 1.1)0.5 ( 1.1)8.1 ( 1.0)data are presented as mean ( sd)different than males : * p < 0.05 ; p < 0.001 study participant demographic data data are presented as mean ( sd ) different than males : * p < 2 ) were apparent on the dxa image in virtually all ( 93 % ) of these volunteers . only 14 of 158 ( 9 % ) of the women and eight of 146 ( 5 % ) of these men had no apparent degenerative changes . as a result , lumbar spine t - scores deviated from normal to a lesser degree than in the radius in both men and women ( p < 0.0001 ; fig . 3 ) . of those 14 individuals without obvious degenerative changes on their dxa image , 59 % were less than age 70.fig . examples of what were considered as obvious degenerative changes on the dxa images are presented here . the image on the left is a 70-year - old man in whom focal abnormalities at l2 and l3 are apparent . the individual vertebral body t - scores are consistent ; from l1 to l4 these values are + 1.1 , + 4.5 , + 4.2 and + 1.7 ; thus , a > 1 t - score difference is present between adjacent vertebrae . the 86-year - old female on the right has apparent degenerative changes at l2 , l3 and l4 with obvious osteophytes at l2 and l3 . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectivelyfig . 3comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . the broad range of lumbar spine t - scores in males is striking with values above + 5 in some men . this was not the case in the one - third radius site examples of spinal degenerative changes . examples of what were considered as obvious degenerative changes on the dxa images are presented here . the image on the left is a 70-year - old man in whom focal abnormalities at l2 and l3 are apparent . the individual vertebral body t - scores are consistent ; from l1 to l4 these values are + 1.1 , + 4.5 , + 4.2 and + 1.7 ; thus , a > 1 t - score difference is present between adjacent vertebrae . the 86-year - old female on the right has apparent degenerative changes at l2 , l3 and l4 with obvious osteophytes at l2 and l3 . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectively comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . the broad range of lumbar spine t - scores in males is striking with values above + 5 in some men . self - reported tallest height was greater than current stadiometer - measured height in 97 % ( 296/304 ) of study participants . males had a larger mean reported height loss than females ( 4.5 vs. 3.7 cm ; p < 0.05 ; table 1 ; fig . use of reported tallest height in the alm / ht ratio reduced this value by a mean of 4.9 % in men and 4.4 % in women ( fig . the distribution curves ( using a normal continuous fit function ) for the amount of reported historical height loss ( a ) shows a mean of 4.5 cm in men and 3.7 cm in women . the variability of height loss is striking with some individuals reporting more than 10 cm of height loss . the negative values ( suggesting height gain ) are evidence of limitations of historical tallest height . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) historical height change and effect on alm / ht ratio . the distribution curves ( using a normal continuous fit function ) for the amount of reported historical height loss ( a ) shows a mean of 4.5 cm in men and 3.7 cm in women . the variability of height loss is striking with some individuals reporting more than 10 cm of height loss . the negative values ( suggesting height gain ) are evidence of limitations of historical tallest height . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) the prevalence of sarcopenia increased with age ( p < 0.01 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarcopenia than the standard criteria ( p < 0.0001 ) . overall , 15 % of this cohort was classified as sarcopenic with standard criteria whereas the sensitive criteria identified 26 % .fig . 5prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . sarcopenia was more common in individuals age 80 + than 7079 and 6069 years ( p < 0.01 ) regardless of criteria used ( a ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . similarly , osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.001 ) regardless of criteria used ( b ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . sarcopenia was more common in individuals age 80 + than 7079 and 6069 years ( p < 0.01 ) regardless of criteria used ( a ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . similarly , osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.001 ) regardless of criteria used ( b ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria osteoporosis prevalence increased with age ( p < 0.001 ) with both the standard and sensitive criteria ( fig . the sensitive criteria identified more individuals with osteoporosis than the standard criteria ( p < 0.0001 ) . overall , 17 % of this cohort was classified as osteoporotic with standard criteria whereas the sensitive criteria identified 29 % . sarco - osteoporosis prevalence increased with age ( p < 0.05 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarco - osteoporosis than the standard criteria ( p < 0.0001 ) . the prevalence of osteoporosis and sarcopenia were similar ; however , the prevalence of sarco - osteoporosis was less than either ( p < 0.0001 ) . specifically , 5 % of the entire cohort was classified as having sarco - osteoporosis with standard criteria whereas the sensitive criteria identified 11 % . in this cross - sectional study of community - dwelling adults age 60 and older , the prevalence of osteoporosis , sarcopenia and sarco - osteoporosis was higher in older individuals . the prevalence of osteoporosis and sarcopenia was similar , whereas sarco - osteoporosis was less common . use of potentially more sensitive criteria ( i.e. , using tallest historical height instead of current height to define sarcopenia and including 1/3 radius bmd t - score in the definition of osteoporosis ) increased the prevalence of sarcopenia , osteoporosis and sarco - osteoporosis . this is not surprising as both degenerative changes , which elevate dxa - measured bmd , and reported height loss were present in virtually all of these individuals . to our knowledge , clearly , the prevalence of sarcopenia , osteoporosis and sarco - osteoporosis will vary depending on the population studied ( i.e. , prevalent hip fracture , included gender , age group and degree of frailty ) . as noted in the introduction , the prevalence of sarco - osteoporosis has been reported as high as 45 % in individuals who already suffered a hip fracture . other authors report percentages closer to those found in this study [ 19 , 30 ] . similar to the results reported here , most studies that divided participants by age observed a higher prevalence in older age groups [ 19 , 2931 ] . age is accepted as a major contributor to fracture risk [ 6 , 12 ] . it is probable that there are many risk factors hidden within age contributing to this increased risk ; evidence is increasing that sarcopenia is one such factor [ 19 , 2528 , 31 , 4547 ] . importantly , sarcopenia is closely linked to falls and increased risk of falling is a well - established contributor to fracture [ 10 , 26 , 48 , 49 ] . in addition to increasing falls risk , sarcopenia might also decrease bone strength by reducing mechanical loading to the skeleton . reduction of mechanical stimulation could result from decreased maximal force that weaker muscles produce and/or less time that the skeleton is loaded due to relative immobility [ 5053 ] . as such , the concept of sarco - osteoporosis , seems likely to facilitate fracture risk estimation [ 27 , 28 , 31 ] . moreover , simple approaches such as described here ( radius bmd and use of historical height ) with potential to enhance diagnostic sensitivity for sarco - osteoporosis , may have direct clinical applicability in improving fracture risk estimation . consideration of clinical risk factors for fracture in addition to bmd has significantly changed osteoporosis treatment decision - making . however , potential limitation of frax ( www.shef.ac.uk/frax ) , relevant to this study is that only femoral neck ( not spine or radius ) bmd is considered . furthermore , sarcopenia , muscle function and falls are not able to be included in this algorithm given limitations of the observational cohorts used to derive frax [ 5456 ] . a second method to estimate fracture risk developed by the garvan institute ( www.garvan.org.au/bone-fracture-risk ) does include history and number of falls within the past year but no measure of muscle mass or function [ 10 , 11 ] . it could be argued that measurement of bmd at additional skeletal sites , i.e. , the forearm , is not necessary when bmd is measured at both the spine and femoral neck . however , lumbar spine bmd is elevated by degenerative changes that are present in virtually all older adults , 93 % in this study . for example , a study of 300 australian men and women age 60 and above found x - ray evidence of osteophytes in 69 % of all males and females , disc narrowing in 67 % and apophyseal osteoarthritis ( posterior element disease ) in 99 % of participants . another study of 600 japanese women age 60 and above found osteoarthritic changes in 96 % of individuals on lumbar spine x - rays . as could be expected , spinal degenerative changes increase lumbar spine bmd measured by dxa ; on average by 1524 % [ 5759 ] . additionally , femoral neck bmd measurement may also be elevated by degenerative changes . other femoral neck bmd measurement confounders include internal artifacts , fat panniculi and anatomic variations [ 60 , 61 ] . it seems likely that such confounders are one contributor to the less than ideal predictive capability of tools such as frax. as such , evaluations of potential approaches to enhance fracture prediction capability , potentially including forearm bmd measurement , are appropriate . limitations of this study include relatively small sample size and cross - sectional nature . furthermore , in this study sarcopenia could only be defined based on dxa measured alm / ht ratio , as muscle function data were not available in all cohorts used for this analysis . current consensus definitions for sarcopenia appropriately use both muscle mass and muscle function parameters as the combination of these measures improve the prediction of poor outcomes [ 20 , 22 ] . additionally , use of self - reported tallest height is inferior to serial height measurements over time as there is risk for recall errors and bias . two systematic reviews have identified a tendency in adults to overestimate historical tallest height , leading to a greater height loss than if prospectively measured . [ 62 , 63 ] despite this , self - reported height loss has apparent clinical applicability and , importantly , predicts clinical outcomes including vertebral fractures , mortality and morbidity [ 62 , 64 , 65 ] . future study to further define the accuracy of height recall , and an additional limitation is that data regarding prior fragility fracture or other outcomes for sarco - osteoporosis are not available in this study . future work is needed in longitudinal studies to evaluate the effect of sarco - osteoporosis diagnosis on risk for fragility fracture and also to explore the utility of using historical tallest height and radius bmd measurement on this diagnosis . in conclusion , sarco - osteoporosis is present in 10 % of adults over age 80 and the prevalence is higher in older people . using simple , but potentially more sensitive criteria , i.e. , adding the one - third radius bmd t - score and using tallest historical height , rather than current height , in the sarcopenia alm / height calculation identifies more individuals as potentially being at risk . assessment of both bone and muscle mass / function in older adults could potentially enhance fracture risk prediction ; research utilizing existing longitudinal studies in which bone and muscle status was carefully defined and fractures rigorously captured is needed to evaluate the potential utility of sarcopenia and sarco - osteoporosis consideration in fragility fracture risk assessment . although no causal attribution is possible in this analysis , sarco - osteoporosis may explain some of the increase in fracture risk currently related to age . bjoern buehring , diane krueger , and neil binkley do not have any potential conflicts of interest .

backgrounda clinical need exists to improve identification of those who will sustain fragility fractures . individuals with both osteoporosis ( op ) and sarcopenia ( sp ) , so - called sarco - osteoporosis ( sop ) , might be at higher fracture risk than those with op or sp alone . approaches to facilitate sop identification , e.g. , use of tallest historical rather than current height and inclusion of radius bone mineral density ( bmd ) measurement , may be of benefit . this study examined the effect of advancing age on sop prevalence with and without use of historical tallest height and radius bmd measurement.methodsadults age 60 + underwent dual - energy x - ray absorptiometry ( dxa ) bmd and total body composition measurement . op and sp were defined using standard criteria : t - score 2.5 at the lumbar spine or hip and appendicular lean mass ( alm)/current height2 < 5.45 kg / m2 ( female ) and < 7.26 kg / m2 ( male ) . proposed sensitive sp criteria used historical tallest height instead of current height , while sensitive op criteria added the 1/3rd radius t - score . the primary outcome was sop prevalence by decade ( 6069 , 7079 , 80+).resultsa total of 304 individuals ( 146 m/158 f ) participated . op , sp and sop prevalence were higher in older adults and increased ( p < 0.05 ) with the sensitive criteria . sop prevalence was lower than that of op or sp and increased ( standard / sensitive ) criteria from 1.1 % / 4.5 % in the 6069 years age group to 10.4 % / 21.9 % in the 80 + years age group.conclusionssop prevalence is higher in older adults . use of historical tallest height and 1/3rd radius bmd increases sop prevalence . future studies need to assess whether having sop increases fracture risk and whether use of tallest height and/or one - third radius bmd improves fracture risk prediction .

Introduction Methods Participants Bone mineral density and body composition measurement Statistical analysis Results Study participants Prevalence of degenerative changes/effect of including one-third radius measurement on T-score distribution Height loss/effect on ALM/height Prevalence of sarcopenia, osteoporosis and sarco-osteoporosis Discussion Conclusion Conflict of interest

a clinical need exists to optimally identify those who will sustain fragility fractures . simply utilizing bone mineral density ( bmd ) measurement alone is not adequate , as the majority of people who sustain osteoporosis - related fractures do not have osteoporosis by bmd t - score [ 14 ] . it is possible that individuals with both osteoporosis and sarcopenia ( sarco - osteoporosis ) are at greatest fracture risk [ 2730 ] . however , spine bmd as measured by dual - energy x - ray absorptiometry ( dxa ) is commonly elevated in older adults by degenerative changes [ 3638 ] . thus , it is possible that routine performance of one - third radius bmd measurement in older adults might improve fracture risk prediction . to this end , we hypothesized that use of historical tallest height in the alm / ht ratio and inclusion of radius bmd t - score might enhance identification of sarcopenia and osteoporosis , respectively , thereby facilitating sarco - osteoporosis recognition and potentially fracture risk estimation in older adults . to test this hypothesis , the aims of this study in a group of adults age 60 + years were to : ( 1 ) evaluate the effect of inclusion of one - third radius bmd measurement on t - score distribution and use of historical height vs. current height on the alm / ht ratio and ( 2 ) define the prevalence of sarcopenia , osteoporosis and sarco - osteoporosis with and without using one - third radius bmd and historical height . here we report the effect of these approaches on prevalence of osteoporosis , sarcopenia and sarco - osteoporosis in a convenience sample of 304 community dwelling adults age 60 years and over . these studies include the midus ( mid - life in the united states ) ii study , precision , a dxa precision research study comparing bmd and body composition between two ge healthcare lunar densitometers , ( idxa and prodigy ) and jump , a study examining jump mechanography as a tool to assess function in older adults . the lowest bmd t - score of the lumbar spine , femoral neck , total proximal femur or one - third radius was chosen to define osteoporosis using the world health organization ( who ) t - score based classification system . , a t - score 2.5 at the lumbar spine or hip and alm / current height < 5.45 kg / m ( female ) and < 7.26 kg / m ( male ) . for the sensitive definitions , a one - third radius t - score ( derived using the ge normative gender matched database ) of 2.5 was considered osteoporosis and the alm / ht ratio utilized historical tallest height and the sarcopenia cut points noted above of 5.45 and 7.26 kg / m were applied.fig . pearson and fisher chi - square statistics ( analyze - it ; leeds uk ) were used to compare the prevalence of osteoporosis , sarcopenia and sarco - osteoporosis using standard or sensitive definitions in the three age groups ( 6069 , 7079 and 80 + years ) . these studies include the midus ( mid - life in the united states ) ii study , precision , a dxa precision research study comparing bmd and body composition between two ge healthcare lunar densitometers , ( idxa and prodigy ) and jump , a study examining jump mechanography as a tool to assess function in older adults . the lowest bmd t - score of the lumbar spine , femoral neck , total proximal femur or one - third radius was chosen to define osteoporosis using the world health organization ( who ) t - score based classification system . , a t - score 2.5 at the lumbar spine or hip and alm / current height < 5.45 kg / m ( female ) and < 7.26 kg / m ( male ) . for the sensitive definitions , a one - third radius t - score ( derived using the ge normative gender matched database ) of 2.5 was considered osteoporosis and the alm / ht ratio utilized historical tallest height and the sarcopenia cut points noted above of 5.45 and 7.26 examples of the bone ( left ) and soft tissue ( right ) dxa image of an individual . pearson and fisher chi - square statistics ( analyze - it ; leeds uk ) were used to compare the prevalence of osteoporosis , sarcopenia and sarco - osteoporosis using standard or sensitive definitions in the three age groups ( 6069 , 7079 and 80 + years ) . bmd t - score was lower ( p < 0.05 ) at all sites in women . participants were stratified by decade of age : 6069 ( n = 89 ) ; 7079 ( n = 119 ) ; and 80 + ( n = 96).table 1study participant demographic dataage ( years)bmi ( kg / m)height loss ( cm)l1l4 t - scorefemoral neck t - scoretotal femur t - scoreone - third radius t - scorealm / height ( kg / m)female n = 15874.5 * ( 7.0)27.1 ( 5.5)3.7 * ( 2.9)0.7 ( 1.7)1.5 * ( 0.9)1.0 * ( 1.1)1.9 ( 1.4)6.5 ( 1.0)male n = 14676.5 ( 7.9)27.2 ( 4.0)4.5 ( 3.1)+0.9 ( 2.1)1.1 ( 1.0)0.7 ( 1.1)0.5 ( 1.1)8.1 ( 1.0)data are presented as mean ( sd)different than males : * p < 0.05 ; p < 0.001 study participant demographic data data are presented as mean ( sd ) different than males : * p < 0.05 ; p < 0.001 obvious degenerative changes ( examples in fig . 3comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectively comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . use of reported tallest height in the alm / ht ratio reduced this value by a mean of 4.9 % in men and 4.4 % in women ( fig . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) the prevalence of sarcopenia increased with age ( p < 0.01 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarcopenia than the standard criteria ( p < 0.0001 ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria osteoporosis prevalence increased with age ( p < 0.001 ) with both the standard and sensitive criteria ( fig . the sensitive criteria identified more individuals with osteoporosis than the standard criteria ( p < 0.0001 ) . sarco - osteoporosis prevalence increased with age ( p < 0.05 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarco - osteoporosis than the standard criteria ( p < 0.0001 ) . specifically , 5 % of the entire cohort was classified as having sarco - osteoporosis with standard criteria whereas the sensitive criteria identified 11 % . bmd t - score was lower ( p < 0.05 ) at all sites in women . participants were stratified by decade of age : 6069 ( n = 89 ) ; 7079 ( n = 119 ) ; and 80 + ( n = 96).table 1study participant demographic dataage ( years)bmi ( kg / m)height loss ( cm)l1l4 t - scorefemoral neck t - scoretotal femur t - scoreone - third radius t - scorealm / height ( kg / m)female n = 15874.5 * ( 7.0)27.1 ( 5.5)3.7 * ( 2.9)0.7 ( 1.7)1.5 * ( 0.9)1.0 * ( 1.1)1.9 ( 1.4)6.5 ( 1.0)male n = 14676.5 ( 7.9)27.2 ( 4.0)4.5 ( 3.1)+0.9 ( 2.1)1.1 ( 1.0)0.7 ( 1.1)0.5 ( 1.1)8.1 ( 1.0)data are presented as mean ( sd)different than males : * p < 0.05 ; p < 0.001 study participant demographic data data are presented as mean ( sd ) different than males : * p < 2 ) were apparent on the dxa image in virtually all ( 93 % ) of these volunteers . as a result , lumbar spine t - scores deviated from normal to a lesser degree than in the radius in both men and women ( p < 0.0001 ; fig . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . t - score differences between adjacent vertebrae were observed with values from l1 through l4 being + 0.2 , + 1.7 , + 3.5 and + 4.1 , respectively comparison of lumbar spine , femur neck and one - third radius t - scores in men and women . depicted are distribution curves ( using a normal continuous fit function ) of lumbar spine , femur neck and one - third radius t - scores in men ( a ) and women ( b ) . in both genders mean bmd t - scores were the higher at lumbar spine compared to the femur neck or the one - third radius site ( all p < 0.0001 ) . mean bmd t - scores at the femur neck were higher than the one - third radius in females but lower in males ( all p < 0.01 ; see table 1 for t - score values and their standard deviations ) . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) historical height change and effect on alm / ht ratio . the distribution curves of percentage change of using reported historical height loss instead of current height show that in both genders the mean alm / height ratio was decreased ( 4.4 % in females and 4.9 % in males ) the prevalence of sarcopenia increased with age ( p < 0.01 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarcopenia than the standard criteria ( p < 0.0001 ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria prevalence of sarcopenia , osteoporosis and sarco - osteoporosis by decade using standard or sensitive criteria . sarcopenia was more common in individuals age 80 + than 7079 and 6069 years ( p < 0.01 ) regardless of criteria used ( a ) . significantly more ( p < 0.0001 ) individuals had sarcopenia using the sensitive criteria ( alm / reported historical tallest height ) compared to standard criteria ( alm / current height ) . significantly more ( p < 0.0001 ) individuals had osteoporosis using the sensitive criteria ( lowest t - score from hip , lumbar spine and one - third radius ) compared to standard criteria ( lowest t - score of hip and lumbar spine only ) . sarco - osteoporosis was more common in individuals age 80 + than 7079 and 6069 ( p < 0.05 ) regardless of criteria used ( c ) . significantly more ( p < 0.0001 ) individuals had sarco - osteoporosis using the sensitive criteria compared to standard criteria osteoporosis prevalence increased with age ( p < 0.001 ) with both the standard and sensitive criteria ( fig . the sensitive criteria identified more individuals with osteoporosis than the standard criteria ( p < 0.0001 ) . sarco - osteoporosis prevalence increased with age ( p < 0.05 ) using either the standard or sensitive criteria ( fig . the sensitive criteria identified more individuals with sarco - osteoporosis than the standard criteria ( p < 0.0001 ) . the prevalence of osteoporosis and sarcopenia were similar ; however , the prevalence of sarco - osteoporosis was less than either ( p < 0.0001 ) . specifically , 5 % of the entire cohort was classified as having sarco - osteoporosis with standard criteria whereas the sensitive criteria identified 11 % . , using tallest historical height instead of current height to define sarcopenia and including 1/3 radius bmd t - score in the definition of osteoporosis ) increased the prevalence of sarcopenia , osteoporosis and sarco - osteoporosis . moreover , simple approaches such as described here ( radius bmd and use of historical height ) with potential to enhance diagnostic sensitivity for sarco - osteoporosis , may have direct clinical applicability in improving fracture risk estimation . future work is needed in longitudinal studies to evaluate the effect of sarco - osteoporosis diagnosis on risk for fragility fracture and also to explore the utility of using historical tallest height and radius bmd measurement on this diagnosis . in conclusion , sarco - osteoporosis is present in 10 % of adults over age 80 and the prevalence is higher in older people . , adding the one - third radius bmd t - score and using tallest historical height , rather than current height , in the sarcopenia alm / height calculation identifies more individuals as potentially being at risk . assessment of both bone and muscle mass / function in older adults could potentially enhance fracture risk prediction ; research utilizing existing longitudinal studies in which bone and muscle status was carefully defined and fractures rigorously captured is needed to evaluate the potential utility of sarcopenia and sarco - osteoporosis consideration in fragility fracture risk assessment .

vertebrate animals affiliate in multiple contexts , but sexual behavior is likely the only context of affiliation that is nearly ubiquitous across vertebrate species . thus , the various forms of social contact that we observe across species are subject to independent evolution in different taxa , although some forms are conserved across very large clades . the provision of maternal care to offspring post - hatching or post - parturition is a good example . this kind of behavior typifies most mammals and most birds , but mammals and birds have evolved extended maternal care independently ( alcock , 2009 ; goodenough et al . , 2010 ) . studies of many vertebrate taxa demonstrate that the neural mechanisms of sexual behavior are strongly conserved ( goodson , 2005 ; martinez - garcia et al . , 2008 ) , and limited evidence suggests that mechanisms of parental care are at least grossly similar in mammals and birds , as well ( buntin et al . , 2006 ) . however , almost no generalizations can be made regarding the mechanisms of evolutionarily labile aspects of social behavior , such as cooperative breeding , mating systems , and grouping . these variables represent major defining features of species - specific social structures and have therefore garnered an extraordinary amount of attention from evolutionary biologists and behavioral ecologists . however , evolutionarily labile behaviors pose fairly extreme challenges for comparative neurobiology , largely for reasons of feasibility . for instance , after two decades of intensive research , the neural mechanisms of selective partner preference ( a hallmark of monogamy ) , paternal care , and alloparental care are reasonably well understood for only a single species , the monogamous prairie vole ( microtus ochrogaster ) , and we know a limited amount about the critical variables that differentiate prairie voles from other non - monogamous vole species ( carter et al . , 2008 ; donaldson and young , 2008 ; aragona and wang , 2009 ; ross and young , 2009 ; mcgraw and young , 2010 ) . what we do not yet know is whether neural mechanisms have evolved convergently in other monogamous or alloparental species , although this is a matter of substantial current interest ( fink et al . , 2006 ; this example points out a fundamental difference in the approaches to social organization that are employed by evolutionary biologists and neuroethologists . evolutionary biologists can often bring extensive information to bear on a given question , perhaps using data from dozens or hundreds of species , and analyze it in a phylogenetically corrected way that allows the extrapolation of general patterns . to some extent , neurobiologists can employ similar techniques if they are based on anatomical characteristics or genes ( northcutt , 2008 ; pollen and hofmann , 2008 ) . nonetheless , we can not make any strong claims about evolutionary patterns of regulation if the experimental approaches of behavioral neuroscience are not employed , and it simply not feasible to replicate experiments in dozens or hundreds of species . for this reason , neuroethological approaches to questions about evolutionarily labile behaviors will likely continue to be somewhat different from evolutionary biological approaches , even when both are employed by the same investigators . however , if neuroethologists want to establish predictive validity for other species , comparative experimentation focused on divergent and convergent evolution is essential . in the sections that follow , we describe a quasi - experimental approach that we have taken to study convergent and divergent evolution in grouping behavior within the estrildid finch family . although only five species have been used ( table 1 ) , they have been carefully selected and our findings should therefore have good predictive validity for other estrildid finches . whether our findings can be extrapolated to other vertebrate taxa is a matter of greater speculation , and a topic that we will return to in the section conclusion . behavioral and ecological characteristics of estrildid finch species that have been used for studies of grouping behavior . for relevant references , see footnote 1 . the genus uraeginthus contains three species of blue waxbill that are each known by multiple common names . thus , although u. angolensis is most typically referred to as simply blue waxbill , we have referred to this species as angolan blue waxbill in our publications in order to clearly differentiate them from other species . this species is commonly encountered in the scientific literature , but under a variety of common names , including spotted munia , nutmeg mannikin , and spice finch . sociality sensu stricto , as defined by grouping behavior ( alexander , 1974 ) , has broad influences on other important variables such as reproductive behavior , disease transmission , resource exploitation , and defense ( moller and birkhead , 1993 ; krause and ruxton , 2002 ; silk , 2007 ) , but has not previously been examined in neurobiological studies , likely because grouping is difficult to isolate from other aspects of ecology and behavior , such as mating system and patterns of parental care . for instance , rodent species that differ in their grouping behavior also differ in whether they are monogamous or polygamous , and whether the father contributes to parental care ( king , 1968 ; tamarin , 1985 ) . however , if we want to examine the neural mechanisms that titrate species - typical group - size preferences , control for such variables is very important , given that shared mechanisms ( particularly neuroendocrine mechanisms ) often regulate numerous aspects of social behavior ( e.g. , pair bonding , parental care , and affiliation ) and related aspects of physiology ( e.g. , hormone levels and stress physiology ; carter et al . birds offer excellent opportunities to study grouping , since we can identify closely related species that are virtually identical in most aspects of behavior and ecology , but that nonetheless exhibit extreme variation in sociality , ranging from territorial pairs to massive flocks . the majority of estrildid species ( approximately 141 total ; clements , 2007 ) form small groups of about 612 birds when they are not breeding , and then loosely distribute themselves for nesting without strong territoriality . defense of a breeding territory has evolved in five to six species across three genera . colonial breeding has also evolved in several genera , although most colonies are small ( perhaps 510 breeding pairs ) . available evidence suggests that all estrildids exhibit biparental care and long - term ( typically life - long ) , socially monogamous pair bonds ( immelmann , 1965 ; goodwin , 1982 ) . we have collected five estrildid finch species that exhibit large and seasonally stable differences in grouping , but are otherwise closely matched in behavior and ecology ( table 1 ) . in addition to being socially monogamous and biparental , as already noted , all of these species live in arid or semi - arid grassland scrub and breed opportunistically or semi - opportunistically in relation to rainfall ( skead , 1975 ; goodwin , 1982 ; zann , 1996 ; goodson et al . , 2006 ) . these include two territorial african species that live as male female pairs year - round ( violet - eared waxbill , uraeginthus granatina , and melba finch , pytilia melba ) ; two highly gregarious , colonially breeding species that exhibit modal group sizes of approximately 100 ( zebra finch , taeniopygia guttata , and spice finch , lonchura punctulata ) ; and a moderately gregarious species , the angolan blue waxbill ( uraeginthus angolensis ) , which exhibits a modal group size of approximately 20 . the two territorial species have evolved their territorial behavior independently and the two colonial species have also evolved their extreme sociality independently ( goodson et al . , 2006 ) . the gregarious angolan blue waxbill is sympatric with both the melba finch and violet - eared waxbill , and our laboratory population of these three species has been established through the breeding of birds that were caught from a single location in the kalahari thornscrub of south africa in 2001 . we have obtained zebra finches and wild - caught spice finches ( likely of an indian subspecies ) from commercial suppliers . the inclusion of the zebra finch is an important asset to our research program because domestic zebra finches are both behaviorally robust and readily available , which makes them an ideal species for extensive laboratory studies . domestic zebra finches are behaviorally indistinguishable from wild - caught zebra finches ( morris , 1958 ) and virtually their full range of social behavior can be observed in the lab . for instance , we are able to quantify over 20 behaviors while zebra finches are interacting in a colony environment , and can readily track the natural formation of monogamous pair bonds ( goodson et al . , 1999 ; vertebrates possess a core social behavior network within the basal ( limbic ) forebrain and midbrain ( figure 1 ) that is evolutionarily conserved across all taxa , but is particularly stable across the amniote clades giving rise to birds and mammals , as demonstrated by a wide range of functional and anatomical studies . this network includes the medial extended amygdala ( including the medial bed nucleus of the stria terminalis , bstm ) , lateral septum ( ls ) , anterior hypothalamus ( ah ) , ventromedial hypothalamus ( vmh ) , midbrain central gray ( cg ; or periaqueductal gray ) , and the ventral tegmental area , although these structures are not strictly social in function and contribute to other core networks for the regulation of behavior and physiology . all of these areas express high densities of sex steroid receptors , and in contrast to most other areas of the brain that influence social behavior , they are absolutely essential for basic functions such as the expression of sexual , aggressive , and parental behaviors , and also for the regulation of anxiety , social recognition , and approach avoidance processes ( newman , 1999 ; goodson , 2005 ) . an evolutionarily conserved suite of brain regions that regulate vertebrate social behavior . ( a ) the core components of the social behavior network include numerous areas of the basal forebrain the medial extended amygdala ( medial amygdala , mea , or taenial amygdala , tna , plus the medial bed nucleus of the stria terminalis , bstm ) , medial preoptic area ( mpoa ) , anterior hypothalamus ( ah ) , ventromedial hypothalamus ( vmh ) , and lateral septum ( ls ) , as well as areas of the midbrain , most notably the central gray ( cg ; or periaqueductal gray , pag ) and the ventral tegmental area ( vta ) . ( b ) a photomontage of a female zebra finch brain at the level of the anterior commissure ( ac ) . immunocytochemical triple - labeling for vasoactive intestinal polypeptide ( vip ) , neuropeptide y ( npy ) , and tyrosine hydroxylase ( th ) shows the location of the ah and multiple zones of the ls , bst , and vmh . . scale bar = 200 m . modified from goodson , 2005 . other abbreviations : bstl , lateral bed nucleus of the stria terminalis ; hp , hippocampus ; lh , lateral hypothalamus ; lsc , caudal division of the lateral septum ( dorsal , ventrolateral , and ventral zones denoted as lsc.d , lsc.vl , and lsc.v , respectively ) ; lsr , rostral division of the lateral septum ; me , median eminence ; ms , medial septum ; msib , internal band of the medial septum ; ot , optic tract ; om , occipitomesencephalic tract ; pvn , paraventricular nucleus of the hypothalamus ; sh , septohippocampal septum ; v , lateral ventricle . birds exhibit mammal - like patterns of immediate early gene ( ieg ) induction in this network across a variety of social contexts , including territorial aggression , a context that is strongly relevant to species differences in sociality . ieg proteins such as fos and egr-1 are rapidly inducible transcription factors that are proxy markers of neuronal activity . following resident intruder encounters in territorial rodents ( kollack - walker et al . , 1997 ; motta et al . , 2009 ) and simulated territorial intrusion ( sti ; playback of song and presentation of a caged decoy male ) in male song sparrows , significant activation is observed in forebrain areas such as the bstm , ls , paraventricular nucleus of the hypothalamus ( pvn ) , ah , and the lateral portion of the vmh ( goodson and evans , 2004 ; goodson et al . , 2005b ) , as well as midbrain areas such as the cg ( maney and ball , 2003 ) . ieg results are extensively comparable in winter and summer sparrows ( goodson and evans , 2004 ; goodson et al . notably , ieg responses of the ah , pvn , and multiple zones of the ls correlate negatively with aggression ( goodson et al . , 2005b ) , and the case is much the same in hamsters and mice , such that fighting - induced fos expression is higher in subordinate versus dominant animals ( kollack - walker et al . , 1997 ; motta et al . , 2009 ) . this applies to most aggression - related areas in the brain , and we have obtained very similar results in the territorial violet - eared waxbill ( j. l. goodson , s. e. schrock , and m. a. kingsbury , unpublished observations ) . neural circuitry that regulates grouping is substantially less well known . as an initial approach to this issue , we examined the neural responses of territorial and gregarious estrildids to same - sex conspecifics ( goodson et al . , 2005a ) . subjects were isolated in a quiet room and then exposed to a same - sex conspecific through a wire barrier . importantly , this manipulation elicits very little overt behavior and thus species differences in neural response should primarily reflect species differences in perceptual or motivational processes . despite the simplicity of this paradigm , fos and egr-1 responses clearly distinguish the territorial from gregarious ( flocking ) species , including differences between the two sympatric uraeginthus species described in the previous section . overall , territorial birds exhibit significantly greater ieg responses in the medial extended amygdala ( especially the bstm ) , ventrolateral ls , ah , lateral vmh , and cg ( and adjacent intercollicular territory that is now known to be homologous to the dorsal and dorsolateral cg of mammals ; goodson et al . this pattern is virtually identical to a pattern associated with aversive social stimulation in rodents ( sheehan et al . , 2001 ; providing a good example of deep functional conservation in the social behavior circuits of vertebrate brains ) , but possible mechanisms of gregariousness were not evident at this level of analysis . this may partially reflect the fact that functionally distinct neurons are intercalated with one another in the various brain areas that influence social behavior ( e.g. , choi et al . , for instance , as described in the next section , fos induction within arginine vasotocin ( vt ) neurons of the bstm is strictly associated with affiliation - related stimuli , and following same - sex exposure , the gregarious species show greater fos induction in the vt neurons than do territorial species ( goodson and wang , 2006 ) . however , as just noted , the overall fos response of the bstm is greater in the territorial birds ( goodson et al . , 2005a ) , suggesting that the vt neurons are intermingled with other neurons that exhibit opposing profiles of response . these hypothetical aversion neurons have not yet been identified , although aromatase - expressing neurons , which are numerous in the bstm and mostly negative for vt ( kabelik et al . , 2010 ) , are possible candidates . consistent with this idea , recent findings in mice suggest that estrogen signaling within the bstm is important for the regulation of resident intruder aggression ( trainor et al . , 2006 , 2007 ) and inhibits affiliative behavior in male prairie voles ( lei et al . , 2010 ) . nonapeptides , are among the most interesting modulators of social behavior identified to date . although many anatomical and functional properties of nonapeptide systems are strongly conserved across all vertebrate taxa ( and in the case of neurosecretory magnocellular neurons , perhaps across all bilaterians ; tessmar - raible et al . , 2007 ) , there is substantial diversity in receptor distributions and nonapeptides have been extensively linked to behavioral variation across individuals , sexes , and species ( goodson and bass , 2001 ; de vries and panzica , 2006 ; donaldson and young , 2008 ; goodson , 2008 ) . duplication of the vt gene in early jawed vertebrates gave rise to two nonapeptide lineages , which include the mammalian peptides arginine vasopressin ( vp ; homolog of vt ) and oxytocin ( ot ) . most non - mammalian vertebrates express vt and an ot - like peptide , such as isotocin , found in ray - finned fishes , or mesotocin ( mt ) , which is ubiquitously expressed in non - mammalian tetrapods ( acher , 1972 ; hoyle , 1999 ) . all jawed vertebrates express their two nonapeptide forms in both magnocellular and parvocellular neurons of the preoptic area and hypothalamus , which in amniotes are located primarily within the supraoptic nucleus of the hypothalamus and the pvn ( moore and lowry , 1998 ; goodson , 2008 ) . in rats , the parvocellular neurons of the pvn give rise to widespread projections in the brain ( de vries and buijs , 1983 ) , and this is almost certainly the same in other vertebrates . lesions of the pvn in rats virtually eliminate vp projections to the caudal brainstem , but not other areas , and eliminate ot projections throughout the brain ( de vries and buijs , 1983 ) . thus , given the strong similarities of mt and ot systems , it is likely the case that extrahypothalamic mt projections in birds are exclusively or almost exclusively derived from the pvn . in addition to these hypothalamic cell groups , most tetrapods exhibit a very unique vt / vp cell group of the bstm , which in some mammals extends into medial amygdala proper . with only a few exceptions , these neurons and their projections to the ls and other basal forebrain areas have been found to be seasonally variable , exquisitely dependent on sex steroids , and sexually dimorphic ( males > females ) . in each of these respects , the extrahypothalamic vt / vp circuitry arising in the bstm is among the most extremely plastic systems in the brain . indeed , as demonstrated for many species of mammals , amphibians , birds , and reptiles , this circuitry typically disappears in animals that are in non - reproductive condition or otherwise deprived of sex steroids ( goodson and bass , 2001 ; de vries and panzica , 2006 ) . however , in estrildids that exhibit opportunistic breeding and have no apparent endogenous reproductive cycles , no such collapse is observed , although basal transcriptional activity [ as measured by constitutive expression of fos protein in vt - immunoreactive ( -ir ) neurons ] is nonetheless regulated by androgens ( kabelik et al . , thus , of the finches that we have examined , large seasonal fluctuations in vt immunoreactivity are found only for the spice finch , a species that exhibits photorefractoriness and endogenous rhythms of reproductive physiology that correlate with monsoon cyclicity ( goodwin , 1982 ; chaturvedi and prasad , 1991 ; sikdar et al . , 1992 ; the vt / vp neurons of the bstm project to numerous other areas of the basal forebrain where vt / vp modulates aggression , parental behavior , social recognition , and various affiliative and anxiety - like behaviors ; and at least in male prairie voles , vp release in the ventral pallidum promotes partner preference ( donaldson and young , 2008 ; veenema and neumann , 2008 ; goodson and thompson , 2010 ; insel , 2010 ) . although these basal forebrain sites likely receive vt / vp from multiple hypothalamic cell groups in addition to the bstm population , the relative contributions of the different cell groups are difficult to ascertain , given the extensive evidence for paracrine signaling and volumetric release from dendrites and soma ( ludwig and leng , 2006 ; goodson and kabelik , 2009 ) . in order to determine the kinds of stimuli that different vt / vp cell groups respond to ( particularly those of the bstm ) , we have conducted several experiments in which we have exposed animals to social stimuli or control conditions , and then sacrificed the animals 90 min later for immunohistochemical colocalization of vt and fos ( see table 2 for a summary of studies ) . experimental induction of fos is still robust at 90 min , but the half - life of fos protein is only 45 min ( herdegen and leah , 1998 ) , and thus if cellular activity is depressed by our social manipulations , we can detect this as a reduction in fos expression . fos colocalization , since mt behavioral functions were unknown at the time , but we are now conducting these analyses with alternate tissue series from the same animals . a summary of main findings from studies of nonapeptide systems in territorial ( t ) , moderately gregarious ( mg ) , and highly gregarious ( hg ) species of estrildid finches . goodson and wang ( 2006 ) , also see goodson et al . goodson ( 1998 ) , goodson and adkins - regan ( 1999 ) , goodson et al . goodson et al . ( 2004 ) , kelly et al . ( 2011 ) . our experiments have demonstrated a remarkable sensitivity of the bstm vt / vp cells to social valence ( figure 2 ) . relative to handled controls , vt - ir neurons in the territorial estrildid species decrease their fos expression in response to same - sex stimuli ( exposed through a wire barrier as described above ) , but increase fos expression in response to their pairbond partner . in contrast , in colonial birds that form mixed - sex flocks , vt - ir cells in the bstm increase their activity in response to both same - sex stimuli and competitive courtship interactions , but not following intense subjugation ( goodson and wang , 2006 ) . no sex differences were observed in any of these results and similar effects were not observed for vt fos colocalization in the pvn ( j. l. goodson and y. wang , unpublished observations ) . notably , although the modestly gregarious angolan blue waxbill shows only a small ( and not significant ) increase in vt fos colocalization following exposure to a same - sex conspecific , the response profile of this species is still significantly different from its territorial congener , the violet - eared waxbill . species differences in vt signaling may be further magnified based on vt - ir cell numbers , which are approximately 10 times more abundant in the colonial species relative to other species ( goodson and wang , 2006 ) . finally , adding onto the remarkable response profile of this cell group , recent experiments in male zebra finches demonstrate that the bstm vt neurons increase their fos expression selectively in response to a positive social stimulus , but show no response to a positive non - social stimulus ( goodson et al . valence sensitivity of vasotocin ( vt ) neurons in the medial bed nucleus of the stria terminalis ( bstm ) , as demonstrated by socially induced changes in the immunocytochemical colocalization of vt and the proxy activity marker fos . ( a ) representative colocalization of vt ( green ) and fos ( red ) in the bstm of a male zebra finch following a courtship interaction . scale bar = 20 m . ( b ) in the zebra finch , which is a highly gregarious species , isolation in a quiet room followed by exposure to a same - sex conspecific through a wire barrier produces a robust increase in vt neuronal activity in both males and females . fos colocalization in the territorial violet - eared waxbill , a species that does not naturally exhibit same - sex affiliation , but exposure to the subject 's pairbond partner ( a presumably positive stimulus ) , produces a robust increase in neuronal activity . ( d ) vt fos colocalization increases in zebra finches following competition with a same - sex individual for courtship access to an opposite - sex bird , but not if the subject is paired with a highly aggressive partner and intensely subjugated . subjugated animals were aggressively displaced or attacked 71210 times during a 10-min interaction , demonstrating that social arousal alone does not increase vt ( 2009b ) ; ( b d ) are modified from goodson and wang ( 2006 ) . our results in birds are consistent with other recent findings in mice , and may therefore represent a common feature of bstm vt / vp neurons across all tetrapods . in male c57bl/6j mice , bstm vp neurons exhibit robust fos responses to copulation ( which is clearly a positive , affiliation - related stimulus ) and very modest responses to non - aggressive same - sex chemoinvestigation , but show no greater fos response to aggressive interactions than simple chemoinvestigation ( ho et al . , 2010 ) . notably , the sensitivity of amygdala neurons to valence has been extensively characterized through neurophysiological and neuroimaging studies in mammals ( nishijo et al . , 1988 ; also see goodson and thompson , 2010 ) , but specific cell types that process valence have not previously been identified . neurophysiological studies in monkeys demonstrate that neurons which exhibit stable preferences for positive or negative stimuli are intercalated ( paton et al . , 2006 ) , and thus the neurochemical identification of valence - sensitive populations represents an important step toward understanding how social value is encoded and used by the brain to regulate behavior . species differences in the response profiles of various brain areas and specific cell group are likely coordinated , at least to an extent , by the differential expression of receptors for neuromodulators . in fact , using the five finch species already introduced , we have obtained good evidence that the distributions of binding sites for vasoactive intestinal polypeptide ( vip ) , vt , and mt all exhibit convergent and divergent evolution in relation to sociality ( based on quantitative autoradiography using i vip and iodinated antagonists of ot and v1a receptors ; goodson et al . , 2006 , 2009c ) . remarkably , binding sites for all three peptides exhibit sociality - related evolution within the ls , indicating that the ls plays a special role in grouping . in order to be interpreted as sociality - related , we require that binding densities differ significantly between the two territorial and three flocking species ( defined as contrast a ) and differ significantly between the two territorial and two colonial species ( contrast b ) , with the modestly gregarious species being intermediate or falling in line with the two colonial species . the most convincing comparisons are those that meet these criteria , with additional significant differences between the territorial and gregarious uraeginthus species , which are sympatric ( contrast c ) . figure 3 shows one such pattern for the density of v1a - like receptor density in the dorsal ls , and similar abc species differences are observed in several other ls zones for both v1a - like , ot - like and vip binding sites ( goodson et al . , 2006 , 2009c ) . binding densities are mostly biased toward the gregarious species , although sociality - related differences in ot - like binding densities reverse along a dorso - ventral gradient , and the relative density between the pallial and subpallial ls provides the clearest differentiation of territorial and flocking species ( goodson et al . , 2009c ; figure 4 ) , suggesting that sociality is reflected in a complex neuromodulatory balance across the ls subnuclei . outside of the ls , sociality - related differences in the finches are observed for vip binding in the bstm , which are biased toward the gregarious species ( goodson et al . , 2006 ) . v1a antagonist binding in the lateral septum ( ls ) reflect evolutionary convergence and divergence in flocking and territoriality . ( a e ) representative i v1a antagonist binding in the ls of the territorial melba finch [ mf ; ( a ) ] , territorial violet - eared waxbill [ vew ; ( b ) ] , moderately gregarious angolan blue waxbill [ abw ; ( c ) ] , colonial spice finch [ sf ; ( d ) ] , and colonial zebra finch [ zf ; ( e ) ] . the scale bar in ( e ) corresponds to 500 m in ( a e ) . ( f , g ) representative sections for a male angolan blue waxbill and male spice finch ( colonial ) , respectively , showing species differences in binding for the nidopallium ( n ) and other areas of the forebrain . the scale bar in ( g ) corresponds to 1 mm in ( f , g ) . v1a antagonist binding in the dorsal ( pallial ) portion of the ls , shown as decompositions per min / mg ( dpm / mg ; means sem ) . different letters above the error bars denote significant species differences ( fisher 's plsd following significant anova ; p < 0.0001 ) . abbreviations : e , entopallium ; ha , apical part of the hyperpallium ; lsc , caudal division of the lateral septum ( dorsal , ventrolateral , and ventral zones denoted as lsc.d , lsc.vl , and lsc.v , respectively ) ; lsr , rostral division of the lateral septum ; lst , lateral striatum ; ms , medial septum ; n , nidopallium ; sh , septohippocampal septum ; teo , optic tectum . species - specific distributions of oxytocin - like binding sites reflect evolutionary convergence and divergence in flocking and territoriality . ( a c ) representative autoradiograms of i ot antagonist binding sites in the caudal ls ( lsc ) in two sympatric , congeneric finches the territorial violet - eared waxbill ( a ) and the gregarious angolan blue waxbill ( b ) , plus the highly gregarious zebra finch ( c ) . ( d ) densities of binding sites in the dorsal ( pallial ) lsc of two territorial species ( melba finch , mf , and violet - eared waxbill , vew ) , a moderately gregarious species ( angolan blue waxbill , abw ) , and two highly gregarious species ( spice finch , sf , and zebra finch , zf ) . different letters above the boxes denote significant species differences ( mann whitney p < ( e ) binding densities tend to reverse in the subpallial lsc ( p = 0.06 ) , suggesting that species differences in sociality are most closely associated with the relative densities of binding sites along a dorso - ventral gradient , as confirmed in the bottom ( f ) using a dorsal : ventral ratio . abbreviations : hp , hippocampus ; lsc.d , dorsal zone of the lsc ; lsc.v , vl , ventral , and ventrolateral zones of the lsc ; n , nidopallium ; plh , posterolateral hypothalamus ; teo , optic tectum . densities of nonapeptide receptors in the ls are also highly variable in mammals , although the relevance of these species differences to behavior has largely been a matter of speculation ( insel et al . however , recent findings suggest that ot receptor densities may reflect species differences in the provision of alloparental care by juvenile females , and alloparental care in female prairie voles is negatively correlated with ot receptor densities in the ls ( olazbal and young , 2006 ) . in male prairie voles , social investigation behavior is positively correlated with v1a receptor densities and negatively with ot receptor densities in the ls ( ophir et al . , the relevance of nonapeptide receptors to grouping behavior has been directly confirmed by a series of recent experiments using central antagonist and antisense manipulations in zebra finches . based on the well known effects of ot and ot receptors on affiliation in mammals ( carter et al . , 2008 ; goodson and thompson , 2010 ) , we began by blocking ot - like receptors ( as in mammals , a single ot - like receptor has been identified in birds , vt3 ; baeyens and cornett , 2006 ) . using the choice apparatus shown in figure 5a , in which subjects can choose between groups of 10 or 2 same - sex conspecifics ( or not spend time near either ) , we first showed that subcutaneous and lateral ventricle infusions of an ot antagonist reduce preferences for the larger group ( figures 5b e ) without influencing the amount of time that subjects spent in close proximity to other birds ( hereafter contact time ) . the peripheral effect was female - specific and ventricular infusions of mt produced a female - specific increase in the amount of time that subjects spent with large group , again with no effect on contact time . infusions of the ot antagonist directly into the ls replicated the peripheral and ventricular antagonist effects in females , whereas control infusions into the medial striatum ( adjacent to the ventricle ) did not ( goodson et al . a 1-m wide testing cage was subdivided into zones by seven perches ( thin lines ) . subjects were considered to be within close proximity when they were within 6 cm of a stimulus cage ( i.e. , on the perches closest to the sides of the testing cage ) . ( b e ) relative to vehicle treatments , subcutaneous ( s.c . ) or intracerebroventricular ( i.c.v . ) nh2 , d(ch2)5[tyr(me ) , thr]ovt ( ota ; 250 ng ) , reduce the amount of time that zebra finches spend in close proximity to the large group ( b , c ) and increase time in close proximity to the small group ( d , e ) . * p < 0.05 , * * * p < 0.001 , main effect of treatment ; # p < 0.5 sex*treatment ; n = 12 m , 12 f. letters above the error bars denote significant within - sex effects . modified from goodson et al . as described in the previous sections , gregarious species show a relatively greater social induction of fos within vt neurons , more vt - ir neurons in the bstm , and a higher density of vt v1a - like receptors in the ls than do territorial species ( goodson and wang , 2006 ; goodson et al . , 2006 ) , suggesting the hypothesis that vt projections from the bstm to the ls promote sociality . indeed , recent experiments in male zebra finches demonstrate that knockdown of vt production in the bstm by antisense oligonucleotides potently reduces gregariousness relative to subjects infused with scrambled oligonucleotides , with a median reduction of 80% . gregariousness in this experiment was defined as the percent of contact time that was spent next to a group of 10 conspecifics versus a group of 2 ( kelly et al . , 2011 ) . surprisingly , this same manipulation increased contact time modestly relative to scrambled oligonucleotide controls ( median difference of 25% ) , although intraseptal infusions of a v1a antagonist produce no effects on contact time ( and convincingly so ) . as with the vt antisense , intraseptal v1a antagonist infusions virtually eliminated subjects preferences for the large group while concomitantly increasing the amount of time that subjects spent with the small group . we have further replicated this effect in both males and females using a novel v1a antagonist that crosses the blood brain barrier ( jnj-17308616 ; j. l. goodson and s. e. schrock , unpublished observations ) . in conjunction with both the antisense and central antagonist studies , we also conducted tests of anxiety - like behavior ( novelty - suppressed feeding and exploration of a novel environment ) , which produced particularly intriguing results : whereas septal vp is usually found to be anxiogenic in rodents , it appears to be strongly anxiolytic in zebra finches ( kelly et al . , 2011 ) whether this difference is the result of unique receptor distributions and/or is an important factor in the social evolution of zebra finches remains to be determined . however , it is interesting in this light to note that septal vp may promote active stress coping in rats ( ebner et al . , 1999 ) , which are more social than laboratory mice and hamsters . using five finch species that are all socially monogamous and biparental , we have shown that ( 1 ) receptor distributions for multiple neuropeptide systems ( vt / mt , vip , and crf ) exhibit divergent and convergent evolution in relation to species - typical group size , particularly within the ls , ( 2 ) vt cells in the bstm exhibit an exquisite sensitivity to the valence of social stimuli , thereby creating differences between gregarious and territorial species in the response of their bstm vt neurons to same - sex conspecifics , ( 3 ) endogenous nonapeptide signaling via v1a- and ot - like receptors in the ls promotes preferences for larger groups in zebra finches without effects on social contact time , and ( 4 ) in male zebra finches , antisense knockdown of vt production in the bstm profoundly reduces gregariousness . although not discussed above , we have also found that the three flocking finch species exhibit relatively more dopamine neurons than do territorial species in a caudal subpopulation of cells in the ventral tegmental area that has also been implicated in appetitive courtship behavior ( goodson et al . , 2009a ) . all of the neurochemical systems just mentioned influence myriad behavioral and physiological functions , thus we might expect that those systems may not evolve in relation to grouping , and in an estrildid - like manner , if other species - specific behavioral and physiological functions constrain the evolutionary process . this may occur if a given neural mechanism is under strong selection in relation to something other than grouping . at the same time , nonapeptides influence basic social behaviors across a wide range of vertebrates , suggesting that they may be common or even ubiquitous targets of selection during social evolution . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

species - typical patterns of grouping have profound impacts on many aspects of physiology and behavior . however , prior to our recent studies in estrildid finches , neural mechanisms that titrate species - typical group - size preferences , independent of other aspects of social organization ( e.g. , mating system and parental care ) , have been wholly unexplored , likely because species - typical group size is typically confounded with other aspects of behavior and biology . an additional complication is that components of social organization are evolutionarily labile and prone to repeated divergence and convergence . hence , we can not assume that convergence in social structure has been produced by convergent modifications to the same neural characters , and thus any comparative approach to grouping must include not only species that differ in their species - typical group sizes , but also species that exhibit convergent evolution in this aspect of social organization . using five estrildid finch species that differ selectively in grouping ( all biparental and monogamous ) we have demonstrated that neural motivational systems evolve in predictable ways in relation to species - typical group sizes , including convergence in two highly gregarious species and convergence in two relatively asocial , territorial species . these systems include nonapeptide ( vasotocin and mesotocin ) circuits that encode the valence of social stimuli ( positive negative ) , titrate group - size preferences , and modulate anxiety - like behaviors . nonapeptide systems exhibit functional and anatomical properties that are biased toward gregarious species , and experimental reductions of nonapeptide signaling by receptor antagonism and antisense oligonucleotides significantly decrease preferred group sizes in the gregarious zebra finch . combined , these findings suggest that selection on species - typical group size may reliably target the same neural motivation systems when a given social structure evolves independently .

Introduction: Evolution and Diversity in Social Phenotypes A Comparative Approach to Grouping Neural Circuits of Territorial Aggression, Aversion, and Sociality in Songbirds Nonapeptide Systems: Phenotypic Diversity and the Species-Specific Assignment of Social Valence Evolutionary Convergence and Divergence in Neuropeptide Receptor Distributions: A Special Status for the Lateral Septum? Nonapeptides Promote Flocking Conclusion Conflict of Interest Statement

, 2008 ) , and limited evidence suggests that mechanisms of parental care are at least grossly similar in mammals and birds , as well ( buntin et al . however , almost no generalizations can be made regarding the mechanisms of evolutionarily labile aspects of social behavior , such as cooperative breeding , mating systems , and grouping . however , evolutionarily labile behaviors pose fairly extreme challenges for comparative neurobiology , largely for reasons of feasibility . for instance , after two decades of intensive research , the neural mechanisms of selective partner preference ( a hallmark of monogamy ) , paternal care , and alloparental care are reasonably well understood for only a single species , the monogamous prairie vole ( microtus ochrogaster ) , and we know a limited amount about the critical variables that differentiate prairie voles from other non - monogamous vole species ( carter et al . , 2006 ; this example points out a fundamental difference in the approaches to social organization that are employed by evolutionary biologists and neuroethologists . evolutionary biologists can often bring extensive information to bear on a given question , perhaps using data from dozens or hundreds of species , and analyze it in a phylogenetically corrected way that allows the extrapolation of general patterns . nonetheless , we can not make any strong claims about evolutionary patterns of regulation if the experimental approaches of behavioral neuroscience are not employed , and it simply not feasible to replicate experiments in dozens or hundreds of species . however , if neuroethologists want to establish predictive validity for other species , comparative experimentation focused on divergent and convergent evolution is essential . in the sections that follow , we describe a quasi - experimental approach that we have taken to study convergent and divergent evolution in grouping behavior within the estrildid finch family . although only five species have been used ( table 1 ) , they have been carefully selected and our findings should therefore have good predictive validity for other estrildid finches . whether our findings can be extrapolated to other vertebrate taxa is a matter of greater speculation , and a topic that we will return to in the section conclusion . behavioral and ecological characteristics of estrildid finch species that have been used for studies of grouping behavior . this species is commonly encountered in the scientific literature , but under a variety of common names , including spotted munia , nutmeg mannikin , and spice finch . sociality sensu stricto , as defined by grouping behavior ( alexander , 1974 ) , has broad influences on other important variables such as reproductive behavior , disease transmission , resource exploitation , and defense ( moller and birkhead , 1993 ; krause and ruxton , 2002 ; silk , 2007 ) , but has not previously been examined in neurobiological studies , likely because grouping is difficult to isolate from other aspects of ecology and behavior , such as mating system and patterns of parental care . for instance , rodent species that differ in their grouping behavior also differ in whether they are monogamous or polygamous , and whether the father contributes to parental care ( king , 1968 ; tamarin , 1985 ) . however , if we want to examine the neural mechanisms that titrate species - typical group - size preferences , control for such variables is very important , given that shared mechanisms ( particularly neuroendocrine mechanisms ) often regulate numerous aspects of social behavior ( e.g. , pair bonding , parental care , and affiliation ) and related aspects of physiology ( e.g. birds offer excellent opportunities to study grouping , since we can identify closely related species that are virtually identical in most aspects of behavior and ecology , but that nonetheless exhibit extreme variation in sociality , ranging from territorial pairs to massive flocks . we have collected five estrildid finch species that exhibit large and seasonally stable differences in grouping , but are otherwise closely matched in behavior and ecology ( table 1 ) . in addition to being socially monogamous and biparental , as already noted , all of these species live in arid or semi - arid grassland scrub and breed opportunistically or semi - opportunistically in relation to rainfall ( skead , 1975 ; goodwin , 1982 ; zann , 1996 ; goodson et al . these include two territorial african species that live as male female pairs year - round ( violet - eared waxbill , uraeginthus granatina , and melba finch , pytilia melba ) ; two highly gregarious , colonially breeding species that exhibit modal group sizes of approximately 100 ( zebra finch , taeniopygia guttata , and spice finch , lonchura punctulata ) ; and a moderately gregarious species , the angolan blue waxbill ( uraeginthus angolensis ) , which exhibits a modal group size of approximately 20 . the gregarious angolan blue waxbill is sympatric with both the melba finch and violet - eared waxbill , and our laboratory population of these three species has been established through the breeding of birds that were caught from a single location in the kalahari thornscrub of south africa in 2001 . domestic zebra finches are behaviorally indistinguishable from wild - caught zebra finches ( morris , 1958 ) and virtually their full range of social behavior can be observed in the lab . , 1999 ; vertebrates possess a core social behavior network within the basal ( limbic ) forebrain and midbrain ( figure 1 ) that is evolutionarily conserved across all taxa , but is particularly stable across the amniote clades giving rise to birds and mammals , as demonstrated by a wide range of functional and anatomical studies . this network includes the medial extended amygdala ( including the medial bed nucleus of the stria terminalis , bstm ) , lateral septum ( ls ) , anterior hypothalamus ( ah ) , ventromedial hypothalamus ( vmh ) , midbrain central gray ( cg ; or periaqueductal gray ) , and the ventral tegmental area , although these structures are not strictly social in function and contribute to other core networks for the regulation of behavior and physiology . ( a ) the core components of the social behavior network include numerous areas of the basal forebrain the medial extended amygdala ( medial amygdala , mea , or taenial amygdala , tna , plus the medial bed nucleus of the stria terminalis , bstm ) , medial preoptic area ( mpoa ) , anterior hypothalamus ( ah ) , ventromedial hypothalamus ( vmh ) , and lateral septum ( ls ) , as well as areas of the midbrain , most notably the central gray ( cg ; or periaqueductal gray , pag ) and the ventral tegmental area ( vta ) . birds exhibit mammal - like patterns of immediate early gene ( ieg ) induction in this network across a variety of social contexts , including territorial aggression , a context that is strongly relevant to species differences in sociality . , 2009 ) and simulated territorial intrusion ( sti ; playback of song and presentation of a caged decoy male ) in male song sparrows , significant activation is observed in forebrain areas such as the bstm , ls , paraventricular nucleus of the hypothalamus ( pvn ) , ah , and the lateral portion of the vmh ( goodson and evans , 2004 ; goodson et al . , 2005b ) , and the case is much the same in hamsters and mice , such that fighting - induced fos expression is higher in subordinate versus dominant animals ( kollack - walker et al . this applies to most aggression - related areas in the brain , and we have obtained very similar results in the territorial violet - eared waxbill ( j. l. goodson , s. e. schrock , and m. a. kingsbury , unpublished observations ) . despite the simplicity of this paradigm , fos and egr-1 responses clearly distinguish the territorial from gregarious ( flocking ) species , including differences between the two sympatric uraeginthus species described in the previous section . overall , territorial birds exhibit significantly greater ieg responses in the medial extended amygdala ( especially the bstm ) , ventrolateral ls , ah , lateral vmh , and cg ( and adjacent intercollicular territory that is now known to be homologous to the dorsal and dorsolateral cg of mammals ; goodson et al . , 2001 ; providing a good example of deep functional conservation in the social behavior circuits of vertebrate brains ) , but possible mechanisms of gregariousness were not evident at this level of analysis . this may partially reflect the fact that functionally distinct neurons are intercalated with one another in the various brain areas that influence social behavior ( e.g. , for instance , as described in the next section , fos induction within arginine vasotocin ( vt ) neurons of the bstm is strictly associated with affiliation - related stimuli , and following same - sex exposure , the gregarious species show greater fos induction in the vt neurons than do territorial species ( goodson and wang , 2006 ) . , 2005a ) , suggesting that the vt neurons are intermingled with other neurons that exhibit opposing profiles of response . although many anatomical and functional properties of nonapeptide systems are strongly conserved across all vertebrate taxa ( and in the case of neurosecretory magnocellular neurons , perhaps across all bilaterians ; tessmar - raible et al . , 2007 ) , there is substantial diversity in receptor distributions and nonapeptides have been extensively linked to behavioral variation across individuals , sexes , and species ( goodson and bass , 2001 ; de vries and panzica , 2006 ; donaldson and young , 2008 ; goodson , 2008 ) . in rats , the parvocellular neurons of the pvn give rise to widespread projections in the brain ( de vries and buijs , 1983 ) , and this is almost certainly the same in other vertebrates . lesions of the pvn in rats virtually eliminate vp projections to the caudal brainstem , but not other areas , and eliminate ot projections throughout the brain ( de vries and buijs , 1983 ) . with only a few exceptions , these neurons and their projections to the ls and other basal forebrain areas have been found to be seasonally variable , exquisitely dependent on sex steroids , and sexually dimorphic ( males > females ) . , thus , of the finches that we have examined , large seasonal fluctuations in vt immunoreactivity are found only for the spice finch , a species that exhibits photorefractoriness and endogenous rhythms of reproductive physiology that correlate with monsoon cyclicity ( goodwin , 1982 ; chaturvedi and prasad , 1991 ; sikdar et al . , 1992 ; the vt / vp neurons of the bstm project to numerous other areas of the basal forebrain where vt / vp modulates aggression , parental behavior , social recognition , and various affiliative and anxiety - like behaviors ; and at least in male prairie voles , vp release in the ventral pallidum promotes partner preference ( donaldson and young , 2008 ; veenema and neumann , 2008 ; goodson and thompson , 2010 ; insel , 2010 ) . in order to determine the kinds of stimuli that different vt / vp cell groups respond to ( particularly those of the bstm ) , we have conducted several experiments in which we have exposed animals to social stimuli or control conditions , and then sacrificed the animals 90 min later for immunohistochemical colocalization of vt and fos ( see table 2 for a summary of studies ) . experimental induction of fos is still robust at 90 min , but the half - life of fos protein is only 45 min ( herdegen and leah , 1998 ) , and thus if cellular activity is depressed by our social manipulations , we can detect this as a reduction in fos expression . a summary of main findings from studies of nonapeptide systems in territorial ( t ) , moderately gregarious ( mg ) , and highly gregarious ( hg ) species of estrildid finches . relative to handled controls , vt - ir neurons in the territorial estrildid species decrease their fos expression in response to same - sex stimuli ( exposed through a wire barrier as described above ) , but increase fos expression in response to their pairbond partner . ( a ) representative colocalization of vt ( green ) and fos ( red ) in the bstm of a male zebra finch following a courtship interaction . ( b ) in the zebra finch , which is a highly gregarious species , isolation in a quiet room followed by exposure to a same - sex conspecific through a wire barrier produces a robust increase in vt neuronal activity in both males and females . fos colocalization in the territorial violet - eared waxbill , a species that does not naturally exhibit same - sex affiliation , but exposure to the subject 's pairbond partner ( a presumably positive stimulus ) , produces a robust increase in neuronal activity . , 2006 ) , and thus the neurochemical identification of valence - sensitive populations represents an important step toward understanding how social value is encoded and used by the brain to regulate behavior . in fact , using the five finch species already introduced , we have obtained good evidence that the distributions of binding sites for vasoactive intestinal polypeptide ( vip ) , vt , and mt all exhibit convergent and divergent evolution in relation to sociality ( based on quantitative autoradiography using i vip and iodinated antagonists of ot and v1a receptors ; goodson et al . in order to be interpreted as sociality - related , we require that binding densities differ significantly between the two territorial and three flocking species ( defined as contrast a ) and differ significantly between the two territorial and two colonial species ( contrast b ) , with the modestly gregarious species being intermediate or falling in line with the two colonial species . figure 3 shows one such pattern for the density of v1a - like receptor density in the dorsal ls , and similar abc species differences are observed in several other ls zones for both v1a - like , ot - like and vip binding sites ( goodson et al . binding densities are mostly biased toward the gregarious species , although sociality - related differences in ot - like binding densities reverse along a dorso - ventral gradient , and the relative density between the pallial and subpallial ls provides the clearest differentiation of territorial and flocking species ( goodson et al . outside of the ls , sociality - related differences in the finches are observed for vip binding in the bstm , which are biased toward the gregarious species ( goodson et al . ( a e ) representative i v1a antagonist binding in the ls of the territorial melba finch [ mf ; ( a ) ] , territorial violet - eared waxbill [ vew ; ( b ) ] , moderately gregarious angolan blue waxbill [ abw ; ( c ) ] , colonial spice finch [ sf ; ( d ) ] , and colonial zebra finch [ zf ; ( e ) ] . ( a c ) representative autoradiograms of i ot antagonist binding sites in the caudal ls ( lsc ) in two sympatric , congeneric finches the territorial violet - eared waxbill ( a ) and the gregarious angolan blue waxbill ( b ) , plus the highly gregarious zebra finch ( c ) . ( d ) densities of binding sites in the dorsal ( pallial ) lsc of two territorial species ( melba finch , mf , and violet - eared waxbill , vew ) , a moderately gregarious species ( angolan blue waxbill , abw ) , and two highly gregarious species ( spice finch , sf , and zebra finch , zf ) . different letters above the boxes denote significant species differences ( mann whitney p < ( e ) binding densities tend to reverse in the subpallial lsc ( p = 0.06 ) , suggesting that species differences in sociality are most closely associated with the relative densities of binding sites along a dorso - ventral gradient , as confirmed in the bottom ( f ) using a dorsal : ventral ratio . however , recent findings suggest that ot receptor densities may reflect species differences in the provision of alloparental care by juvenile females , and alloparental care in female prairie voles is negatively correlated with ot receptor densities in the ls ( olazbal and young , 2006 ) . , the relevance of nonapeptide receptors to grouping behavior has been directly confirmed by a series of recent experiments using central antagonist and antisense manipulations in zebra finches . , 2008 ; goodson and thompson , 2010 ) , we began by blocking ot - like receptors ( as in mammals , a single ot - like receptor has been identified in birds , vt3 ; baeyens and cornett , 2006 ) . as described in the previous sections , gregarious species show a relatively greater social induction of fos within vt neurons , more vt - ir neurons in the bstm , and a higher density of vt v1a - like receptors in the ls than do territorial species ( goodson and wang , 2006 ; goodson et al . in conjunction with both the antisense and central antagonist studies , we also conducted tests of anxiety - like behavior ( novelty - suppressed feeding and exploration of a novel environment ) , which produced particularly intriguing results : whereas septal vp is usually found to be anxiogenic in rodents , it appears to be strongly anxiolytic in zebra finches ( kelly et al . however , it is interesting in this light to note that septal vp may promote active stress coping in rats ( ebner et al . using five finch species that are all socially monogamous and biparental , we have shown that ( 1 ) receptor distributions for multiple neuropeptide systems ( vt / mt , vip , and crf ) exhibit divergent and convergent evolution in relation to species - typical group size , particularly within the ls , ( 2 ) vt cells in the bstm exhibit an exquisite sensitivity to the valence of social stimuli , thereby creating differences between gregarious and territorial species in the response of their bstm vt neurons to same - sex conspecifics , ( 3 ) endogenous nonapeptide signaling via v1a- and ot - like receptors in the ls promotes preferences for larger groups in zebra finches without effects on social contact time , and ( 4 ) in male zebra finches , antisense knockdown of vt production in the bstm profoundly reduces gregariousness . although not discussed above , we have also found that the three flocking finch species exhibit relatively more dopamine neurons than do territorial species in a caudal subpopulation of cells in the ventral tegmental area that has also been implicated in appetitive courtship behavior ( goodson et al . all of the neurochemical systems just mentioned influence myriad behavioral and physiological functions , thus we might expect that those systems may not evolve in relation to grouping , and in an estrildid - like manner , if other species - specific behavioral and physiological functions constrain the evolutionary process . this may occur if a given neural mechanism is under strong selection in relation to something other than grouping .

telemedicine is the use of information technology and telecommunications to support healthcare at a distance . in this review , the main issues of geriatrics with regard to scope , disease burden geriatrics is a subspecialty of internal medicine and primary care that was named in 1909 by ignatz leo nascher . the specialty was recognized by a certificate of special qualifications under the internal medicine board certification by the american board of medical specialties in 1988 . that is of course a very recent date and indicates the late recognition of the care of the aging as a special matter . children 's hospitals became prominent in the 19th century , but nothing comparable happened at that time in the care of the elderly . there were hardly enough old people to attract attention ! in 1900 , most newborns could not be expected to live past 50 . now , life expectancy at birth exceeds 80 years in many countries , and this demographic trend is continuing . in 2010 , there were perhaps 524 million people over 65 years in the world , whereas in 2050 that number will be 1.5 billion . interestingly , most of that increase will be in the developing world . in about five more years , people in the world older than 65 years will exceed the number of people under age 5 . mortality in older populations has been sharply reduced with great inroads in relation to cardiovascular disease , cancer , and diabetes . however , unlike infections , these conditions are not cured ; they are curtailed . today the main threats are non - communicable diseases , including heart , stroke , cancer , diabetes , hypertension , and dementia . the previous model of healthcare throughout the world might be termed ' incident care ' . in this model , someone became aware of a health problem , sought the advice of a physician , received a diagnosis , and usually some treatment converted the condition into a short - term illness . however , among older patients , 92% have at least one chronic disease , and 77% suffer from at least two . in other words , there has been a huge increase in older people who are always sick rather than intermittently sick . the objective of care and management is to reduce the morbidity and slow the progression . thus , longitudinal management of progressive disease and anticipation of complications are the hallmarks of successful geriatric practice . they may have only one grandchild ; thus , the pool of family members to care for them is limited . furthermore , they tend to live so long that the intimacy of family may have passed . as an offset , men are gaining in the longevity statistics and may survive with their spouses for mutual support . preference for independent living is growing as children seem to have more career demands with both man and wife employed . for older patients , the chronic diseases they carry may progress at varying rates , and the possibility of a devastating stroke , cardiac decompensation , or diabetic complications may suddenly take away the possibility of independent living . in the united states , an older person has a fall every 15 seconds requiring a hospital visit , and every 28 minutes there is a fatal fall . then there is the terrible problem if dementia . in a study among the countries of the organization for economic cooperation and development , the disease burden of alzheimer disease / dementia was some 30 million . we just know that a typical chronically ill person has a limited network of family for care , tries to live independently , and is subject to dementia that will eventually make them incompetent for independent living . in summary , geriatrics is a medical specialty which with primary care is struggling with an ever increasing population of chronically ill patients who may live a long time with care but are subject to myriad complications . the objective of longitudinal care is to limit disease progress , diagnose problems such as cancer early , look for early signs of deterioration , and support healthy lifestyles . rather like parents in the case of children , the care of older patients often involves family members as caregivers . from the standpoint of social planning , the paramount fiscal issue is the huge increase in the cost of patients who will require frequent interventions , long - term hospital care , and institutional care . patients may perceive the desperation of the situation in that the largest incidence for suicide in the united states is among men past age 85 . the current mode of health care involving visits to clinics at rare intervals is an overwhelming challenge of expense and personnel , and there is no resource in sight for meeting the demand . the crucial expectation is for medical care to anticipate the crises , avoid them , limit office visits , limit emergency visits , anticipate mental decline , intervene only when needed , and support the social net . however , the ability of the health community to respond to this huge challenge is not apparent or sufficient . in the united states , there are 784,633 physicians in practice for a population of some 320 million . in the 1981 work force ( graduate medical education national advisory committee ) report , an excess of physicians was perceived and medical education was limited . by 2000 , it was clear that something in the calculation was wrong . with no increase in physician output in 20 years , the shortfall for older patients is said to be 70,000 . in the united states , the steady - state output of physicians was only 16,000 per year for a population that had increased to 320 million . if all medical graduates committed to geriatrics , it would take many years to train enough of them for the task . in fact , the number of trainees in geriatrics is declining , and in 2013 there were only 259 new fellows in the entire country . one possibility is that any adult primary care physician could fill the gap ; however , primary care is not doing very well in the united states either . the association of american medical colleges has recommended a 30% increase in the number of enrollees and an additional 30 medical schools . yet despite the need for increased care of geriatric patients , the american geriatric society has only 6,000 members . thus , the number of physicians in the world is not going to remotely meet the geriatric need . however , if the shortage of physicians is deep in the developed world , the shortage of nurses for home health is hopeless . we can not send out an army of health professionals to meet with patients often enough to avert health crises . in the united states , a 17% expenditure of the gdp on healthcare simply is not going to expand by say 25% to account for our current expectations for delivery of care to the elderly . our aim has been to reduce healthcare expenditures ; however , demographics are contrary to that expectation . furthermore , as patients become more and more frail , they are less able to come on their own to intermittent care visits . unfortunately , when they are in nursing facilities , access to specialist care is further confounded by transportation challenges . in conclusion , there is a growing crisis in providing proper longitudinal health care to the elderly in our current practice modes . telemedicine was given its name by kenneth bird in 1975 , and the american telemedicine association was established in 1994 . e - health , telehealth , telecare , and other terms are in reality synonyms . the use of telecommunications to support healthcare came first with videoconference access to specialty care . the notion of using telecommunications for medicine has a history as long as any form of distant communication . however , analog television communication was terribly expensive . the introduction of satellite connectivity in 1962 made the distance less prohibitive but the cost exploded . the practicality of telemedicine shares a history with the transistor , which was commercialized by bell laboratories and was the subject of the nobel prize in physics awarded to shockley , bardeen , and houses in 1956 . the vast composite of the human condition could be stored and transmitted in digital form across any telecommunication medium . the application of transistors to personal computing has a long history , but for the purpose of timelines , the introduction of the apple computer in 1976 was seminal . thereafter , management of data in small , compact , highly effective modules became an everyday practice . today , the storage of huge amounts of patient data , including images and live transmission is a simple and cheap process . the combination of telecommunications and information management constitutes a tool inviting the imagination of practitioners and attacking the very basic issues of cost and access . any practitioner can work from a desk to any point of need and can provide coherent and definitive diagnosis and management expertise . the growth of telemedicine has been greatly accelerated by wireless communication for devices , cellular telephony , fiberoptic connectivity , and digital records . the us american recovery and reinvestment act of 2009 had many components to stimulate the economy after the great recession crisis . among these were the broadband initiative , which was intended to bring great bandwidth to almost every village and school in the country . also , the act included the health information technology for economic and clinical health ( hitech ) provisions , requiring the staged expansion of electronic medical records , which were effective , and through such a system , healthcare providers could communicate with one another throughout the us in meaningful ways . in fact , the current surge of telemedicine would not have happened without the ubiquity of the internet , which came into large - scale use in 1994 . the rapid expansion of internet protocol version 6 since 2008 means that there are enough internet protocol unique addresses for every person and device on the planet with ample room for growth . thus , we have the internet of things , and this makes telemedicine so much more feasible . the drop in cost from required but fabulously expensive satellite connectivity to internet connectivity is simply astounding . these innovations and applications come at a time when the costs of medical care are soaring and the geriatric challenge is upon us . please note that the increase of healthcare costs is upward and exponential while the cost of telecommunication and electronic support is declining in the opposite direction . there is no better match in healthcare for improvement and cost saving through telemedicine than the care of the elderly . the population has a huge disease burden , which is not sustainable with the cost constraints of traditional incidental care . the provision of telemedicine services for the elderly is not particularly different from any other medical matters . there is a common belief that high - end electronics and telecommunications can not be used by the elderly because they have limited computer and technology skills . in the developed world , the current rapid growth in the aging population has occurred as a result of what has been dubbed the baby boom . the people entering the ranks of the elderly now are not the same as those very elderly who were born before the second world war . they suffered thorough the technical frustrations of floppy disks and every iteration of windows software . this group followed and cheered the magic of the space program and invented the information age . perhaps they are not actively interested in every nuance of innovation , but in general , they are technically competent and savvy about applications they perceive as having value for them . unless they have cognitive difficulties , the upcoming elderly will be the vanguard of electronic medicine in geriatrics if we invite them and listen to them without prejudice . therefore , there is a need for telemedicine in the elderly , and they are competent to participate in the abundant technology available . telemedicine has been assessed in many ways , but a brief review might be helpful . the american telemedicine association has issued 14 practice guidelines for telemedicine in a variety of applications from pathology to dermatology to diabetes . now , publications are heavily weighted to randomized controlled trials and telemedicine can be studied as a matter of meta - analysis in the cochrane database . in the early days of telemedicine , the cynical view was that a telemedicine program would close when the grant was spent . as a corollary , telemedicine would not have any inherent value until someone finally paid for it . the veterans health system has 70,000 telemedicine visits per year and they declare how much it saves money in the largest health system in the united states . more than half the states now have laws requiring private insurers to pay for telemedicine for its subscribers demonstrations were carried out in remote parts of the world , and its relevance to highly developed communities was not clear . however , in the past 15 years , telemedicine has been used in all manner of healthcare delivery , and has been integrated into the fabric of overall healthcare . when telemedicine was a desperate measure for access , equivalence to standard office care was not an issue . currently , equivalence is of the utmost importance , and demonstration of at least non - inferiority has been crucial to acceptance by patients , physicians , regulators , and payers . videoconference encounters have been shown in every way to be comparable to face - to - face in terms of diagnostic accuracy , interrater variation , patient acceptance , and cost . the best survey in this regard is in the definitive textbook by bashshur and shannon . with regard to image transfer standards , radiology can demand the highest dicom standard from transmission technology and is not compromised . images produced by smartphone videoconferencing , microscopy , dermatology , and pathology are completely satisfactory . these facts are really technical matters and are not the subject of this clinical review . since the elderly have an excess burden of chronic disease , what is the evidence that longitudinal management by telemedicine is helpful ? an extensive review by bashshur et al . has been used by us congressional committees in policy decisions . the data clearly indicates that the use of telemedicine is superior to office visits in terms of emergency visits , hospitalizations , complications , and quality of life for diabetes , hypertension , pain , congestive heart failure , cancer care , rehabilitation after stroke , and dementia . the best place for older people to live is where they want to live , and that is almost always at home . yet when they are frail and forgetful , and have limited mobility , home may be a very dangerous place . extending the practice of their medical team to the home is a great strategy for retention of independence and early recognition of problems . simple monitoring devices can assess gait , pace , activity in general , and adherence to a medical regimen . monitoring of blood pressure , pulse , oxygen saturation , and weight can be done with non - invasive sensors , and the data can be transferred wirelessly to a computer for transmission to a health management office . the management site can look for alerts and trends with a response that is anywhere from a call to a summon of emergency personnel to the home , to making a small change in medication with a message back to the patient . cheap sensors , either passive or interactive , can capture the essence of a health situation at home and the managing physician can embrace that home with much of the immediate care available otherwise only in the office or hospital . instead of a visit on demand from time to time or regularly every month , surveillance can inform and empower the physician about the situation and allow timely and effective intervention . a smart home with sensors and alerts can assess the environment for such subtleties as a running tub , the temperature of the water , the age of materials in the refrigerator , the consumption of food , water , and medications as well as the urgent situations indicated by the presence of smoke , carbon monoxide , or excess heat . mobile phones can provide information support and alerts to the home bound in ways that were hard to imagine five years ago . there are thousands of medial apps for the smartphones that can be incorporated into comprehensive plans for home health by a managing physician with the fullest consent of the patient . home healthcare performed in person by skilled nursing teams is certainly well established and broadly practiced . however , that intervention can not be provided hourly or after hours . furthermore , the home health nurse alone when confronted by a worrisome change can only order transfer to definitive diagnosis and care or communicate by phone with the managing physician . instead , telemedicine as an augmentation to the physical visit , can be used by nurses to transfer images to evaluate skin lesions or gait , to discuss telemetry data , and receive new instructions which otherwise would only be possible after a visit by the patient to the physician 's office . another use of telemedicine in home healthcare is the critical support of home caregivers who are subject to exhaustion , terrible stress , and a strong urge at times to give up and send the house - bound patient to a facility . telemedicine creates a virtual team that can include the home caregiver and dispel the sense of isolation . the alternative of travel to a fixed health facility is tedious , labor intensive , perhaps painful , and expensive . smartphone programs are excellent for behavior modification in terms of weight , diet , smoking cessation , alcohol abuse , medication , and exercise . the impact , according to published reports , has largely been confined to teenagers , caregivers , and veterans suffering from post traumatic stress disorder . however , the applicability to the elderly has no apparent barrier , and there are examples of success in the elderly . increasingly , medical practices are using social media to enrich medical practice through enhanced communication and reminders to reinforce a regimen . the loss of memory and neural plasticity is perhaps the most difficult matter in the care of the elderly . considerable research is ongoing to find the basis of alzheimer disease and dementia in general . there are numerous devices and programs available on hand held devices to exercise the memory and perhaps slow the progress of dementia . the value is yet to be determined ; however , it is likely that such programs will be integral to health maintenance in the future . in a typical nursing home , the population is generally elderly , and nursing care is of paramount importance . it is carefully monitored by a physician , and outcomes are carefully analyzed by authorities . however , there is usually no physician present , and if there is any medical question , the first resort is transfer to an emergency medical facility . the regular use of telemedicine to bring the center into close contact with a medical unit can reduce visits dramatically . since the elderly have an excess burden of chronic disease , what is the evidence that longitudinal management by telemedicine is helpful ? an extensive review by bashshur et al . has been used by us congressional committees in policy decisions . the data clearly indicates that the use of telemedicine is superior to office visits in terms of emergency visits , hospitalizations , complications , and quality of life for diabetes , hypertension , pain , congestive heart failure , cancer care , rehabilitation after stroke , and dementia . the best place for older people to live is where they want to live , and that is almost always at home . yet when they are frail and forgetful , and have limited mobility , home may be a very dangerous place . extending the practice of their medical team to the home is a great strategy for retention of independence and early recognition of problems . simple monitoring devices can assess gait , pace , activity in general , and adherence to a medical regimen . monitoring of blood pressure , pulse , oxygen saturation , and weight can be done with non - invasive sensors , and the data can be transferred wirelessly to a computer for transmission to a health management office . the management site can look for alerts and trends with a response that is anywhere from a call to a summon of emergency personnel to the home , to making a small change in medication with a message back to the patient . cheap sensors , either passive or interactive , can capture the essence of a health situation at home and the managing physician can embrace that home with much of the immediate care available otherwise only in the office or hospital . instead of a visit on demand from time to time or regularly every month , surveillance can inform and empower the physician about the situation and allow timely and effective intervention . a smart home with sensors and alerts can assess the environment for such subtleties as a running tub , the temperature of the water , the age of materials in the refrigerator , the consumption of food , water , and medications as well as the urgent situations indicated by the presence of smoke , carbon monoxide , or excess heat . mobile phones can provide information support and alerts to the home bound in ways that were hard to imagine five years ago . there are thousands of medial apps for the smartphones that can be incorporated into comprehensive plans for home health by a managing physician with the fullest consent of the patient . home healthcare performed in person by skilled nursing teams is certainly well established and broadly practiced . furthermore , the home health nurse alone when confronted by a worrisome change can only order transfer to definitive diagnosis and care or communicate by phone with the managing physician . instead , telemedicine as an augmentation to the physical visit , can be used by nurses to transfer images to evaluate skin lesions or gait , to discuss telemetry data , and receive new instructions which otherwise would only be possible after a visit by the patient to the physician 's office . another use of telemedicine in home healthcare is the critical support of home caregivers who are subject to exhaustion , terrible stress , and a strong urge at times to give up and send the house - bound patient to a facility . telemedicine creates a virtual team that can include the home caregiver and dispel the sense of isolation . the alternative of travel to a fixed health facility is tedious , labor intensive , perhaps painful , and expensive . smartphone programs are excellent for behavior modification in terms of weight , diet , smoking cessation , alcohol abuse , medication , and exercise . the impact , according to published reports , has largely been confined to teenagers , caregivers , and veterans suffering from post traumatic stress disorder . however , the applicability to the elderly has no apparent barrier , and there are examples of success in the elderly . increasingly , medical practices are using social media to enrich medical practice through enhanced communication and reminders to reinforce a regimen . the loss of memory and neural plasticity is perhaps the most difficult matter in the care of the elderly . considerable research is ongoing to find the basis of alzheimer disease and dementia in general . there are numerous devices and programs available on hand held devices to exercise the memory and perhaps slow the progress of dementia . the value is yet to be determined ; however , it is likely that such programs will be integral to health maintenance in the future . in a typical nursing home , the population is generally elderly , and nursing care is of paramount importance . it is carefully monitored by a physician , and outcomes are carefully analyzed by authorities . however , there is usually no physician present , and if there is any medical question , the first resort is transfer to an emergency medical facility . the regular use of telemedicine to bring the center into close contact with a medical unit can reduce visits dramatically . the impact on practice has proven far more challenging than the technology or patient acceptance . why is telemedicine a threat to physicians ? in most countries physicians have a very high workload , and the idea of adding any more is just not possible . also in many venues , the payment for these services is not clear or not adequate . in the ideal situation , without cost or staff concerns , the impact is very positive because the patient care is so much better . in the veterans health system in the united states , doctors are all on staff , and their attraction to telemedicine is that it actually takes less time ! a well coordinated telemedicine clinic is very efficient with almost no delay between patients . furthermore , in such a system all records are immediately available by computer . for an office physician who has proper access to technology , the overhead costs of telemedicine visits are in fact negligible compared to maintaining an office , parking , many waiting rooms , staff , etc . telemedicine must be integrated into the workflow of physicians and staff in such a way as to make it an improvement and not an add - on . if telemedicine is left to the singular efforts of a physician , the task is very difficult . in the united states , there is a strong movement for telemedicine to be freestanding and not managed by the personal physician . this can certainly be seen as a threat to the sanctity of physician - patient relations . however , the attraction of this innovation to patients is undeniable , and the success is such that there are considerable efforts to accredit the freestanding activities also the best practice standards possible . the american telemedicine association is active in this area . certainly not ! the notion that some electronic beast is taking away our patients is a bleak one and contrary to the best traditions of medicine . the approach for concerned and conscientious physicians is to learn about the matter , to become leaders rather than followers , and to bring this tool into the daily practice of medicine rather than having a disruptive technology eroding our basic bond of patient care . the question regularly arises as to whether or not telemedicine is in fact a medical specialty . the challenge for biomedical engineers , software engineers , and the telecommunications community is to make the technology as transparent as possible such that the average physician and patient will not find it intimidating . the challenge to physicians is to acquire sufficient facility with the technology as to be a competent user not subject to a profound reliance on technical personnel . finally , the physician should know enough to be a prudent user of new technology . telemedicine like any other tool in medicine can be poorly used , and people can be hurt , or certainly , they can be poorly served . the good practitioner will understand the tools , their application , safeguards and nuances for best practice . there are many courses available and a little time to become comfortable is time well invested in the future of practice . this brief review has shown that our current practice of care for the elderly is simply not going to succeed as the numbers of elderly patients continues to grow . some of the evidence for the value of telemedicine as a tool for physicians and healthcare systems was presented . the use of telemedicine is a fait accompli in much of the world , and it continues to have an increasing role that is deeply imbedded in our electronic practices coupled with social media . this is a great opportunity for medical practice to evolve to new levels of engagement with our patients and new levels of attainment in terms of quality care .

objectivesthe global population of elderly people is increasing at a remarkable rate , which may be expected to continue for some time . older patients require more care , and with the current model of care delivery , the costs may be expected to rise , although higher cost is unsustainable . for this reason , a new pattern of practice is needed . telemedicine will be presented as a highly effective and necessary tool in geriatrics.methodsthis review will present some of the background and evidence for telemedicine as a way to address the challenges of geriatrics through geriatric telemedicine . some of the evidence for the value of telemedicine as a tool for physicians and healthcare systems is presented.resultstelemedicine offers many means to address the problems of geriatric care in creative ways . the use of electronic medicine , telecommunications , and information management has now found its way into the very fabric of health care . the use of telemedicine is a fait accompli in much of the world , and it continues to have an increasing role deeply imbedded in our electronic practices coupled with social media.conclusionsthe evidence for successful incorporation of telemedicine into practice is abundant and continues to accrue . this is a great opportunity for medical practice to evolve to new levels of engagement with patients and new levels of attainment in terms of quality care .

I. Introduction II. Geriatric Issues III. Telemedicine in General IV. Geriatric Telemedicine V. Clinical Matters in Geriatric Telemedicine 1. Chronic Disease 2. Home Care 3. Health Maintenance 4. Nursing Home Care VI. Impact on Practice VII. Conclusion

telemedicine is the use of information technology and telecommunications to support healthcare at a distance . in this review , the main issues of geriatrics with regard to scope , disease burden geriatrics is a subspecialty of internal medicine and primary care that was named in 1909 by ignatz leo nascher . that is of course a very recent date and indicates the late recognition of the care of the aging as a special matter . children 's hospitals became prominent in the 19th century , but nothing comparable happened at that time in the care of the elderly . in 1900 , most newborns could not be expected to live past 50 . now , life expectancy at birth exceeds 80 years in many countries , and this demographic trend is continuing . in 2010 , there were perhaps 524 million people over 65 years in the world , whereas in 2050 that number will be 1.5 billion . interestingly , most of that increase will be in the developing world . in about five more years , people in the world older than 65 years will exceed the number of people under age 5 . mortality in older populations has been sharply reduced with great inroads in relation to cardiovascular disease , cancer , and diabetes . today the main threats are non - communicable diseases , including heart , stroke , cancer , diabetes , hypertension , and dementia . the previous model of healthcare throughout the world might be termed ' incident care ' . in this model , someone became aware of a health problem , sought the advice of a physician , received a diagnosis , and usually some treatment converted the condition into a short - term illness . however , among older patients , 92% have at least one chronic disease , and 77% suffer from at least two . the objective of care and management is to reduce the morbidity and slow the progression . they may have only one grandchild ; thus , the pool of family members to care for them is limited . preference for independent living is growing as children seem to have more career demands with both man and wife employed . for older patients , the chronic diseases they carry may progress at varying rates , and the possibility of a devastating stroke , cardiac decompensation , or diabetic complications may suddenly take away the possibility of independent living . in the united states , an older person has a fall every 15 seconds requiring a hospital visit , and every 28 minutes there is a fatal fall . in a study among the countries of the organization for economic cooperation and development , the disease burden of alzheimer disease / dementia was some 30 million . we just know that a typical chronically ill person has a limited network of family for care , tries to live independently , and is subject to dementia that will eventually make them incompetent for independent living . in summary , geriatrics is a medical specialty which with primary care is struggling with an ever increasing population of chronically ill patients who may live a long time with care but are subject to myriad complications . the objective of longitudinal care is to limit disease progress , diagnose problems such as cancer early , look for early signs of deterioration , and support healthy lifestyles . rather like parents in the case of children , the care of older patients often involves family members as caregivers . from the standpoint of social planning , the paramount fiscal issue is the huge increase in the cost of patients who will require frequent interventions , long - term hospital care , and institutional care . patients may perceive the desperation of the situation in that the largest incidence for suicide in the united states is among men past age 85 . the current mode of health care involving visits to clinics at rare intervals is an overwhelming challenge of expense and personnel , and there is no resource in sight for meeting the demand . the crucial expectation is for medical care to anticipate the crises , avoid them , limit office visits , limit emergency visits , anticipate mental decline , intervene only when needed , and support the social net . however , the ability of the health community to respond to this huge challenge is not apparent or sufficient . in the united states , there are 784,633 physicians in practice for a population of some 320 million . with no increase in physician output in 20 years , the shortfall for older patients is said to be 70,000 . in the united states , the steady - state output of physicians was only 16,000 per year for a population that had increased to 320 million . if all medical graduates committed to geriatrics , it would take many years to train enough of them for the task . in fact , the number of trainees in geriatrics is declining , and in 2013 there were only 259 new fellows in the entire country . yet despite the need for increased care of geriatric patients , the american geriatric society has only 6,000 members . thus , the number of physicians in the world is not going to remotely meet the geriatric need . however , if the shortage of physicians is deep in the developed world , the shortage of nurses for home health is hopeless . we can not send out an army of health professionals to meet with patients often enough to avert health crises . in the united states , a 17% expenditure of the gdp on healthcare simply is not going to expand by say 25% to account for our current expectations for delivery of care to the elderly . in conclusion , there is a growing crisis in providing proper longitudinal health care to the elderly in our current practice modes . telemedicine was given its name by kenneth bird in 1975 , and the american telemedicine association was established in 1994 . e - health , telehealth , telecare , and other terms are in reality synonyms . the use of telecommunications to support healthcare came first with videoconference access to specialty care . the practicality of telemedicine shares a history with the transistor , which was commercialized by bell laboratories and was the subject of the nobel prize in physics awarded to shockley , bardeen , and houses in 1956 . the vast composite of the human condition could be stored and transmitted in digital form across any telecommunication medium . the application of transistors to personal computing has a long history , but for the purpose of timelines , the introduction of the apple computer in 1976 was seminal . thereafter , management of data in small , compact , highly effective modules became an everyday practice . today , the storage of huge amounts of patient data , including images and live transmission is a simple and cheap process . the combination of telecommunications and information management constitutes a tool inviting the imagination of practitioners and attacking the very basic issues of cost and access . the growth of telemedicine has been greatly accelerated by wireless communication for devices , cellular telephony , fiberoptic connectivity , and digital records . among these were the broadband initiative , which was intended to bring great bandwidth to almost every village and school in the country . also , the act included the health information technology for economic and clinical health ( hitech ) provisions , requiring the staged expansion of electronic medical records , which were effective , and through such a system , healthcare providers could communicate with one another throughout the us in meaningful ways . in fact , the current surge of telemedicine would not have happened without the ubiquity of the internet , which came into large - scale use in 1994 . these innovations and applications come at a time when the costs of medical care are soaring and the geriatric challenge is upon us . there is no better match in healthcare for improvement and cost saving through telemedicine than the care of the elderly . the population has a huge disease burden , which is not sustainable with the cost constraints of traditional incidental care . the provision of telemedicine services for the elderly is not particularly different from any other medical matters . there is a common belief that high - end electronics and telecommunications can not be used by the elderly because they have limited computer and technology skills . in the developed world , the current rapid growth in the aging population has occurred as a result of what has been dubbed the baby boom . the people entering the ranks of the elderly now are not the same as those very elderly who were born before the second world war . this group followed and cheered the magic of the space program and invented the information age . unless they have cognitive difficulties , the upcoming elderly will be the vanguard of electronic medicine in geriatrics if we invite them and listen to them without prejudice . therefore , there is a need for telemedicine in the elderly , and they are competent to participate in the abundant technology available . the american telemedicine association has issued 14 practice guidelines for telemedicine in a variety of applications from pathology to dermatology to diabetes . now , publications are heavily weighted to randomized controlled trials and telemedicine can be studied as a matter of meta - analysis in the cochrane database . in the early days of telemedicine , the cynical view was that a telemedicine program would close when the grant was spent . as a corollary , telemedicine would not have any inherent value until someone finally paid for it . more than half the states now have laws requiring private insurers to pay for telemedicine for its subscribers demonstrations were carried out in remote parts of the world , and its relevance to highly developed communities was not clear . however , in the past 15 years , telemedicine has been used in all manner of healthcare delivery , and has been integrated into the fabric of overall healthcare . currently , equivalence is of the utmost importance , and demonstration of at least non - inferiority has been crucial to acceptance by patients , physicians , regulators , and payers . videoconference encounters have been shown in every way to be comparable to face - to - face in terms of diagnostic accuracy , interrater variation , patient acceptance , and cost . images produced by smartphone videoconferencing , microscopy , dermatology , and pathology are completely satisfactory . since the elderly have an excess burden of chronic disease , what is the evidence that longitudinal management by telemedicine is helpful ? the data clearly indicates that the use of telemedicine is superior to office visits in terms of emergency visits , hospitalizations , complications , and quality of life for diabetes , hypertension , pain , congestive heart failure , cancer care , rehabilitation after stroke , and dementia . the best place for older people to live is where they want to live , and that is almost always at home . yet when they are frail and forgetful , and have limited mobility , home may be a very dangerous place . extending the practice of their medical team to the home is a great strategy for retention of independence and early recognition of problems . simple monitoring devices can assess gait , pace , activity in general , and adherence to a medical regimen . monitoring of blood pressure , pulse , oxygen saturation , and weight can be done with non - invasive sensors , and the data can be transferred wirelessly to a computer for transmission to a health management office . cheap sensors , either passive or interactive , can capture the essence of a health situation at home and the managing physician can embrace that home with much of the immediate care available otherwise only in the office or hospital . a smart home with sensors and alerts can assess the environment for such subtleties as a running tub , the temperature of the water , the age of materials in the refrigerator , the consumption of food , water , and medications as well as the urgent situations indicated by the presence of smoke , carbon monoxide , or excess heat . there are thousands of medial apps for the smartphones that can be incorporated into comprehensive plans for home health by a managing physician with the fullest consent of the patient . furthermore , the home health nurse alone when confronted by a worrisome change can only order transfer to definitive diagnosis and care or communicate by phone with the managing physician . instead , telemedicine as an augmentation to the physical visit , can be used by nurses to transfer images to evaluate skin lesions or gait , to discuss telemetry data , and receive new instructions which otherwise would only be possible after a visit by the patient to the physician 's office . another use of telemedicine in home healthcare is the critical support of home caregivers who are subject to exhaustion , terrible stress , and a strong urge at times to give up and send the house - bound patient to a facility . the alternative of travel to a fixed health facility is tedious , labor intensive , perhaps painful , and expensive . smartphone programs are excellent for behavior modification in terms of weight , diet , smoking cessation , alcohol abuse , medication , and exercise . the impact , according to published reports , has largely been confined to teenagers , caregivers , and veterans suffering from post traumatic stress disorder . however , the applicability to the elderly has no apparent barrier , and there are examples of success in the elderly . increasingly , medical practices are using social media to enrich medical practice through enhanced communication and reminders to reinforce a regimen . the loss of memory and neural plasticity is perhaps the most difficult matter in the care of the elderly . the value is yet to be determined ; however , it is likely that such programs will be integral to health maintenance in the future . in a typical nursing home , the population is generally elderly , and nursing care is of paramount importance . it is carefully monitored by a physician , and outcomes are carefully analyzed by authorities . however , there is usually no physician present , and if there is any medical question , the first resort is transfer to an emergency medical facility . the regular use of telemedicine to bring the center into close contact with a medical unit can reduce visits dramatically . since the elderly have an excess burden of chronic disease , what is the evidence that longitudinal management by telemedicine is helpful ? the data clearly indicates that the use of telemedicine is superior to office visits in terms of emergency visits , hospitalizations , complications , and quality of life for diabetes , hypertension , pain , congestive heart failure , cancer care , rehabilitation after stroke , and dementia . the best place for older people to live is where they want to live , and that is almost always at home . yet when they are frail and forgetful , and have limited mobility , home may be a very dangerous place . extending the practice of their medical team to the home is a great strategy for retention of independence and early recognition of problems . simple monitoring devices can assess gait , pace , activity in general , and adherence to a medical regimen . monitoring of blood pressure , pulse , oxygen saturation , and weight can be done with non - invasive sensors , and the data can be transferred wirelessly to a computer for transmission to a health management office . cheap sensors , either passive or interactive , can capture the essence of a health situation at home and the managing physician can embrace that home with much of the immediate care available otherwise only in the office or hospital . a smart home with sensors and alerts can assess the environment for such subtleties as a running tub , the temperature of the water , the age of materials in the refrigerator , the consumption of food , water , and medications as well as the urgent situations indicated by the presence of smoke , carbon monoxide , or excess heat . there are thousands of medial apps for the smartphones that can be incorporated into comprehensive plans for home health by a managing physician with the fullest consent of the patient . furthermore , the home health nurse alone when confronted by a worrisome change can only order transfer to definitive diagnosis and care or communicate by phone with the managing physician . instead , telemedicine as an augmentation to the physical visit , can be used by nurses to transfer images to evaluate skin lesions or gait , to discuss telemetry data , and receive new instructions which otherwise would only be possible after a visit by the patient to the physician 's office . another use of telemedicine in home healthcare is the critical support of home caregivers who are subject to exhaustion , terrible stress , and a strong urge at times to give up and send the house - bound patient to a facility . the alternative of travel to a fixed health facility is tedious , labor intensive , perhaps painful , and expensive . smartphone programs are excellent for behavior modification in terms of weight , diet , smoking cessation , alcohol abuse , medication , and exercise . the impact , according to published reports , has largely been confined to teenagers , caregivers , and veterans suffering from post traumatic stress disorder . however , the applicability to the elderly has no apparent barrier , and there are examples of success in the elderly . increasingly , medical practices are using social media to enrich medical practice through enhanced communication and reminders to reinforce a regimen . the loss of memory and neural plasticity is perhaps the most difficult matter in the care of the elderly . the value is yet to be determined ; however , it is likely that such programs will be integral to health maintenance in the future . in a typical nursing home , the population is generally elderly , and nursing care is of paramount importance . it is carefully monitored by a physician , and outcomes are carefully analyzed by authorities . however , there is usually no physician present , and if there is any medical question , the first resort is transfer to an emergency medical facility . the regular use of telemedicine to bring the center into close contact with a medical unit can reduce visits dramatically . in most countries physicians have a very high workload , and the idea of adding any more is just not possible . also in many venues , the payment for these services is not clear or not adequate . in the ideal situation , without cost or staff concerns , the impact is very positive because the patient care is so much better . in the veterans health system in the united states , doctors are all on staff , and their attraction to telemedicine is that it actually takes less time ! for an office physician who has proper access to technology , the overhead costs of telemedicine visits are in fact negligible compared to maintaining an office , parking , many waiting rooms , staff , etc . telemedicine must be integrated into the workflow of physicians and staff in such a way as to make it an improvement and not an add - on . if telemedicine is left to the singular efforts of a physician , the task is very difficult . in the united states , there is a strong movement for telemedicine to be freestanding and not managed by the personal physician . this can certainly be seen as a threat to the sanctity of physician - patient relations . however , the attraction of this innovation to patients is undeniable , and the success is such that there are considerable efforts to accredit the freestanding activities also the best practice standards possible . the notion that some electronic beast is taking away our patients is a bleak one and contrary to the best traditions of medicine . the approach for concerned and conscientious physicians is to learn about the matter , to become leaders rather than followers , and to bring this tool into the daily practice of medicine rather than having a disruptive technology eroding our basic bond of patient care . the question regularly arises as to whether or not telemedicine is in fact a medical specialty . the challenge for biomedical engineers , software engineers , and the telecommunications community is to make the technology as transparent as possible such that the average physician and patient will not find it intimidating . the challenge to physicians is to acquire sufficient facility with the technology as to be a competent user not subject to a profound reliance on technical personnel . finally , the physician should know enough to be a prudent user of new technology . telemedicine like any other tool in medicine can be poorly used , and people can be hurt , or certainly , they can be poorly served . there are many courses available and a little time to become comfortable is time well invested in the future of practice . this brief review has shown that our current practice of care for the elderly is simply not going to succeed as the numbers of elderly patients continues to grow . some of the evidence for the value of telemedicine as a tool for physicians and healthcare systems was presented . the use of telemedicine is a fait accompli in much of the world , and it continues to have an increasing role that is deeply imbedded in our electronic practices coupled with social media . this is a great opportunity for medical practice to evolve to new levels of engagement with our patients and new levels of attainment in terms of quality care .

from the mid-1970s south africa grew increasingly isolated within the international community , including the medical community , and in 1976 the medical association of south africa ( masa ) decided to withdraw from the world medical association ( wma ) because diplomatic pressure had prevented it from attending two of the international organisation s world medical assemblies . yet despite an international academic boycott , even as late as the 1980s , some south african civil society organisations with cordial relations with the country s government continued to enjoy membership of certain prestigious international organisations , such as the wma . indeed , in august 1980 the american medical association ( ama ) launched a campaign for their south african counterparts in the masa to be readmitted to the wma . the transnational anti - apartheid activists who opposed the masa s readmission to the world body argued that , in their exoneration of the doctors who had failed to treat the fatally injured steve biko whilst he was in police detention in september 1977 , the masa had failed to uphold the principles enshrined in the wma s tokyo declaration against torture and the geneva declaration ( an updated version of the hippocratic oath ) . the wma readmitted the south africans at its meeting in lisbon in september 1981 . anti - apartheid health activists within south africa and their international allies , continued to campaign for the masa s expulsion , however , and framed its readmission to the wma as a matter of conscience. by contrast , their opponents in the wma derided them as individuals concerned with anti - apartheid activists efforts in this regard were unsuccessful as the masa was not subsequently expelled from the wma . its defiance of the international academic boycott against south africa did , however , cost the wma its relationship with the world health organisation ( who ) and meant that many national medical associations disaffiliated from the former . south africa s membership of the wma only ceased to be a serious politically difficult issue for the international medical association when the country became democratic in 1994 . this article explores the paradox of south africa s readmission to the wma in the wake of the biko doctors scandal , despite an international academic boycott . it describes the ways in which anti - apartheid activism led to internal rifts within the wma . the activists alleged that the wma had engaged in unprincipled , racist and pro - free - market behaviour . this activism came to diminish the moral reputation and the number of national member organisations of the wma , which was an international health organisation ( iho ) . the activists characterised the wma as having conferred moral authority on the masa , which was closely aligned to the national party government , which followed a racially discriminatory policy of apartheid . they also held that in a cold war context , the renewal of the masa s wma membership was aimed at bolstering the ama s influence in the international organisation . these activists believed that this placed the ama in an excellent position to promote free market - focused approaches to health care delivery , internationally . the topic of steve biko s death in detention has received considerable attention and it has served as an important case study in discussions of medical ethics and the nature of racial discrimination in apartheid era medicine . similarly , the diverse array of anti - apartheid health - related non - governmental organisations has also been an area of interest to historians of medicine . biko s death in detention and the contours of health politics within south africa in the period examined in this article are very important topics which continue to merit attention in their own rights . this paper , however , takes a different , transnational historical angle on these events and , instead , focuses on the impacts of racism in south african medicine upon the internal politics of an iho . while there is a small literature dealing with the history of the wma , scholars have tended not to discuss its controversial stance during the 197694 apartheid era in detail . instead , studies have predominantly focused on how the organisation s ethical projects were shaped by post - war revelations that some european physicians had perpetrated nazi atrocities , as well as on its declarations and role in formalising ethical standards for research involving human subjects . a critical exception to this is laurel baldwin - ragaven et al.s book an ambulance of the wrong colour , which is based upon testimony given at the health sector hearings of south africa s truth and reconciliation commission ( trc ) in 1997 . briefly mention the controversy around the masa s readmission to the wma in 1981 , they do not provide a detailed analysis of the development of domestic and transnational advocacy against it . such multi - country advocacy can be understood within a wider framework of transnational activism , which margaret keck and kathryn sikkink have termed activism beyond borders. according to keck and sikkink such transnational activism occurs when activists based in different countries communicate , share resources and work together to press for changes to policies of which they are critical . there is a rich literature on transnational anti - apartheid activism , which points to the importance of shared , morally resonant framings of apartheid in generating support for the anti - apartheid movement in diverse countries . for example , hkan thrn has argued that exchanges of information , knowledge and symbolic practices between activists in different countries were key activities in the transnational anti - apartheid movement . similarly , audie klotz has argued that the enforcement of an international norm of racial equality promoted by anti - apartheid activists focusing on the influence of transnational anti - apartheid health activism on the internal politics of the wma , this article develops the literature on transnational anti - apartheid activism in general by describing activists roles in opposing racism in international medicine . the transnational anti - apartheid health activism discussed here is relevant to our understanding of ihos in the enforcement of medical ethics . in particular , it points to the deficiencies of an international medical professional association such as the wma as an adjudicator of last resort in a case where serious human rights violations had been perpetrated by physicians . the masa was a founder member association and attended the wma s first general assembly in 1947 . this iho had a range of objectives including upholding the reputation and interests of the medical profession and assisting the world s people in attaining an improved state of health . the fledgling association quickly developed codes of medical ethics and established a relationship with the who . from its earliest days , the wma was dependent upon funding from the american medical association ( ama ) for its financial survival and a substantial proportion of this came from the leaders of us pharmaceutical companies . among the principles of social security the wma adopted in 1947 were the ideas that all medical services should be controlled by physicians , and that doctors should not be full - time salaried servants of the government or social security bodies a position which was very similar to that taken by the ama in the 1950s and 1960s . the ama was also plagued by racial conflict in this period : its 1968 conference was interrupted by civil rights activists who expressed their opposition to the exclusion of black physicians from membership of some southern chapters . it voted to end such discrimination at the same gathering , however , perceptions that racism lingered in the organisation persisted among many black physicians . in 1972 the ama left the international medical body because of what tessa richards has framed as disagreements over funding and voting strengths. the withdrawal of the americans from the organisation caused a crisis of legitimacy the soviet union had never been a member and the people s republic of china had no presence in the organisation and so with the ama s exit , it did not include doctors from three of the world s great powers . the departure of the americans also influenced the canadians in their decision to leave . in 1974 the organisation moved its secretariat from new york to ferney - voltaire in france . from the mid-1970s there had been increasing efforts by certain governments to isolate south african professional organisations , including its medical association because of its government s policy of apartheid . in 1975 the japanese government refused south african delegates visas to visit the country to attend the world medical assembly which was held in tokyo that year . the masa expressed its fury at its representatives being declined visas by the japanese government by arguing that as a founder member of the wma , in good standing it had an absolute right to be allowed to attend all world medical assemblies. the following year , the ghanaian government followed suit when ghana hosted the world medical assembly . that same year , 1976 , the masa resigned from the wma in disgust at this diplomatic pressure . in 1979 , after negotiations with the belgian surgeon dr andr wynen , who was then the part - time secretary - general of the wma , the americans re - joined , on condition that the international medical body changed its bylaws so that the number of votes a national body had within the iho depended upon the number of members it had declared i.e. paid for . this meant that a wealthier nation such as the us with more doctors who were potential members of its medical association ( in this case , the ama ) had far more votes at the wma than a less affluent country such as nigeria , with a medical association which was poorer by virtue of having fewer members . by 1983 the usa had thirty five votes , west germany fourteen , japan fourteen and the rest of the forty - seven member countries had only one or two votes each . voting strengths within the wma was controversial in the case of the vote to readmit the masa to the international association . the dispute over the role of physicians in relation to biko s maltreatment and death in detention was central to the development of a rift within the wma over apartheid . the soweto uprising of 1976 was inspired by biko s black consciousness ( bc ) writings . biko had studied at natal university s medical school , whose student - body was all black . this legislation broadly criminalised extra - parliamentary opposition as it defined terrorism as consisting of any act aimed at changing the economic or social system or fostering animosity between the races . biko died from brain injuries at a pretoria hospital twenty - six days into his detention . the depth of the international outrage over biko s death in detention and the wider crackdown on anti - apartheid opposition which had occurred post - soweto was also reflected in the united nations security council s unanimous vote in favour of resolution 418 which instructed states to stop supplying armaments to south africa . in november 1977 an inquest was held into biko s death in detention in which no one was found responsible for the loss of his life . the presiding magistrate referred part of the evidence which had been presented to the south african medical and dental council ( samdc , hereafter referred to as the medical and dental council ) . in terms of section 45 of the medical , 56 of 1974 , courts were to send the council evidence which appeared to implicate doctors in having engaged in improper or disgraceful professional conduct . a group of physicians also lodged a complaint with the medical and dental council about the behaviour of the two doctors , who had treated biko in detention . this group consisted of members of the black transvaal medical society and physicians with relevant specialist expertise who supported the organisation , including the head of a renal unit , a general medical lecturer and a neurosurgical registrar . the transvaal medical society was a voluntary organisation of black medical doctors , dentists , pharmacists , nurses and paramedics. their complaint was largely based upon evidence given at the inquest by the doctors who had treated biko . the black medical society and their physician supporters argued that a reading of the inquest record clearly shows a prima facie case of improper and/or disgraceful conduct on the part of lang and tucker , the doctors who had treated biko in detention . three years after biko s death , the medical and dental council reached a decision on the anti - apartheid doctors complaint . on 24 april 1980 , the committee of preliminary inquiry of the medical and dental council found that there was no prima facie evidence of disgraceful conduct by the doctors involved in biko s care . jonathan gluckman , a private pathologist based in johannesburg , had performed a post - mortem examination on biko s body at the request of his family . gluckman was also a member of the federal council of the masa , to which he sent a letter signed by thirty - eight of its members calling for it to conduct an inquiry into whether tucker was fit to remain a member of the organisation . a critical component of the dispute which evolved over the medical association s presence in the wma was the decision the south africans subsequently took on this issue . the cape midlands branch of the masa discussed the issue and found that a charge of unethical conduct against dr tucker could not be sustained and ordered that the case be closed . following the cape midlands branch s decision on tucker , the federal council of the masa decided that as far as it was concerned the case was also constitutionally and legally closed . in august 1980 , a month after the masa closed the case against tucker , the ama extended an invitation to their south african counterparts to attend their annual meeting in chicago . dr marais viljoen , the secretary - general of the masa was quoted in a south african newspaper as having said that the friendly invitation of the a.m.a . to attend their meeting in chicago later this year is an indication of the acceptance of south africa in medical circles. the same report stated that there were also indications that the ama would be sending a delegation to south africa in the near future to examine the system of medical services there. according to an article in one of the masa s publications , dr wynen , the head of the wma , also apparently offered his support for the masa . masa representatives who attended the chicago meeting found that the only false note sounded during the meeting , as far as sa [ south africa ] is concerned , took place during a meeting of the american medical association s board of trustees which had been requested by the secretary of the nigerian medical association , dr beko ransome - kuti , during which he criticised sa [ south africa ] for its alleged policies of discrimination against blacks in general and black doctors in particular . the criticism was , however , short - lived when the ama trustees pointed out to him that many of them had been to sa and that his facts were incorrect . ransome - kuti of the nigerian medical association was the brother of the famous musician fela kuti , and publicly shared his anti - apartheid views . like his musician brother , the doctor was involved in broader activism for human rights and democracy in nigeria . both came from a family tradition of vocal civil society advocacy : their mother campaigned against unfair colonial taxation of women and their father had been an anglican priest and founder of the nigerian union of teachers . in february 1981 dr jack sammons , the executive vice - president of the ama said that the world s best medical services were to be found in the us , south africa , canada and australia , with west germany following closely. an article translated from the largely pro - government afrikaans - language die burger by anti - apartheid health activists provided a similar picture of dr sammons s impressions of south africa during his visit and quoted him as having said in south africa we can learn a lot about various aspects of medical care , such as financing , manpower utilisation and the organisation of a complex such as groote schuur [ a large teaching - hospital in cape town ] , which has many services. such a position bore critical similarities to the concept of constructive engagement which was an approach to us foreign policy towards south africa developed by dr chester crocker , the assistant secretary of state for african affairs in the reagan administration . at the core of constructive engagement was crocker s optimism that the administration of president p. w. botha was meaningfully and incrementally reforming the apartheid system , developments which were thought to be deserving of encouragement by means of maintaining a friendly relationship with leaders of the south african government . this policy was criticised by its opponents as overly circumspect about the possibility of offending pretoria and insufficiently informed of the demands of the black opposition . it was doubtless also shaped by the fact that south africa was deemed by hawks in washington dc to be a strategically important bulwark against the infiltration of foreign soviet - aligned forces into the region of southern africa . an article in a south african journal by and for anti - apartheid health activists also published in february 1981 was much more critical of the ama s trip to the country . it noted that the visit by the ama delegates was in spite of an academic boycott on links with south africa , a boycott which had been called by various international organisations including the united nations general assembly , the commonwealth conference and the organisation of african unity . the aim of such boycotts was to isolate south africa financially , militarily , academically and in the arena of sports , and to thereby exert peaceful pressure on south africa to end apartheid. anti - apartheid health activists who contributed to and edited the journal feared that the ama delegation s visit could have been a prelude to m.a.s.as attempt to gain readmission to the world medical association and part of south africa s policy to seek credibility and acceptance in the international community. the ama s visit would thus give the impression that south africa and its health care are not so bad and that international contact will help promote change in this country. the south african anti - apartheid health activists feared that subsequent to the visit , the ama would probably claim to have conducted its own unprejudiced examination of medical care in south africa and would offer to exert pressure on the south african doctors to make positive changes to the health system . they correctly thought that the visit would focus on the adequacy of south africa s training of physicians and the quality of care provided in hospitals and private practice and not on whether the country s health system met the needs of all its people . the anti - apartheid health workers suspected that the ama delegation would not come into contact with the migrant labour system , forced population relocation , the bantustan policy and the oppression and unemployment that are the background to health problems in south africa. any report which would have resulted from the visit would have been inaccurate in the activists view and , therefore , they saw meaningful change resulting from the americans visit which they condemned as a breach of the academic boycott. in the light of the biko controversy , the anti - apartheid health workers were dismayed that the [ ama ] group notably declined to comment on south african hospital overcrowding or the treatment of detainees , because of their lack of knowledge [ my emphases ] . this was the type of response we expected from this group and it is obvious they ignored ( or were not shown ) the desperate lack of health facilities in rural areas and black urban areas . in august 1981 , with the september meeting of world medical assembly in lisbon a mere month away , anti - apartheid activists from various countries swung into action to counter - act what they saw as the ama s campaign for the readmission of the south africans to the wma . the anti - apartheid american committee on africa sent a memorandum to the british anti - apartheid movement s health committee on the issue in august 1981 , in which they outlined their opposition to the south africans proposed readmission to the wma . they pointed out that the country s medical and dental council had taken two and a half years to reach a ruling on the biko doctors and had seen no reason for disciplinary action against them . then they asked for their british counterparts to join them in opposing the ama s expression of support for the south africans readmission and argued that to allow them back into the world body would be to condone both racism and the operation of a vicious double standard in the application of the hippocratic oath. similarly , in august 1981 , the nigerian medical association had lodged a complaint with the australian embassy in london about the latter s national medical association s support for their south african counterparts . the australian government joined the anti - apartheid groups in opposing the ama s support for south africa s readmission to the wma . the australian medical association s president dr lionel wilson was quoted as having said of their support for the south africans bid for readmission to the wma that it was a difficult decision to make but the australian body had decided to support colleagues in south africa because they believed that solutions to sa s [ south africa s ] racial problems were most likely to come through the efforts of compassionate , educated people who have been exposed to world opinion. indeed , wilson argued that we [ the australian medical association ] believe masa is one of the few liberal organisations in sa [ south africa]. in september 1981 it was reported that the british anti - apartheid movement had sent to the portuguese embassy in london a memorandum written by fifteen anti - apartheid organisations and addressed to all the wma s members , which had had to be smuggled out of south africa . according to the report , when the wma considered the proposal to admit the transkei association and readmit the south africans , the medical associations of nigeria , ghana and liberia threatened to leave the world medical body . the strength of opposition to the south africans readmission by many africans from other nations on the continent was demonstrated by a position paper on the issue which was written by two nigerian physicians , dr o. o. adekunle and dr beko ransome - kuti and sent to the british anti - apartheid movement . adekunle and ransome - kuti contrasted the opposition of the nigerian , liberian and ghanaian medical associations with the visit to south africa of the ama delegation who , they thought , were perfectly happy with the conditions there [ in south africa]. they then moved on to note that in their view there was a critical disjuncture between the wma s declarations and the masa s behaviour . by contrast , the masa had been silent on human rights violations in the country , and , the nigerian doctors went on to state that there is a saying that silence means consent all evidence points to its acquiescence with discriminatory health policies of the apartheid government . the m.a.s.a . is obviously towing the line of its sister organisation the south african medical and dental council which itself has only two coloured members in a country of over 80% non - white [ people ] . one of the memoranda sent by south african opponents of the masa s readmission to the wma was penned by the natal health workers association . it outlined the role of masa in directly and indirectly implementing the state s policy of apartheid and thereby perpetuating this form of oppression against the majority of its citizens ; the same policy of apartheid which the international community had committed itself to eradicate . apartheid medicine on the majority of our people [ black south africans] and thereby violating all codes of medical ethics and negating all considerations of human rights. its memo went on to describe key facets of apartheid medicine : systematic racial discrimination in medical training ( fewer black doctors were trained ) ; racial disparities in rates of mortality and disease and an unduly high death rate among black patients due to substandard provision of medical care for them , partly due to a shortage of health professionals who were employed to cater for their needs . uphold the highest traditions and ethics of the medical profession and to ensure racial non - discrimination in health care . therefore , it called on the wma and all its progressive and democratic members to reject the application by masa for membership. masa office - bearers hit back against their black critics who opposed the association s readmission to the wma by attacking their credibility . in the days leading up to the vote on south africa s readmission to the world body , dr marais viljoen , the secretary - general of the masa was quoted as having asked of the black health groups who are they ? what are their objectives? he apparently claimed that his organisation did not condone the findings of the medical and dental council regarding the conduct of the doctors treating mr biko , but noted the council s findings. dr viljoen was also said to have rejected allegations that the medical association had not maintained its objectives or upheld the highest traditions and the ethics of the medical profession. this transnational campaign against the south africans readmission to the wma proved unsuccessful and the breakdown of votes on 25 september 1981 was reported as follows : australia , belgium , brazil , cuba , taiwan , west germany , italy , japan , portugal and the united states voted in favour of south africa s readmission.france , korea and spain abstained.ten votes recorded against the masa which were mostly african and asian countries , including india . australia , belgium , brazil , cuba , taiwan , west germany , italy , japan , portugal and the united states voted in favour of south africa s readmission . ten votes recorded against the masa which were mostly african and asian countries , including india . professor guy de klerk , speaking on behalf of the masa , said that it welcomed its readmission to the wma as a recognition of the high standards of medical ethics and care in the country. anti - apartheid activists in britain swung into action to denounce the outcome of the wma s vote . this was yet another example of transnational anti - apartheid activists challenging the moral authority of the nationalist government and civil society groups perceived as being aligned to it . in late september 1981 , dr johnny fluxman of the british anti - apartheid movement s health committee argued that the wma voting system enables a handful of western countries to dominate the organisation and stifle any criticism by third world countries. he also pointed out that it did not represent any african , scandinavian or socialist countries , and that countries such as taiwan and transkei , not recognised by the un were members . fluxman added that fifteen anti - apartheid organisations within south africa had written to the wma asking it not to readmit the country s medical association but , instead approval has been given to masa and its cover - up of the murder of steve biko , and to south africa s bantustan policy the transkei bantustan has been admitted as a member alongside masa. he concluded his letter by calling upon the british medical association ( bma ) to resign from the wma , noting that they had already stated their opposition to the south africans readmission . meanwhile , within south africa , there were reports in early october that groups such as the black transvaal medical society feared that the acceptance of masa into the wma would lead to other south african organisations clamouring for international recognition putting up a relentless fight. for black medical bodies such as the transvaal medical society , apartheid and oppression violated all codes of medical ethics and the masa , being a predominantly white body , had directly and indirectly condoned this state of affairs. the society also held that the wma s re - acceptance of the masa would forever be regarded a breakthrough for apartheid and oppression of the black majority of the country and a damning and adverse blow to resistance against the status quo and domination of man by man. the newly readmitted masa covered the issue in an editorial in its journal , the south african medical journal , published on 10 october 1981 . the journal reported that the breakdown of votes was seventy - seven in favour of its readmission , with ten against , and eight abstentions were registered . an assembly of doctors concerned with the practice of medicine and not infrequently in conflict with politicians ( as papers read at the scientific sessions later in the week amply demonstrated ) . we are sure that the masa has a contribution to make to this body and that the wma will in no way be weakened by its presence. as the controversy over the south africans involvement in the wma deepened , the idea that a cordon sanitaire could be imposed between medical ethics and politics , would continue to be asserted by the masa and their international supporters . four months later , in january 1982 , the issue was considered by the executive board of the world health organisation ( who ) . the who was one of many un agencies which had restricted south africa s membership since 1960 after agitation by the newly independent african states . on 20 january 1982 , h.e . alhaji yusuff maitama - sule , nigeria s representative to the un who also chaired its special committee on apartheid ( established in 1964 ) sent a telegram to the executive board of the who on the matter of the masa s readmission to the wma . maitama - sule accused the wma of violating article ii of the international convention on the suppression and punishment of the crime of apartheid of 1973 : this convention defined apartheid as being a crime against humanity. the nigerian diplomat held that apartheid was an evil system which the non - white world had played a leading role in opposing because it represented an affront to their recently won freedom , independence , and i dare say , human dignity. he went on to add , let me take advantage of this appearance before who and eminent doctors to state that we in the special committee , and i might add in the organisation of african unity , consider the role being played by the masa and wma as not being too different from the role played by many nazi doctors during the second world war. maitama - sule then said that when the time of retribution came , the example of nuremberg would not be lost on the united nations and the african people. he ended by calling on the who executive board to terminate its relationship with the masa and the so - called transkei. an african diplomat to the un was publicly equating the actions of the wma , an organisation which was set up in the wake of the nuremberg trials and which had the promotion of medical ethics as one of its core aims , with those of nazi doctors . the wma s credibility as the keeper of the medical ethical creed was under serious attack at a critical international health institution . the who s executive board voted to discontinue official relations with the wma on 27 january 1982 by twenty - seven votes in favour , one vote against ( the us ) and one abstention . this decision was reversible provided the wma expelled the south africans . in subsequent years , this decision would be cited repeatedly by anti - apartheid activists to show that the wma had suffered a loss of prestige and was out of step with established international norms of racial equality in relation to how to deal with its south african member organisation . by this period , there was an increasing enforcement of such norms , internationally . on 5 february 1982 , dr neil aggett a young physician and trade union organiser died in johannesburg after having spent seventy days in detention without trial . meanwhile , shortly after their readmission to the wma , the south africans were soon nominated and elected for some of its key positions . ( marais ) viljoen the masa s secretary - general was elected to the council of the wma and nominated to its medical ethics committee : a nomination he described as of major importance to the south african medical profession , whose medical ethics have frequently been questioned at an international level. this was a position to which he was appointed in 1983 . viljoen was alleged by anti - apartheid activists to be both a national party supporter and a long - time member of its inner conclave , the secretive broederbond . with an unsuccessful campaign against the south africans readmission behind them , the health workers association ( formerly the transvaal medical society ) re - strategised and also began to campaign against the wma itself . at its national meeting in may 1982 , an anonymous activist gave a speech on the wma s history , a summary of which was kept by the organisation . the full , written version of the speech is worth discussing at some length as it reveals what some anti - apartheid health workers based within the country thought the socio - economic and political forces were behind the ama s campaign for the readmission of their south african counterparts . the activist cited an article published in the south african medical journal in 1951 to claim that the best financial support for the wma had come from the great pharmaceutical firms of the united states many of which , as we have seen , had close ties with the ama . usa through the ama , attempted to impose its hegemony on the wma since its inception . the global advantages for the multinational pharmaceutical industry of an american dominated wma are obvious . their close association with ama would enable them to come into contact with the medical profession from many countries to whom they could promote their drugs . furthermore , health programmes and policies that were compatible with the financial interests of american capitalism would be promoted at a global level in the wma . the anti - apartheid health activist went on to argue that part of the impetus behind forming national medical associations was to block or slow the formation of national health services . the author also argued that exiled cuban doctors in miami had represented cuba in the wma since 1959 . in the document s conclusions the anti - apartheid health activist claimed that the wma was an organisation which was not truly representative of the world medical profession because less than a third of the world s countries had medical associations affiliated to it . the activist went on to argue that the wma only allowed associations which were independent of governments as its members and that this was intended to exclude socialist governments from joining it. the article went on to add that this concept of independent is farcical as in most countries the medical associations are closely aligned with the state. this author again compared the wma to the who , by stating that while the who has made a significant contribution at an international level to the promotion of health care , wma s contribution to this sphere has been negligible. without significant reform to the wma s structure it was unlikely to make a significant contribution , in the author s view . reactionary organisation which could not be changed from within as the british medical association had apparently realised by this stage . the health workers association s new strategy on this issue had to be to oppose both the masa and the wma at national and international levels . by july 1982 the british medical association ( bma ) had officially terminated its relationship with the masa . in a letter to the editor of the south african medical journal , dr jonathan gluckman outlined his version of what had caused the termination of the relationship . as we have seen gluckman had performed a post - mortem examination on biko s body and was also a member of the executive committee of the masa and he met with the bma council s executive committee to discuss its decision to terminate its relationship with the masa . gluckman was a fierce opponent of the termination of the bma s relationship with the masa and following his meeting with representatives of its council he claimed that the british association which [ he said ] was part of the trades union congress was dominated by the politics of the extreme left in britain , and that the effect of this is to erode the structures of the bma to the same extent as it has eroded the structure of great britain. he claimed that so vicious had been the attitude of the council of the bma , that its representatives for the then upcoming meeting of the wma had been instructed to vote against the readmission of the south africans and not to be influenced by any debate or arguments to the contrary which might have been forthcoming [ his emphasis]. in july 1983 dr antonio gentil martins , the portuguese president of the wma was one of the guests of honour at the fifty - fourth meeting of the masa in cape town . also in attendance were representatives from the american medical association ( the ama ) and the west german medical association . the presence of these representatives of the wma reflected the fact that two years earlier the south africans had been readmitted to the international medical association . following his visit to south africa , dr martins spoke highly of the south african medical profession , and , according to an anti - apartheid activist newsletter , he had said , we found that the quality of health care available to all races was completely equal. he then apparently went on to argue that providing medical infrastructure was a political issue and not the responsibility of the profession. this was dr martins s second visit to south africa . on his first visit to the country , the portuguese surgeon had been quoted as having defended the masa s controversial approach to the scandal surrounding biko s death in detention discussed above . the only member of masa involved in the affair had been exonerated of all blame . masa therefore could not be held responsible for the treatment of mr biko. he claimed to be familiar with the problem of overcrowding in south african facilities and apparently said that providing the infrastructure for adequate medical services was a political issue and not the responsibility of masa. martins had seen the same technology available in hospitals serving black people and those serving whites and he apparently claimed that i saw no difference as far as the quality of care was concerned. he was also quoted as having argued that the low standard of education and lack of family planning among black people were among the worst problems facing south african medicine. martins apparently went on to say that no country can afford to provide hospital beds for a population growing as rapidly as that of south africa s blacks. such a racist , neo - malthusian argument that the poverty experienced by africans was their own fault due to their supposed uncontrolled fertility was a key element of apartheid thinking , as evident in the south african government s disproportionate spending on the promotion and provision of family planning when compared to their spending on the other health services for black people , including maternal and child health services . the masa had invited representatives to attend the same 1983 congress , and the president of the ama , dr frank j. jirka said that health services in this country [ south africa ] compared favourably with those in the united states of america. one of the delegates was dr james h sammons ( the executive vice president of the ama , as mentioned above ) who was named the leading medical personality in the usa for 1983 by the times us news and world report , inter alia , referred to the masa s report on the medical treatment of prisoners and detainees . superb job and he hoped that the recommendations would be favourably considered by the authorities. dr sammons argued that the maltreatment of prisoners also occurred in america and he said unfortunately it happens far too often , and although there are problems , it is not excused by the problems. there was yet another call by the masa s supporters to separate politics. dr horst bourmer of the west german medical association apparently held that doctors , as doctors , should never become involved in politics , but should concern themselves with the improvement of medical care only. bourmer called for governments to grant medical associations autonomy on professional matters and then was quoted as having said that on the other hand i believe that [ the ] exclusion of medical associations from international medical politics because of the policies of their governments is discrimination at its worst . humanity and fraternity should be the motto of all who belong to the medical profession . at its october 1983 meeting in vienna , the wma decided to hold its 1985 assembly in cape town on a clear day robben island a potent symbol of racial discrimination robben island contained a prison where several opponents of apartheid remained detained and whose most famous resident just over a year before had been nelson mandela , who had recently been moved to pollsmoor prison on the mainland , just outside cape town . in this context , at its first annual national conference in durban from 5 to 6 december 1983 , the new anti - apartheid national medical and dental association ( namda ) resolved to oppose cape town s hosting of the wma . the new association aimed to unite all south african doctors and dentists opposed to apartheid in one nationwide organisation . unlike the masa , namda was affiliated with the united democratic front ( udf ) , a national anti - apartheid civil society coalition . on 5 january 1984 eroded by a series of events which had cast doubt on the ability and willingness of wma to provide an international , representative forum for the resolution of important medical , professional and ethical issues. the press release mentioned that an undemocratic voting block system had resulted in the south africans having been readmitted to the international medical association against the wishes of the majority of countries belonging to wma , even though certain important issues affecting its application for re - admission had not at that time been resolved . these issues included the failure of the masa to adequately investigate the conduct of certain doctors who had examined steve biko before his death in police custody . the statement noted that the british association had tried to reform the wma constitution from within in the years subsequent to south africa s readmission . it also noted that the who had withdrawn consultative status from the wma over the issue and questioned the representative legitimacy of members such as the transkei association and the cubans who were represented by the free doctors of cuba based in miami . but british doctors were far from unanimous in supporting their national medical association s stance on this issue . the lancet s brief coverage of the withdrawal of the british medical association from the wma stated that its undemocratic voting system was at the heart of its decision on the issue . it also reported that a group of british doctors had formed who continued to support the wma . such dissenters within bma on south africa issue held views which were in keeping with a substantial slice of british public opinion . the conservative party government led by margaret thatcher had a policy which was in many respects similar to the constructive engagement of their american counterparts . moreover , in the early 1980s , cultural and economic ties between britain and south africa remained strong , despite calls for sanctions . back in south africa , dr r.d . le roex , chairman of the federal council of the country s medical association mentioned in his 1984 report for that body that it had hosted dr lionel l. wilson , the wma s council s former chairman and past president of the australian medical association . le roex expressed his appreciation for the role played by the masa in supporting of improvement of medical care for south african political detainees . he stated how much he valued the south africans ongoing membership of the wma , as such international contact was essential if the lofty ideals of the wma , viz . to achieve the highest international standards of medical education , medical science , medical art and medical ethics , and health care for all people of the world , are to be attained. le roex said that at the wma s meeting in singapore the south africans had had the chance to meet both those who were well informed and well disposed towards south africa and its health services and those with the opposite opinion . in his view their opponents were frankly hostile to the system of government in south africa and consequently also to our health care system and to the masa , a position which he viewed as being based on ignorance or misinformation , much of which is deliberately disseminated from this country by misguided colleagues [ namda]. he saw namda s call for a boycott of the cape town assembly as based upon a malicious misrepresentation as the invitation was not a political statement of any kind. by contrast , the masa s chairman said that the south african government s only involvement would be the granting of visas to bona fide delegates. in late 1984 there were dramatic broader developments in the country s popular politics , which had also important effects on anti - apartheid activism abroad . there was a new wave of popular protests in several townships across the country which involved the civic associations and activists aligned to the udf and in many countries in the west images of police brutality towards the demonstrators were broadcast on television news , which broadened opposition to apartheid . in november 1984 the anglican archbishop desmond tutu tutu made a strong impression in the united states and his nobel win was associated with more radical and popular american anti - apartheid activism as represented by the civil disobedience actions of groups such as transafrica led by randall robinson . on 5 february 1985 , three hundred physicians and other health professionals marched on the south african embassy in washington dc five of whom were arrested and later released . some of the thinking behind this growing us opposition to apartheid in south african medicine is suggested by a guest editorial published in the journal of the national medical association in july 1985 . the national medical association was an organisation which represented african - american physicians and members of the communities that they served . charles h. wright began his article stating that dr philip m. smith , the president of the national medical association , had asked members to join him in protesting the proposed cape town meeting of the wma . he then moved on to discuss the scandal around biko s physicians collusion in his torture in detention and noted that while the national medical association s journal had covered the issue and while many of the world s medical societies reacted with revulsion towards south africa s efforts to ignore and cover up this event , the american medical association s officials reacted as if it was a non - event . the journal of the american medical association did not mention the controversy. wright pointed to the ama representatives two visits to south africa and also mentioned that in 1981 the american association had cast all its votes in favour of the country s readmission . he then stated his view that while a campaign for a cancellation of the world medical assembly in cape town was laudatory , a letter - writing campaign to the ama would not have intrude[d ] on its loyalty to its colleagues in south africa. he then called for national medical association members to stop paying for ama membership because a more certain way to make sure that biko , a former medical student did not die in vain is to divest in ama. he ended by stating that his only regret was that he could not join in such a protest as he had already divested from the ama in the late 1950s when the ama showed more concern for hungarian and cuban exiles than for black doctors . the wma s membership dwindled significantly because of its position on south africa . in 1985 doubtless at least partially due to such civil society pressure on the issue , the thirty - seventh ( 1985 ) world medical assembly was moved to brussels , belgium at short notice . meanwhile , a breakaway group was formed which consisted of the medical associations of denmark , finland , iceland , ireland , the netherlands , new zealand , norway and sweden , later joined by the british , canadians and jamaicans . according to an article published in the british medical journal in 1994 , the group met annually and campaigned around demands such as : member associations of the wma should be truly representative of the medical profession in their country.member associations should be politically independent of their government.the wma needed to have a more democratic voting system. member associations of the wma should be member associations should be politically independent of their government . the wma needed to have a more democratic voting system. after a 1987 meeting of this group in canada , the group became known as the toronto group. in johannesburg , in november 1985 , litigation launched by a group of medical academics at the university of the witwatersrand resulted in a supreme court decision which forced the south african medical and dental council to reconsider the case of drs tucker and lang in relation to their treatment of biko . lang continued to practise , however , and was promoted to the position of chief district surgeon in port elizabeth . for the rest of the 1980s the namda and the masa remained bitter foes because apartheid itself remained at the very heart of their dispute . however , the international medical association apparently responded to some of the toronto group s criticisms by implementing certain reforms such as agreeing with the principle that member associations should be truly representative of doctors in their own countries . it also switched to granting each member country one vote per 10 000 members instead of the previous one vote per five thousand members and resolutions on ethical issues now required a two - thirds majority of delegates present to pass . the nordic countries and britain had only just rejoined as late as 1995 when a dr anders milton a swedish nephrologist became new chairman of the wma and stated his relief that with the country s democratic transition it no longer had the political problem of south africa. milton acknowledged that this had been the main reason why the british and swedish associations had left and pledged to reach out to more countries and ensure not only that ethical declarations are taught at medical schools and discussed by practising doctors all over the world but that they are in daily use. transnational anti - apartheid activists efforts to isolate south africa and morally delegitimise the wma for failing to do so , therefore , had had a real impact on physicians support for an iho in a number of countries . in post - apartheid south africa , the issue of the biko doctors was re - examined in the truth and reconciliation commission s health sector hearings in 1997 . in the spirit of nation reconciliation the namda and the masa merged in 1998 to become the south african medical association ( sama ) . also in 1997 , the south african medical and dental council ( samdc ) became the health professions council of south africa ( hpcsa ) as part of a broader package of reforms to the bring the regulation of doctors activities into alignment with the human rights values espoused in the country s new constitution ( 1996 ) . today , the wma is an iho best known for its ethical declarations , including the declaration of tokyo on physicians and torture . yet , one of the clearest examples of the violation of this declaration occurred almost immediately after it was passed the maltreatment of south african activist steve biko by his doctors , physicians whom the country s medical association and medical and dental council were extremely hesitant to discipline until the end of the main period discussed in this article . less documented have been the circumstances around the readmission of the masa and the subsequent exodus of a substantial group of the wma s members because of perceptions that it had exonerated apartheid medicine , including physician collusion in torture a lacuna in existing literature that this article has aspired to make modest progress in addressing . the moral authority of the wma around the world also dwindled as physicians who were anti - apartheid activists in britain , the united states and south africa repeatedly denounced the organisation as racist and campaigned against it in the same breath as they opposed apartheid medicine . these transnational activists or activists beyond borders shared information with each other , including information which helped to generate morally influential framings of racial discrimination in south african medicine . yet they were ultimately unable to successfully press for the wma to reject the masa as a member organisation . what they were , however , able to accomplish was a diminution in the moral authority of the iho both among many physicians in different countries and several international health officials . they successfully drew attention to the fact that in the late twentieth century , senior office - bearers of an organisation fundamentally shaped to combat the horrors of nazi medicine had condoned medical aspects of a system of racial discrimination which had been cast in international law as being yet another crime against humanity. these activists , thereby , managed to reduce the number of member associations and press the who to end its relationship with the wma . the wma and its supporters on this issue derided their opponents as politicians concerned with ethics. anti - apartheid activists rejected such a division by pointing to the racism in south africa s health system and a lack of political freedom to highlight it . indeed , they drew attention to the fact that a former medical student ( steve biko ) and a doctor ( neil aggett ) were among the many activists tortured - to - death in detention . for such activists , these cases demonstrated that even health workers could not freely highlight issues of terrorists with all the dire consequences that entailed . the full reasons why the wma not only bucked international trends to isolate south africa by welcoming its medical association back into its fold but also promptly thereafter invited one the south africans to sit on its ethics committee have yet to be explored in further research . what is hard to dispute is that the wma and the ama ( its member with the largest number of votes ) generated perceptions among opponents of apartheid that it had welcomed the south africans back as part of a wider effort to pack the body with members who would uncritically support the ama an organisation with stances very different to those of the who on the ideal roles of physicians and the private sector in health systems . some african - american physicians opposed to apartheid saw the controversy as the latest chapter in a long history of institutionalised anti - black racism within the american member organisation . the most charitable interpretation of why some national associations chose to support south africa s readmission is that they might have hoped to have won them over to incremental liberal reforms to end apartheid in the manner of crocker s constructive engagement . apartheid s demise occurred almost two decades ago , but the notions that medical ethics should be depoliticised remain current . this article has discussed an important , under - examined , late twentieth - century example of racism in international medicine . medical ethicists have observed that the wma s role as an international adjudicator is limited by the voluntary nature of its international professional guidelines. the history offered in this article , therefore , suggests that transnational civil society actors must remain permanently vigilant to ensure that justice prevails in cases where physicians are accused of human rights violations .

this article describes the role of transnational anti - apartheid activism in south africa , britain and the united states in generating international moral outrage over the readmission of the medical association of south africa ( masa ) to the world medical association ( wma ) , which had taken place in 1981 after it had withdrawn from that body in 1976 . it discusses an example of a controversy where an international health organisation ( iho ) lost moral authority as a result of being accused of white supremacy and a pro - american engagement in cold war politics . at the time of its readmission to the wma , the masa was controversial because of its failure to strike off its membership roll one of the doctors implicated the death in detention of black consciousness leader steve biko in 1977 . it details how these activists viewed the american medical association as having campaigned for the masa s readmission . the wma s readmission of the masa cost the former its relationships with the world health organisation ( who ) and the british medical association a dispute which continued until south africa s democratic transition of 1994 . with its focus on transnational activism in relation to the wma and the effects of activists allegations of racism on its internal politics , this article contributes to the literature on the history of ihos . ultimately , this controversy shows the deficiency of international medical professional associations as ethical arbitrators of last resort .

Introduction Background: The MASAs Resignation from the WMA in 1976 The Biko Doctors Controversy within South Africa The AMAs Campaign for the South Africans to Rejoin the WMA The South Africans Re-enter and the British Exit The Campaign Against the 1985 Cape Town World Medical Assembly Conclusion: Ethics and Medical Associations in South Africa and the World

from the mid-1970s south africa grew increasingly isolated within the international community , including the medical community , and in 1976 the medical association of south africa ( masa ) decided to withdraw from the world medical association ( wma ) because diplomatic pressure had prevented it from attending two of the international organisation s world medical assemblies . indeed , in august 1980 the american medical association ( ama ) launched a campaign for their south african counterparts in the masa to be readmitted to the wma . the transnational anti - apartheid activists who opposed the masa s readmission to the world body argued that , in their exoneration of the doctors who had failed to treat the fatally injured steve biko whilst he was in police detention in september 1977 , the masa had failed to uphold the principles enshrined in the wma s tokyo declaration against torture and the geneva declaration ( an updated version of the hippocratic oath ) . anti - apartheid health activists within south africa and their international allies , continued to campaign for the masa s expulsion , however , and framed its readmission to the wma as a matter of conscience. its defiance of the international academic boycott against south africa did , however , cost the wma its relationship with the world health organisation ( who ) and meant that many national medical associations disaffiliated from the former . south africa s membership of the wma only ceased to be a serious politically difficult issue for the international medical association when the country became democratic in 1994 . this article explores the paradox of south africa s readmission to the wma in the wake of the biko doctors scandal , despite an international academic boycott . this activism came to diminish the moral reputation and the number of national member organisations of the wma , which was an international health organisation ( iho ) . the activists characterised the wma as having conferred moral authority on the masa , which was closely aligned to the national party government , which followed a racially discriminatory policy of apartheid . while there is a small literature dealing with the history of the wma , scholars have tended not to discuss its controversial stance during the 197694 apartheid era in detail . briefly mention the controversy around the masa s readmission to the wma in 1981 , they do not provide a detailed analysis of the development of domestic and transnational advocacy against it . there is a rich literature on transnational anti - apartheid activism , which points to the importance of shared , morally resonant framings of apartheid in generating support for the anti - apartheid movement in diverse countries . similarly , audie klotz has argued that the enforcement of an international norm of racial equality promoted by anti - apartheid activists focusing on the influence of transnational anti - apartheid health activism on the internal politics of the wma , this article develops the literature on transnational anti - apartheid activism in general by describing activists roles in opposing racism in international medicine . in particular , it points to the deficiencies of an international medical professional association such as the wma as an adjudicator of last resort in a case where serious human rights violations had been perpetrated by physicians . from its earliest days , the wma was dependent upon funding from the american medical association ( ama ) for its financial survival and a substantial proportion of this came from the leaders of us pharmaceutical companies . the masa expressed its fury at its representatives being declined visas by the japanese government by arguing that as a founder member of the wma , in good standing it had an absolute right to be allowed to attend all world medical assemblies. in 1979 , after negotiations with the belgian surgeon dr andr wynen , who was then the part - time secretary - general of the wma , the americans re - joined , on condition that the international medical body changed its bylaws so that the number of votes a national body had within the iho depended upon the number of members it had declared i.e. this meant that a wealthier nation such as the us with more doctors who were potential members of its medical association ( in this case , the ama ) had far more votes at the wma than a less affluent country such as nigeria , with a medical association which was poorer by virtue of having fewer members . the dispute over the role of physicians in relation to biko s maltreatment and death in detention was central to the development of a rift within the wma over apartheid . the depth of the international outrage over biko s death in detention and the wider crackdown on anti - apartheid opposition which had occurred post - soweto was also reflected in the united nations security council s unanimous vote in favour of resolution 418 which instructed states to stop supplying armaments to south africa . the black medical society and their physician supporters argued that a reading of the inquest record clearly shows a prima facie case of improper and/or disgraceful conduct on the part of lang and tucker , the doctors who had treated biko in detention . a critical component of the dispute which evolved over the medical association s presence in the wma was the decision the south africans subsequently took on this issue . according to an article in one of the masa s publications , dr wynen , the head of the wma , also apparently offered his support for the masa . masa representatives who attended the chicago meeting found that the only false note sounded during the meeting , as far as sa [ south africa ] is concerned , took place during a meeting of the american medical association s board of trustees which had been requested by the secretary of the nigerian medical association , dr beko ransome - kuti , during which he criticised sa [ south africa ] for its alleged policies of discrimination against blacks in general and black doctors in particular . an article translated from the largely pro - government afrikaans - language die burger by anti - apartheid health activists provided a similar picture of dr sammons s impressions of south africa during his visit and quoted him as having said in south africa we can learn a lot about various aspects of medical care , such as financing , manpower utilisation and the organisation of a complex such as groote schuur [ a large teaching - hospital in cape town ] , which has many services. it noted that the visit by the ama delegates was in spite of an academic boycott on links with south africa , a boycott which had been called by various international organisations including the united nations general assembly , the commonwealth conference and the organisation of african unity . anti - apartheid health activists who contributed to and edited the journal feared that the ama delegation s visit could have been a prelude to m.a.s.as attempt to gain readmission to the world medical association and part of south africa s policy to seek credibility and acceptance in the international community. the south african anti - apartheid health activists feared that subsequent to the visit , the ama would probably claim to have conducted its own unprejudiced examination of medical care in south africa and would offer to exert pressure on the south african doctors to make positive changes to the health system . the anti - apartheid health workers suspected that the ama delegation would not come into contact with the migrant labour system , forced population relocation , the bantustan policy and the oppression and unemployment that are the background to health problems in south africa. in august 1981 , with the september meeting of world medical assembly in lisbon a mere month away , anti - apartheid activists from various countries swung into action to counter - act what they saw as the ama s campaign for the readmission of the south africans to the wma . the anti - apartheid american committee on africa sent a memorandum to the british anti - apartheid movement s health committee on the issue in august 1981 , in which they outlined their opposition to the south africans proposed readmission to the wma . the australian government joined the anti - apartheid groups in opposing the ama s support for south africa s readmission to the wma . the australian medical association s president dr lionel wilson was quoted as having said of their support for the south africans bid for readmission to the wma that it was a difficult decision to make but the australian body had decided to support colleagues in south africa because they believed that solutions to sa s [ south africa s ] racial problems were most likely to come through the efforts of compassionate , educated people who have been exposed to world opinion. in september 1981 it was reported that the british anti - apartheid movement had sent to the portuguese embassy in london a memorandum written by fifteen anti - apartheid organisations and addressed to all the wma s members , which had had to be smuggled out of south africa . one of the memoranda sent by south african opponents of the masa s readmission to the wma was penned by the natal health workers association . in the days leading up to the vote on south africa s readmission to the world body , dr marais viljoen , the secretary - general of the masa was quoted as having asked of the black health groups who are they ? this transnational campaign against the south africans readmission to the wma proved unsuccessful and the breakdown of votes on 25 september 1981 was reported as follows : australia , belgium , brazil , cuba , taiwan , west germany , italy , japan , portugal and the united states voted in favour of south africa s readmission.france , korea and spain abstained.ten votes recorded against the masa which were mostly african and asian countries , including india . australia , belgium , brazil , cuba , taiwan , west germany , italy , japan , portugal and the united states voted in favour of south africa s readmission . professor guy de klerk , speaking on behalf of the masa , said that it welcomed its readmission to the wma as a recognition of the high standards of medical ethics and care in the country. this was yet another example of transnational anti - apartheid activists challenging the moral authority of the nationalist government and civil society groups perceived as being aligned to it . fluxman added that fifteen anti - apartheid organisations within south africa had written to the wma asking it not to readmit the country s medical association but , instead approval has been given to masa and its cover - up of the murder of steve biko , and to south africa s bantustan policy the transkei bantustan has been admitted as a member alongside masa. he concluded his letter by calling upon the british medical association ( bma ) to resign from the wma , noting that they had already stated their opposition to the south africans readmission . four months later , in january 1982 , the issue was considered by the executive board of the world health organisation ( who ) . alhaji yusuff maitama - sule , nigeria s representative to the un who also chaired its special committee on apartheid ( established in 1964 ) sent a telegram to the executive board of the who on the matter of the masa s readmission to the wma . maitama - sule then said that when the time of retribution came , the example of nuremberg would not be lost on the united nations and the african people. an african diplomat to the un was publicly equating the actions of the wma , an organisation which was set up in the wake of the nuremberg trials and which had the promotion of medical ethics as one of its core aims , with those of nazi doctors . in subsequent years , this decision would be cited repeatedly by anti - apartheid activists to show that the wma had suffered a loss of prestige and was out of step with established international norms of racial equality in relation to how to deal with its south african member organisation . meanwhile , shortly after their readmission to the wma , the south africans were soon nominated and elected for some of its key positions . ( marais ) viljoen the masa s secretary - general was elected to the council of the wma and nominated to its medical ethics committee : a nomination he described as of major importance to the south african medical profession , whose medical ethics have frequently been questioned at an international level. the full , written version of the speech is worth discussing at some length as it reveals what some anti - apartheid health workers based within the country thought the socio - economic and political forces were behind the ama s campaign for the readmission of their south african counterparts . by july 1982 the british medical association ( bma ) had officially terminated its relationship with the masa . he claimed that so vicious had been the attitude of the council of the bma , that its representatives for the then upcoming meeting of the wma had been instructed to vote against the readmission of the south africans and not to be influenced by any debate or arguments to the contrary which might have been forthcoming [ his emphasis]. in july 1983 dr antonio gentil martins , the portuguese president of the wma was one of the guests of honour at the fifty - fourth meeting of the masa in cape town . also in attendance were representatives from the american medical association ( the ama ) and the west german medical association . on his first visit to the country , the portuguese surgeon had been quoted as having defended the masa s controversial approach to the scandal surrounding biko s death in detention discussed above . the masa had invited representatives to attend the same 1983 congress , and the president of the ama , dr frank j. jirka said that health services in this country [ south africa ] compared favourably with those in the united states of america. one of the delegates was dr james h sammons ( the executive vice president of the ama , as mentioned above ) who was named the leading medical personality in the usa for 1983 by the times us news and world report , inter alia , referred to the masa s report on the medical treatment of prisoners and detainees . in this context , at its first annual national conference in durban from 5 to 6 december 1983 , the new anti - apartheid national medical and dental association ( namda ) resolved to oppose cape town s hosting of the wma . the lancet s brief coverage of the withdrawal of the british medical association from the wma stated that its undemocratic voting system was at the heart of its decision on the issue . le roex , chairman of the federal council of the country s medical association mentioned in his 1984 report for that body that it had hosted dr lionel l. wilson , the wma s council s former chairman and past president of the australian medical association . in late 1984 there were dramatic broader developments in the country s popular politics , which had also important effects on anti - apartheid activism abroad . he then moved on to discuss the scandal around biko s physicians collusion in his torture in detention and noted that while the national medical association s journal had covered the issue and while many of the world s medical societies reacted with revulsion towards south africa s efforts to ignore and cover up this event , the american medical association s officials reacted as if it was a non - event . he then stated his view that while a campaign for a cancellation of the world medical assembly in cape town was laudatory , a letter - writing campaign to the ama would not have intrude[d ] on its loyalty to its colleagues in south africa. the nordic countries and britain had only just rejoined as late as 1995 when a dr anders milton a swedish nephrologist became new chairman of the wma and stated his relief that with the country s democratic transition it no longer had the political problem of south africa. also in 1997 , the south african medical and dental council ( samdc ) became the health professions council of south africa ( hpcsa ) as part of a broader package of reforms to the bring the regulation of doctors activities into alignment with the human rights values espoused in the country s new constitution ( 1996 ) . yet , one of the clearest examples of the violation of this declaration occurred almost immediately after it was passed the maltreatment of south african activist steve biko by his doctors , physicians whom the country s medical association and medical and dental council were extremely hesitant to discipline until the end of the main period discussed in this article . less documented have been the circumstances around the readmission of the masa and the subsequent exodus of a substantial group of the wma s members because of perceptions that it had exonerated apartheid medicine , including physician collusion in torture a lacuna in existing literature that this article has aspired to make modest progress in addressing . the moral authority of the wma around the world also dwindled as physicians who were anti - apartheid activists in britain , the united states and south africa repeatedly denounced the organisation as racist and campaigned against it in the same breath as they opposed apartheid medicine . anti - apartheid activists rejected such a division by pointing to the racism in south africa s health system and a lack of political freedom to highlight it . indeed , they drew attention to the fact that a former medical student ( steve biko ) and a doctor ( neil aggett ) were among the many activists tortured - to - death in detention . what is hard to dispute is that the wma and the ama ( its member with the largest number of votes ) generated perceptions among opponents of apartheid that it had welcomed the south africans back as part of a wider effort to pack the body with members who would uncritically support the ama an organisation with stances very different to those of the who on the ideal roles of physicians and the private sector in health systems .

the american joint committee on cancer / union for international cancer control ( ajcc / uicc ) tumor - nodes - metastasis ( tnm ) staging system provides the most reliable guidelines for the prognostication and treatment of carcinomas . in fact , clinical outcome can signicantly vary among patients within the same tnm stage . some patients with advanced - stage cancer may remain stable for years , and although rare , partial or full regression of metastatic tumors may occur spontaneously ( 1,2 ) . in contrast , relapse , rapid tumor progression , and patient death occur in approximately 10 - 25% of patients with tnm i / ii stage cancers , despite performing complete surgical resection and even though there is no evidence of residual tumor burden or distant metastasis ( 2,3 ) . the tnm system is based on the tumors ' biological behaviors , without considering host responses . it is becoming increasingly evident that immune response against cancers is an important factor in deciding the clinical outcomes ( 4 - 7 ) . multiple inflammatory cells , especially lymphocytes and macrophages commonly infiltrate into solid tumor tissues . rather than considering a change in the expression of a single immunological marker , the change in a combined index or the immune score ( i m ) would better represent the immune response . the i m system , which provides an i m ranging from 0 to 4 ( im0 to im4 ) , was initially suggested and developed for colorectal cancers ( 8,9 ) , and is based on the enumeration of two lymphocyte populations ( cd3/cd45ro , cd3/cd8 ) , both in the core of the tumor and in the invasive margin of tumors . the i m appears to be the strongest prognostic factor for disease - free survival ( dfs ) , disease specic survival , and overall survival ( os ) in colorectal cancers ( 10 - 13 ) . in this study , we developed a new i m system considering that the immune response consists of multiple effector cells and immune cells infiltrating tumor tissue , each of which may act as promoters or inhibitors of tumor progression , depending on the tumor microenvironment ( 4,14,15 ) . the i m system included t cells , b cells , and myeloid - derived suppressor cells ( mdscs ) , and high - mobility group protein b1 ( hmgb1 ) expression profile , to assay the prognostic role of the immune response in gastric cancers . between 2003 and 2006 , 100 samples of pathologically confirmed cancer tissue were obtained from patients with stage iiia ( 2009 ajcc / uicc staging system ) gastric cancer at the sun yat - sen university cancer center . this study was conducted in accordance with the helsinki declaration , and all patients signed a consent form approved by the research ethics committee of the sun yat - sen university cancer center . paraffin - embedded tissues were sectioned continuously with a thickness of 4 m and baked for 1 h at 65 . briefly , the sections were de - paraffinized using xylene and then rehydrated with graded alcohol to distilled water . the sections were immersed in edta antigen retrieval buffer ( ph 8.0 ) , placed under high pressure for 3 minutes for antigen retrieval , and then allowed to cool to room temperature . after blocking with sheep serum , the sections were incubated overnight at 4 with either a rabbit polyclonal antibody against human hmgb1 at a dilution of 1:1,000 ( abcam , cambridge , ma , usa ) or a mouse monoclonal antibody against human cluster of differentiation 8 ( cd8 ) and cd20 ( zymed , san diego , ca , usa ) , all of which were diluted 1:400 . following incubation with the secondary antibodies , the sections were developed using diaminobenzidine tetrahydrochloride and counterstained with hematoxylin . co - expression of cd33 and phospho - signal transducers and activators of transcription ( p - stat1 ) were detected by sequential , double - immunohistochemical staining using the double - staining en vison g/2 doublestain system ( dakocytomation , glostrup , denmark ) , according to the manufacturer 's instructions and our previous report ( 6 ) . endogenous peroxidases and alkaline phosphatase enzymes were blocked with the dual endogenous enzyme blocking reagent provided in the double - stain kit ; the sections were treated with normal goat serum for 20 min to reduce nonspecific binding , and incubated overnight at 4 with rabbit polyclonal anti - cd33 antibody ( 1:100 ; protein tech group , chicago , usa ) and rabbit monoclonal anti - p - stat1 ( 1:400 ; cell signaling , boston , usa ) . staining was visualized with diaminobenzidine ( brown ) and permanent red ( red ) . as a negative control , the density of immune cells within the tumor specimens and the expression of hmgb1 in cancer cells were scored according to our previous report ( 7 ) . rui - qing peng 's method ( 7 ) was used to score the density of tils ( tumor - infiltrating lymphocytes ) as follows : ( i ) immune cells were counted in at least ten different fields of each section , the areas of highest density were chosen before cell counting ; ( ii ) the cells were counted in the intratumoral compartment ( within the tumor cell nests ) ; ( iii ) necrotic areas were excluded ; ( iv ) two observers counted the cells at the same time , in the same field , using a multiple - lens microscope ; ( v ) the results were expressed as the mean standard error of the mean . the expression of hmgb1 was interpreted via immunoreactivity using the 0 - 4 semi - quantitative scoring systems for both the intensity of staining and the percentage of positive cells ( labeling frequency percentage ) ( 7 ) . the intensity of staining was grouped into the following four categories : no staining / background of negative controls ( score = 0 ) , weak staining detectable above background ( score = 1 ) , moderate staining ( score = 2 ) , and intense staining ( score = 3 ) . the labeling frequency was scored as follows : 0 ( 1% ) , 1 ( 1 - 24% ) , 2 ( 25 - 49% ) , 3 ( 50 - 74% ) , and 4 ( 75% ) . the sum index was obtained by totaling the intensity and percentage scores , as follows : ( - ) , ( + ) , ( + + ) , and ( + + + ) indicated the sum - indices of 0 - 1 , 2 - 3 , 4 - 5 , and 6 - 7 , respectively ; ( - ) and ( + ) were defined as no or modest expression , respectively , and ( + + ) and ( + + + ) were defined as strong expression . if there was an inconsistency in the scoring , a third pathologist was consulted to achieve a consensus . the median value was used as a cut off between the different groups of all immunohistochemical variables in our results . the wilcoxon - mann - whitney test was used to identify markers with significantly different expression among patient groups . the chi - squared test was used to analyze the relationship between hmgb1 expression , cd33p - stat1 expression , and clinicopathological characteristics . the os was defined as death from any cause , and dfs was defined as the time prior to relapse of the primary tumor . survival curves were calculated using the kaplan - meier method and analyzed by the log - rank test . the multivariate cox proportional hazards regression model was applied to analyze hazard ratios ; a two - sided p<0.05 was considered statistically significant . between 2003 and 2006 , 100 samples of pathologically confirmed cancer tissue were obtained from patients with stage iiia ( 2009 ajcc / uicc staging system ) gastric cancer at the sun yat - sen university cancer center . this study was conducted in accordance with the helsinki declaration , and all patients signed a consent form approved by the research ethics committee of the sun yat - sen university cancer center . paraffin - embedded tissues were sectioned continuously with a thickness of 4 m and baked for 1 h at 65 . briefly , the sections were de - paraffinized using xylene and then rehydrated with graded alcohol to distilled water . the sections were immersed in edta antigen retrieval buffer ( ph 8.0 ) , placed under high pressure for 3 minutes for antigen retrieval , and then allowed to cool to room temperature . after blocking with sheep serum , the sections were incubated overnight at 4 with either a rabbit polyclonal antibody against human hmgb1 at a dilution of 1:1,000 ( abcam , cambridge , ma , usa ) or a mouse monoclonal antibody against human cluster of differentiation 8 ( cd8 ) and cd20 ( zymed , san diego , ca , usa ) , all of which were diluted 1:400 . following incubation with the secondary antibodies , the sections were developed using diaminobenzidine tetrahydrochloride and counterstained with hematoxylin . co - expression of cd33 and phospho - signal transducers and activators of transcription ( p - stat1 ) were detected by sequential , double - immunohistochemical staining using the double - staining en vison g/2 doublestain system ( dakocytomation , glostrup , denmark ) , according to the manufacturer 's instructions and our previous report ( 6 ) . endogenous peroxidases and alkaline phosphatase enzymes were blocked with the dual endogenous enzyme blocking reagent provided in the double - stain kit ; the sections were treated with normal goat serum for 20 min to reduce nonspecific binding , and incubated overnight at 4 with rabbit polyclonal anti - cd33 antibody ( 1:100 ; protein tech group , chicago , usa ) and rabbit monoclonal anti - p - stat1 ( 1:400 ; cell signaling , boston , usa ) . staining was visualized with diaminobenzidine ( brown ) and permanent red ( red ) . as a negative control , the density of immune cells within the tumor specimens and the expression of hmgb1 in cancer cells were scored according to our previous report ( 7 ) . rui - qing peng 's method ( 7 ) was used to score the density of tils ( tumor - infiltrating lymphocytes ) as follows : ( i ) immune cells were counted in at least ten different fields of each section , the areas of highest density were chosen before cell counting ; ( ii ) the cells were counted in the intratumoral compartment ( within the tumor cell nests ) ; ( iii ) necrotic areas were excluded ; ( iv ) two observers counted the cells at the same time , in the same field , using a multiple - lens microscope ; ( v ) the results were expressed as the mean standard error of the mean . the expression of hmgb1 was interpreted via immunoreactivity using the 0 - 4 semi - quantitative scoring systems for both the intensity of staining and the percentage of positive cells ( labeling frequency percentage ) ( 7 ) . the intensity of staining was grouped into the following four categories : no staining / background of negative controls ( score = 0 ) , weak staining detectable above background ( score = 1 ) , moderate staining ( score = 2 ) , and intense staining ( score = 3 ) . the labeling frequency was scored as follows : 0 ( 1% ) , 1 ( 1 - 24% ) , 2 ( 25 - 49% ) , 3 ( 50 - 74% ) , and 4 ( 75% ) . the sum index was obtained by totaling the intensity and percentage scores , as follows : ( - ) , ( + ) , ( + + ) , and ( + + + ) indicated the sum - indices of 0 - 1 , 2 - 3 , 4 - 5 , and 6 - 7 , respectively ; ( - ) and ( + ) were defined as no or modest expression , respectively , and ( + + ) and ( + + + ) were defined as strong expression . if there was an inconsistency in the scoring , a third pathologist was consulted to achieve a consensus . the median value was used as a cut off between the different groups of all immunohistochemical variables in our results . the wilcoxon - mann - whitney test was used to identify markers with significantly different expression among patient groups . the chi - squared test was used to analyze the relationship between hmgb1 expression , cd33p - stat1 expression , and clinicopathological characteristics . the os was defined as death from any cause , and dfs was defined as the time prior to relapse of the primary tumor . survival curves were calculated using the kaplan - meier method and analyzed by the log - rank test . the multivariate cox proportional hazards regression model was applied to analyze hazard ratios ; a two - sided p<0.05 was considered statistically significant . among 100 patients , there were 72 men and 28 women , and the median age was 59.5 years ( range from 29 to 82 years ) . based on the borrmann classification , 55 patients ( 55% ) had type ii lesions , 38 had type iii , and only7 had type i and iv lesions . all the patients presented with lymph node metastasis before treatment , 36 patients had n1 stage , and 34 and 30 patients had n2 and n3 stages , respectively . based on the national comprehensive cancer network guidelines , 5-fluorouracil - based adjuvant therapy was administered . of the total number of patients , 54 had died and 44 presented with disease progression during the follow - up . strong membrane staining was observed for cd8 and cd20 lymphoid cells ( figure 1a - d ) . moreover , the hmgb1 showed both membrane and nuclear staining within the tumor cells ( figure 1e , f ) . to characterize mdsc infiltration in gastric cancer tissue , we defined mdscs as cd33/p - stat1 double - positive staining cells ( 6 ) . these cd33/p - stat1 double - positive cells have been found in most gastric cancer tissue but not in the non - tumorous stomach . immunostaining revealed cytomembrane staining for cd33 , and nuclear staining for p - stat1 ( figure 2 ) . cd33/p - stat1 double - positive cells were observed in a subset of cells around the tumor nests . ( a , b ) cd8 t lymphocytes in the gastric cancer tissue ( a , 200 ; b , 400 ) ; ( c , d ) cd20 b lymphocytes in the gastric cancer tissue ( c , 200 ; d , 400 ) ; ( e , f ) the expression of hmgb1 in the gastric cancer tissue ( e , 200 ; f , 400 ) . cd8 , cluster of differentiation 8 ; hmgb1 , high - mobility group protein b1 . ( a , b ) double - stained with cd33 ( red ) and p - stat1 ( brown ) cells in gastric cancer tissue ( a , 400 ; b , 1,000 ) ; ( c , d ) cd33 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( c , 400 ; d , 1,000 ) ; ( e , f ) p - stat1 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( e , 400 ; f , 1,000 ) . the median follow - up time for the 100 cases was 36.5 months , with a range from 2 to 88 months . at the completion of the study , 47 patients were alive , and 53 patients had died . fifty - one deaths were cancer - related , and two deaths were due to causes unrelated to cancer . the estimated 5-year survival and 5-year recurrent free survival rates were 32% and 28% , respectively . univariate analysis showed that the age and immunological parameters were statistically significant prognostic factors for os and dfs ( table 1 ) . however , clinical prognosis was not associated with sex , tumor location , borrmann classification , tumor size and the expression of estrogen receptor ( er ) , progesterone receptor ( pr ) , p53 and carcinoembryonic antigen ( cea ) in tumor tissue . univariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters to quantify the risk of relapse in individual patients , we calculated the " i m " based on the density of cd8 , cd20 , cd33/p - stat1 and the expression of hmgb1 . a score1 was computed on the basis of lymphocyte infiltration ( cd8 t lymphocytes and cd20 b lymphocytes ) ( 18 ) . patients with two low ( 2-lo ) densities of cd8 and cd20 in the intratumoral compartment were classified as having a score of 0 . patients with one high ( 1-hi ) density for one marker were classified as having a score of 1 . patients with two high ( 2-hi ) densities for the markers were classified as having a score of 2 . in contrast to the density of cd8 t cells and cd20 b lymphocytes , the density of cd33/p - stat1 cells and the expression of hmgb1 in cancer cells were found to negatively influence the os and dfs ( table 1 ) . therefore , a score 2 was inversely assigned based on the density of cd33/p - stat1 cells and the expression of hmgb1 . patients with a 2-hi expression of cd33/p - stat1 and hmgb1 [ ( + + ) and ( + + + ) ] in the intratumoral compartment were classified as having a score of 0 . patients with a 1-hi expression for one marker were classified as having a score of 1 . patients with a 2-lo expression for the marker were classified as having a score of 0 . the i m , which ranged from im0 to im4 , was calculated by combining score 1 with score 2 ( figure 3 ) . a patient with 2-hi densities of cd8 and cd20 and with 2-lo expression of cd33/p - stat1 and hmgb1 in contrast , a patient with 2-lo densities of cd8 and with 2-hi expression of cd33/p - stat1 and hmgb1 was assigned a score of im0 . according to these criteria , 21% , 17% , 13% , 29% , and 20% of patients were classified into the subgroups of im0 , im1 , im2 , im3 , and im4 , respectively . next , we evaluated whether the i m was associated with patient prognosis . as shown in figure 4a , the os durations were significantly different among the i m groups ( p<0.001 ) . the cumulative 5-year survival rates were 70.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the survival rates were only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . kaplan - meier analysis of the disease - free survival ( a ) and overall survival rates ( b ) corresponding to each immune score ( i m ) group . a higher i m ( im3 or im4 ) was correlated with longer overall survival and disease - free survival than a lower i m ( im0 , im1 , or im2 ) . similarly , there were significant differences in the dfs durations among the i m subgroups , as shown in figure 4b ; the median dfs time was 33 months in this group of patients , with a range of 1 - 88 months . the cumulative 5-year dfs was 75% and 44.8% among the patients with im4 and im3 , respectively . conversely , all the patients belonging to the im0 , im1 , and im2 subgroups showed a 5-year dfs rate of 0% . the cox multivariate regression analysis was performed by entering the density of cd8 , cd20 , and cd33/p - stat1 cells , the expression of hmgb1 , and i m into a model . only the i m remained significantly associated with os and dfs ( table 2 ) . in addition , we built a final model combining the i m with the n stage , age , sex , tumor location , borrmann classification , tumor size , and the expression of the er , pr , and p53 , as well as immunological parameters . this model showed that only the i m remained significantly associated with os and dfs ( table 2 ) . in addition , the i m remained significantly correlated with os and dfs upon multivariate analysis , whereas other clinical parameters were not significant . multivariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters among 100 patients , there were 72 men and 28 women , and the median age was 59.5 years ( range from 29 to 82 years ) . based on the borrmann classification , 55 patients ( 55% ) had type ii lesions , 38 had type iii , and only7 had type i and iv lesions . all the patients presented with lymph node metastasis before treatment , 36 patients had n1 stage , and 34 and 30 patients had n2 and n3 stages , respectively . based on the national comprehensive cancer network guidelines , 5-fluorouracil - based adjuvant therapy was administered . of the total number of patients , strong membrane staining was observed for cd8 and cd20 lymphoid cells ( figure 1a - d ) . the infiltrating lymphoid cells in the intratumoral compartment moreover , the hmgb1 showed both membrane and nuclear staining within the tumor cells ( figure 1e , f ) . to characterize mdsc infiltration in gastric cancer tissue , we defined mdscs as cd33/p - stat1 double - positive staining cells ( 6 ) . these cd33/p - stat1 double - positive cells have been found in most gastric cancer tissue but not in the non - tumorous stomach . immunostaining revealed cytomembrane staining for cd33 , and nuclear staining for p - stat1 ( figure 2 ) . cd33/p - stat1 double - positive cells were observed in a subset of cells around the tumor nests . ( a , b ) cd8 t lymphocytes in the gastric cancer tissue ( a , 200 ; b , 400 ) ; ( c , d ) cd20 b lymphocytes in the gastric cancer tissue ( c , 200 ; d , 400 ) ; ( e , f ) the expression of hmgb1 in the gastric cancer tissue ( e , 200 ; f , 400 ) . cd8 , cluster of differentiation 8 ; hmgb1 , high - mobility group protein b1 . ( a , b ) double - stained with cd33 ( red ) and p - stat1 ( brown ) cells in gastric cancer tissue ( a , 400 ; b , 1,000 ) ; ( c , d ) cd33 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( c , 400 ; d , 1,000 ) ; ( e , f ) p - stat1 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( e , 400 ; f , 1,000 ) . the median follow - up time for the 100 cases was 36.5 months , with a range from 2 to 88 months . at the completion of the study , 47 patients were alive , and 53 patients had died . fifty - one deaths were cancer - related , and two deaths were due to causes unrelated to cancer . the estimated 5-year survival and 5-year recurrent free survival rates were 32% and 28% , respectively . univariate analysis showed that the age and immunological parameters were statistically significant prognostic factors for os and dfs ( table 1 ) . however , clinical prognosis was not associated with sex , tumor location , borrmann classification , tumor size and the expression of estrogen receptor ( er ) , progesterone receptor ( pr ) , p53 and carcinoembryonic antigen ( cea ) in tumor tissue . univariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters to quantify the risk of relapse in individual patients , we calculated the " i m " based on the density of cd8 , cd20 , cd33/p - stat1 and the expression of hmgb1 . a score1 was computed on the basis of lymphocyte infiltration ( cd8 t lymphocytes and cd20 b lymphocytes ) ( 18 ) . patients with two low ( 2-lo ) densities of cd8 and cd20 in the intratumoral compartment were classified as having a score of 0 . patients with one high ( 1-hi ) density for one marker were classified as having a score of 1 . patients with two high ( 2-hi ) densities for the markers were classified as having a score of 2 . in contrast to the density of cd8 t cells and cd20 b lymphocytes , the density of cd33/p - stat1 cells and the expression of hmgb1 in cancer cells were found to negatively influence the os and dfs ( table 1 ) . therefore , a score 2 was inversely assigned based on the density of cd33/p - stat1 cells and the expression of hmgb1 . patients with a 2-hi expression of cd33/p - stat1 and hmgb1 [ ( + + ) and ( + + + ) ] in the intratumoral compartment were classified as having a score of 0 . patients with a 1-hi expression for one marker were classified as having a score of 1 . patients with a 2-lo expression for the marker were classified as having a score of 0 . the i m , which ranged from im0 to im4 , was calculated by combining score 1 with score 2 ( figure 3 ) . a patient with 2-hi densities of cd8 and cd20 and with 2-lo expression of cd33/p - stat1 and hmgb1 in contrast , a patient with 2-lo densities of cd8 and with 2-hi expression of cd33/p - stat1 and hmgb1 was assigned a score of im0 . according to these criteria , 21% , 17% , 13% , 29% , and 20% of patients were classified into the subgroups of im0 , im1 , im2 , im3 , and im4 , respectively . next , we evaluated whether the i m was associated with patient prognosis . as shown in figure 4a , the os durations were significantly different among the i m groups ( p<0.001 ) . the cumulative 5-year survival rates were 70.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the survival rates were only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . kaplan - meier analysis of the disease - free survival ( a ) and overall survival rates ( b ) corresponding to each immune score ( i m ) group . a higher i m ( im3 or im4 ) was correlated with longer overall survival and disease - free survival than a lower i m ( im0 , im1 , or im2 ) . similarly , there were significant differences in the dfs durations among the i m subgroups , as shown in figure 4b ; the median dfs time was 33 months in this group of patients , with a range of 1 - 88 months . the cumulative 5-year dfs was 75% and 44.8% among the patients with im4 and im3 , respectively . conversely , all the patients belonging to the im0 , im1 , and im2 subgroups showed a 5-year dfs rate of 0% . the cox multivariate regression analysis was performed by entering the density of cd8 , cd20 , and cd33/p - stat1 cells , the expression of hmgb1 , and i m into a model . only the i m remained significantly associated with os and dfs ( table 2 ) . in addition , we built a final model combining the i m with the n stage , age , sex , tumor location , borrmann classification , tumor size , and the expression of the er , pr , and p53 , as well as immunological parameters . this model showed that only the i m remained significantly associated with os and dfs ( table 2 ) . in addition , the i m remained significantly correlated with os and dfs upon multivariate analysis , whereas other clinical parameters were not significant . multivariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters we observed that the os and dfs of stage iiia gastric cancer patients were significantly correlated with the i m . a lower i m was associated with poorer clinical outcomes , implicating that the i m system was a useful prognostic tool for gastric cancer patients , supplementing the tnm staging system . t cells mediated adaptive immunity play a major role in antitumor immunity ( 19,20 ) . most study showed that high densities of cytotoxic t cells and memory t cells are associated with the favorable prognosis in gastric cancer ( 21,22 ) . cd8 t cell are the main effector cells , and the cd4 t cell also can induce and activate cd8 t cell in tumor microenvironment . previous studies have focused on the role of cd8 and cd45ro + t cells in an i m system . the rationale for compiling t cells , b cells , mdscs , and hmgb1expression into this i m system was based on the observation that the immune response , which includes both cellular and humoral immunity , is essential in controlling cancer progression ; thus , both the tumor - inhibitory and tumor - promotional factors were compiled into this i m system . in general , among the immune cell subtypes , cd8 t cells , cd45ro + t cells , and th1 cells show anti - cancerous potential . conversely , the th2 , th17 , and treg cells exert a more complicated influence , depending on the tumor types ( 4,10,15,23 ) . furthermore , in contrast to the b cells present in draining lymph nodes , the cd20 til cells represent anti - cancer immunity for melanomas , ovarian , breast , and head and neck cancers ( 24 - 27 ) . in this study , the densities of both cd8 t cells and cd20 b cells were associated with a better prognosis of gastric cancer , and were represented as positive i m scores . several other subtypes of immune cells , such as the mdscs and tumor - associated macrophages ( tams ) , are connected with the tumor progression . the mdscs originate from myeloid cells , which are transformed into potent immunosuppressive cells upon being recruited to the tumor microenvironment . circulating mdscs have a negative prognostic role in multiple solid tumors ( 28 - 30 ) . although mdscs subtypes are heterogeneous , we have previously identified the cd33 and p - stat1 double - positive cells as a specific type of mdscs ; we observed that the density of cd33 and p - stat1 double - positive cells was associated with a poor prognosis of gastric cancer ( 6 ) . as tams are known to be associated with mdscs , and mdscs are responsible for amplifying the immunosuppressive activity of macrophages , mdscs were compiled into this scoring system instead of tams as the representatives of tumor - promotional immunity ( 31,32 ) . with the exception of immune cells , damage - associated molecular patterns ( damps ) are known to participate during the prime phase of the immune response . some damps are actively secreted by cells undergoing immunogenic cell death ( e.g. , calreticulin and adenosine triphosphate ) , whereas others are emitted passively ( e.g. , hmgb1 ) . the same damps may contribute to both , the inhibition or progression of cancer ( 33 ) . as the immune staining for hmgb1 exhibited more reproducibility than the staining for calreticulin and adenosine triphosphate , hmgb1 expression was selected as a represent active immunological modulator in order to enhance the prognostic potential of immune cells including t cells , b cells , and mdscs . the overexpression of hmgb1 was observed in tumor cells derived from colon , breast , lung , cervical , hepatocellular , and gastric cancers . higher levels of hmgb1 have been associated with greater tumor angiogenesis , growth , invasion , metastasis , and immunosuppressive activity ( 34,35 ) . in this study , the expression of hmgb1 was inversely associated with the prognosis , and was characterized by negative i m scores . using the tnm staging system , gastric cancer patients with iiia stage have an overall 5-year survival rate of 19.8% ( 36 ) . in this study , although the 5-year survival rate was 32% , the os time varied significantly among the i m subgroups . according to the immune score system , the patients can be divided into two groups , the high and low immune score group . the high immune score group include im3 and im4 , and low immune score group include im0 , im1 and im2 . the os and dfs time varied significantly between high group and low group , especially the im0 and im4 . the cumulative 5-year survival rate was 80.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the 5-year survival rate was only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . the cumulative 5-year dfs was 75% , and 44.8% among the patients with im4 and im3 , respectively . however , patients belonging to the im0 , im1 , and im2 subgroups showed a 5-year dfs rate of 0% . these data imply that the clinical outcome was heterogeneous , even within the same tnm stage . this type of heterogeneity should have a considerable influence when the role of adjuvant therapy is assayed . this i m system might be beneficial while selecting patients for adjuvant therapy , especially for adjuvant immunotherapy . this study has the following limitations : first , it included only 100 patients with stage iiia cancer ; a larger number of patients are needed for validating these observations . second , the selection of the hot spots of immune cell infiltration was arbitrary ; therefore , a digital method might improve the reproducibility . in conclusion , this study proved the existence of prognostic heterogeneity among stage iiia gastric patients ; an i m system was designed that might be helpful in identifying such types of heterogeneity .

objective there is heterogeneity in the prognosis of gastric cancers staged according to the tumornodes- metastasis ( tnm ) system . this study evaluated the prognostic potential of an immune score system to supplement the tnm staging system . methods an immunohistochemical analysis was conducted to assess the density of t cells , b cells , and myeloid - derived suppressor cells ( mdscs ) in cancer tissues from 100 stage iiia gastric cancer patients ; the expression of the high - mobility group protein b1 ( hmgb1 ) was also evaluated in cancer cells . the relationship between the overall survival ( os ) , disease - free survival ( dfs ) , and immunological parameters was analyzed . results an immune score system was compiled based on the prognostic role of the density of t cells , b cells , mdscs , and the expression of hmgb1 in cancer tissues . the median 5-year survival of this group of patient was 32% . however , the 5-year survival rates of 80.0% , 51.7% , 0% , 5.8% , and 0% varied among the patients with an immune score of 4 to those with an immune score of 0 based on the immune score system , respectively . similarly , differences in dfs rates were observed among the immune score subgroups . conclusions an immune score system could effectively identify the prognostic heterogeneity within stage iiia gastric cancer patients , implying that this immune score system may potentially supplement the tnm staging system , and help in identifying a more homogeneous group of patients who on the basis of prognosis can undergo adjuvant therapy .

Introduction Materials and Methods Tissue specimens Immunohistochemistry and scoring systems Statistical analysis Results Patient characteristics The density of CD8 Univariate analyses of the relationship between OS and DFS, and clinical and immunological parameters among stage IIIA gastric cancer patients Compilation of the Im Relationship between the Im and patient survival and recurrence Discussion Conclusions

the american joint committee on cancer / union for international cancer control ( ajcc / uicc ) tumor - nodes - metastasis ( tnm ) staging system provides the most reliable guidelines for the prognostication and treatment of carcinomas . in contrast , relapse , rapid tumor progression , and patient death occur in approximately 10 - 25% of patients with tnm i / ii stage cancers , despite performing complete surgical resection and even though there is no evidence of residual tumor burden or distant metastasis ( 2,3 ) . the tnm system is based on the tumors ' biological behaviors , without considering host responses . rather than considering a change in the expression of a single immunological marker , the change in a combined index or the immune score ( i m ) would better represent the immune response . the i m system , which provides an i m ranging from 0 to 4 ( im0 to im4 ) , was initially suggested and developed for colorectal cancers ( 8,9 ) , and is based on the enumeration of two lymphocyte populations ( cd3/cd45ro , cd3/cd8 ) , both in the core of the tumor and in the invasive margin of tumors . the i m appears to be the strongest prognostic factor for disease - free survival ( dfs ) , disease specic survival , and overall survival ( os ) in colorectal cancers ( 10 - 13 ) . the i m system included t cells , b cells , and myeloid - derived suppressor cells ( mdscs ) , and high - mobility group protein b1 ( hmgb1 ) expression profile , to assay the prognostic role of the immune response in gastric cancers . between 2003 and 2006 , 100 samples of pathologically confirmed cancer tissue were obtained from patients with stage iiia ( 2009 ajcc / uicc staging system ) gastric cancer at the sun yat - sen university cancer center . this study was conducted in accordance with the helsinki declaration , and all patients signed a consent form approved by the research ethics committee of the sun yat - sen university cancer center . co - expression of cd33 and phospho - signal transducers and activators of transcription ( p - stat1 ) were detected by sequential , double - immunohistochemical staining using the double - staining en vison g/2 doublestain system ( dakocytomation , glostrup , denmark ) , according to the manufacturer 's instructions and our previous report ( 6 ) . as a negative control , the density of immune cells within the tumor specimens and the expression of hmgb1 in cancer cells were scored according to our previous report ( 7 ) . rui - qing peng 's method ( 7 ) was used to score the density of tils ( tumor - infiltrating lymphocytes ) as follows : ( i ) immune cells were counted in at least ten different fields of each section , the areas of highest density were chosen before cell counting ; ( ii ) the cells were counted in the intratumoral compartment ( within the tumor cell nests ) ; ( iii ) necrotic areas were excluded ; ( iv ) two observers counted the cells at the same time , in the same field , using a multiple - lens microscope ; ( v ) the results were expressed as the mean standard error of the mean . the expression of hmgb1 was interpreted via immunoreactivity using the 0 - 4 semi - quantitative scoring systems for both the intensity of staining and the percentage of positive cells ( labeling frequency percentage ) ( 7 ) . the sum index was obtained by totaling the intensity and percentage scores , as follows : ( - ) , ( + ) , ( + + ) , and ( + + + ) indicated the sum - indices of 0 - 1 , 2 - 3 , 4 - 5 , and 6 - 7 , respectively ; ( - ) and ( + ) were defined as no or modest expression , respectively , and ( + + ) and ( + + + ) were defined as strong expression . between 2003 and 2006 , 100 samples of pathologically confirmed cancer tissue were obtained from patients with stage iiia ( 2009 ajcc / uicc staging system ) gastric cancer at the sun yat - sen university cancer center . this study was conducted in accordance with the helsinki declaration , and all patients signed a consent form approved by the research ethics committee of the sun yat - sen university cancer center . co - expression of cd33 and phospho - signal transducers and activators of transcription ( p - stat1 ) were detected by sequential , double - immunohistochemical staining using the double - staining en vison g/2 doublestain system ( dakocytomation , glostrup , denmark ) , according to the manufacturer 's instructions and our previous report ( 6 ) . as a negative control , the density of immune cells within the tumor specimens and the expression of hmgb1 in cancer cells were scored according to our previous report ( 7 ) . rui - qing peng 's method ( 7 ) was used to score the density of tils ( tumor - infiltrating lymphocytes ) as follows : ( i ) immune cells were counted in at least ten different fields of each section , the areas of highest density were chosen before cell counting ; ( ii ) the cells were counted in the intratumoral compartment ( within the tumor cell nests ) ; ( iii ) necrotic areas were excluded ; ( iv ) two observers counted the cells at the same time , in the same field , using a multiple - lens microscope ; ( v ) the results were expressed as the mean standard error of the mean . the expression of hmgb1 was interpreted via immunoreactivity using the 0 - 4 semi - quantitative scoring systems for both the intensity of staining and the percentage of positive cells ( labeling frequency percentage ) ( 7 ) . the sum index was obtained by totaling the intensity and percentage scores , as follows : ( - ) , ( + ) , ( + + ) , and ( + + + ) indicated the sum - indices of 0 - 1 , 2 - 3 , 4 - 5 , and 6 - 7 , respectively ; ( - ) and ( + ) were defined as no or modest expression , respectively , and ( + + ) and ( + + + ) were defined as strong expression . among 100 patients , there were 72 men and 28 women , and the median age was 59.5 years ( range from 29 to 82 years ) . all the patients presented with lymph node metastasis before treatment , 36 patients had n1 stage , and 34 and 30 patients had n2 and n3 stages , respectively . of the total number of patients , 54 had died and 44 presented with disease progression during the follow - up . ( a , b ) cd8 t lymphocytes in the gastric cancer tissue ( a , 200 ; b , 400 ) ; ( c , d ) cd20 b lymphocytes in the gastric cancer tissue ( c , 200 ; d , 400 ) ; ( e , f ) the expression of hmgb1 in the gastric cancer tissue ( e , 200 ; f , 400 ) . cd8 , cluster of differentiation 8 ; hmgb1 , high - mobility group protein b1 . ( a , b ) double - stained with cd33 ( red ) and p - stat1 ( brown ) cells in gastric cancer tissue ( a , 400 ; b , 1,000 ) ; ( c , d ) cd33 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( c , 400 ; d , 1,000 ) ; ( e , f ) p - stat1 single - stained cells in parallel with cd33/p - stat1 double - positive cells ( e , 400 ; f , 1,000 ) . the estimated 5-year survival and 5-year recurrent free survival rates were 32% and 28% , respectively . however , clinical prognosis was not associated with sex , tumor location , borrmann classification , tumor size and the expression of estrogen receptor ( er ) , progesterone receptor ( pr ) , p53 and carcinoembryonic antigen ( cea ) in tumor tissue . univariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters to quantify the risk of relapse in individual patients , we calculated the " i m " based on the density of cd8 , cd20 , cd33/p - stat1 and the expression of hmgb1 . patients with two low ( 2-lo ) densities of cd8 and cd20 in the intratumoral compartment were classified as having a score of 0 . in contrast to the density of cd8 t cells and cd20 b lymphocytes , the density of cd33/p - stat1 cells and the expression of hmgb1 in cancer cells were found to negatively influence the os and dfs ( table 1 ) . therefore , a score 2 was inversely assigned based on the density of cd33/p - stat1 cells and the expression of hmgb1 . patients with a 2-hi expression of cd33/p - stat1 and hmgb1 [ ( + + ) and ( + + + ) ] in the intratumoral compartment were classified as having a score of 0 . according to these criteria , 21% , 17% , 13% , 29% , and 20% of patients were classified into the subgroups of im0 , im1 , im2 , im3 , and im4 , respectively . the cumulative 5-year survival rates were 70.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the survival rates were only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . kaplan - meier analysis of the disease - free survival ( a ) and overall survival rates ( b ) corresponding to each immune score ( i m ) group . a higher i m ( im3 or im4 ) was correlated with longer overall survival and disease - free survival than a lower i m ( im0 , im1 , or im2 ) . similarly , there were significant differences in the dfs durations among the i m subgroups , as shown in figure 4b ; the median dfs time was 33 months in this group of patients , with a range of 1 - 88 months . the cumulative 5-year dfs was 75% and 44.8% among the patients with im4 and im3 , respectively . conversely , all the patients belonging to the im0 , im1 , and im2 subgroups showed a 5-year dfs rate of 0% . the cox multivariate regression analysis was performed by entering the density of cd8 , cd20 , and cd33/p - stat1 cells , the expression of hmgb1 , and i m into a model . in addition , we built a final model combining the i m with the n stage , age , sex , tumor location , borrmann classification , tumor size , and the expression of the er , pr , and p53 , as well as immunological parameters . multivariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters among 100 patients , there were 72 men and 28 women , and the median age was 59.5 years ( range from 29 to 82 years ) . of the total number of patients , strong membrane staining was observed for cd8 and cd20 lymphoid cells ( figure 1a - d ) . the infiltrating lymphoid cells in the intratumoral compartment moreover , the hmgb1 showed both membrane and nuclear staining within the tumor cells ( figure 1e , f ) . ( a , b ) cd8 t lymphocytes in the gastric cancer tissue ( a , 200 ; b , 400 ) ; ( c , d ) cd20 b lymphocytes in the gastric cancer tissue ( c , 200 ; d , 400 ) ; ( e , f ) the expression of hmgb1 in the gastric cancer tissue ( e , 200 ; f , 400 ) . cd8 , cluster of differentiation 8 ; hmgb1 , high - mobility group protein b1 . the estimated 5-year survival and 5-year recurrent free survival rates were 32% and 28% , respectively . however , clinical prognosis was not associated with sex , tumor location , borrmann classification , tumor size and the expression of estrogen receptor ( er ) , progesterone receptor ( pr ) , p53 and carcinoembryonic antigen ( cea ) in tumor tissue . univariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters to quantify the risk of relapse in individual patients , we calculated the " i m " based on the density of cd8 , cd20 , cd33/p - stat1 and the expression of hmgb1 . patients with two low ( 2-lo ) densities of cd8 and cd20 in the intratumoral compartment were classified as having a score of 0 . in contrast to the density of cd8 t cells and cd20 b lymphocytes , the density of cd33/p - stat1 cells and the expression of hmgb1 in cancer cells were found to negatively influence the os and dfs ( table 1 ) . therefore , a score 2 was inversely assigned based on the density of cd33/p - stat1 cells and the expression of hmgb1 . patients with a 2-hi expression of cd33/p - stat1 and hmgb1 [ ( + + ) and ( + + + ) ] in the intratumoral compartment were classified as having a score of 0 . patients with a 2-lo expression for the marker were classified as having a score of 0 . according to these criteria , 21% , 17% , 13% , 29% , and 20% of patients were classified into the subgroups of im0 , im1 , im2 , im3 , and im4 , respectively . the cumulative 5-year survival rates were 70.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the survival rates were only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . kaplan - meier analysis of the disease - free survival ( a ) and overall survival rates ( b ) corresponding to each immune score ( i m ) group . a higher i m ( im3 or im4 ) was correlated with longer overall survival and disease - free survival than a lower i m ( im0 , im1 , or im2 ) . similarly , there were significant differences in the dfs durations among the i m subgroups , as shown in figure 4b ; the median dfs time was 33 months in this group of patients , with a range of 1 - 88 months . the cumulative 5-year dfs was 75% and 44.8% among the patients with im4 and im3 , respectively . conversely , all the patients belonging to the im0 , im1 , and im2 subgroups showed a 5-year dfs rate of 0% . the cox multivariate regression analysis was performed by entering the density of cd8 , cd20 , and cd33/p - stat1 cells , the expression of hmgb1 , and i m into a model . in addition , we built a final model combining the i m with the n stage , age , sex , tumor location , borrmann classification , tumor size , and the expression of the er , pr , and p53 , as well as immunological parameters . multivariate analyses of os and dfs among patients with uicc - tnm stage iiia gastric cancer according to clinical and immune parameters we observed that the os and dfs of stage iiia gastric cancer patients were significantly correlated with the i m . a lower i m was associated with poorer clinical outcomes , implicating that the i m system was a useful prognostic tool for gastric cancer patients , supplementing the tnm staging system . the rationale for compiling t cells , b cells , mdscs , and hmgb1expression into this i m system was based on the observation that the immune response , which includes both cellular and humoral immunity , is essential in controlling cancer progression ; thus , both the tumor - inhibitory and tumor - promotional factors were compiled into this i m system . in general , among the immune cell subtypes , cd8 t cells , cd45ro + t cells , and th1 cells show anti - cancerous potential . conversely , the th2 , th17 , and treg cells exert a more complicated influence , depending on the tumor types ( 4,10,15,23 ) . furthermore , in contrast to the b cells present in draining lymph nodes , the cd20 til cells represent anti - cancer immunity for melanomas , ovarian , breast , and head and neck cancers ( 24 - 27 ) . in this study , the densities of both cd8 t cells and cd20 b cells were associated with a better prognosis of gastric cancer , and were represented as positive i m scores . although mdscs subtypes are heterogeneous , we have previously identified the cd33 and p - stat1 double - positive cells as a specific type of mdscs ; we observed that the density of cd33 and p - stat1 double - positive cells was associated with a poor prognosis of gastric cancer ( 6 ) . as tams are known to be associated with mdscs , and mdscs are responsible for amplifying the immunosuppressive activity of macrophages , mdscs were compiled into this scoring system instead of tams as the representatives of tumor - promotional immunity ( 31,32 ) . as the immune staining for hmgb1 exhibited more reproducibility than the staining for calreticulin and adenosine triphosphate , hmgb1 expression was selected as a represent active immunological modulator in order to enhance the prognostic potential of immune cells including t cells , b cells , and mdscs . the overexpression of hmgb1 was observed in tumor cells derived from colon , breast , lung , cervical , hepatocellular , and gastric cancers . in this study , the expression of hmgb1 was inversely associated with the prognosis , and was characterized by negative i m scores . using the tnm staging system , gastric cancer patients with iiia stage have an overall 5-year survival rate of 19.8% ( 36 ) . in this study , although the 5-year survival rate was 32% , the os time varied significantly among the i m subgroups . according to the immune score system , the patients can be divided into two groups , the high and low immune score group . the cumulative 5-year survival rate was 80.0% and 51.7% among the patients with im4 and im3 , respectively . conversely , the 5-year survival rate was only 0% , 5.8% , and 0% among patients with im0 , im1 , and im2 , respectively . the cumulative 5-year dfs was 75% , and 44.8% among the patients with im4 and im3 , respectively . this study has the following limitations : first , it included only 100 patients with stage iiia cancer ; a larger number of patients are needed for validating these observations . in conclusion , this study proved the existence of prognostic heterogeneity among stage iiia gastric patients ; an i m system was designed that might be helpful in identifying such types of heterogeneity .

carcinoma of the prostate is the most common malignancy in men with increased incidence rates owing to the population ageing and the improvement of diagnostic procedures . the early detection of the bone metastases is of value in making decision regarding the treatment plan , which may vary extremely according to the bone status . the likelihood of the bone metastases at the first diagnosis varies with the histological score and serum level of prostate - specific antigen , and it is possible to some extent to estimate the degree of tumor spread in the light of the psa value . bone involvement is much less frequently involved with low psa level , so that the bone staging is not recommended with psa less than 10 ng / ml , except if there are known bone disorders which may later cause false positive findings [ 13 ] . however , in patients with psa relapse after primary therapy it is not easy to be guessed based on psa values whether the bone involvement or another involvement is behind a psa rising . in established skeletal involvement , there is a need of effective imaging method to monitor the status progress . bone scan is the widely used screening technique for assessing the bone status in the most malignancies , and it shows mostly high sensitivity in detecting the bone involvement . pet / ct with f - fluorodeoxyglucose ( fdg ) is known to have general limitation in prostate cancer . pet with radiolabeled choline such as f - fech - pet was found to be an effective method in diagnosis of primary and recurrent pca tumors as well [ 4 , 5 ] . two possible mechanisms have been proposed to explain the increased choline uptake in prostate cancer cells . the first is increased cell proliferation in tumors , because choline is a precursor for the biosynthesis of phosphatidylcholine and other phospholipids , the major components of the cell membrane . the second explanation is the overproduction of choline kinase in cancer cells , which was experimentally confirmed in human - derived prostate cancer [ 68 ] . although , f - fech - pet is being increasingly used primarily in patients with prostate cancer and for followup , its efficacy in detecting the bone involvement still needs further investigations in comparison with the established method ( bone scan ) . to our knowledge we aim in this study mainly to assess the value of f - fech - pet / ct in detecting the bone metastases and to compare the results with that of bone scan . 37 patients ( mean age 69 7 ) had been referred in our department for re - staging purposes due to biochemical recurrence , psa median 2.6 ng / dl ( range 0.321 ) . gleason 's score ranges among 3 and 9 : gs.7 ( n = 12 ) , gs.8 ( n = 8) , gs.9 ( n = 7 ) , gs . 3 ( n = 1 ) , and gs 5 ( n = 1 ) . the patients underwent a wide variety of initial therapies including radical prostatectomy ( n = 11 ) , radiotherapy ( n = 7 ) , and pure hormonotherapy in patients with locally advanced disease ( n = 3 ) . a number of patients underwent combined therapy due to previous slight psa increase such as radical prostatectomy followed by salvage radiotherapy ( n = 12 ) , radical prostatectomy followed by salvage antihormonal therapy ( n = 3 ) , or radical prostatectomy followed by hifu ( n = 1 ) . the study was approved by the ethics committee of the heidelberg university , and a written consent was obtained from all patients . bone scintigraphy was performed using a modern double - head gamma - camera ( ecam , siemens medical solution ) equipped with low - energy , high - resolution collimators . whole - body images were obtained 2 - 3 h after intravenous injection of 700 mbq ( 19 mci ) of tcm - mdp at the scan speed of 15 cm / min in the anterior and posterior projection ; additional imaging were acquired , when required . all pet studies were carried out using a biograph 6 pet / ct ( siemens / cti ) . imaging was started as dynamic imaging immediately parallel to administration of a standard of 250 mbq of f - ethylcholine for 10 min before proceeding to whole body imaging . for attenuation correction of the pet scan , a low - dose ct ( 130 kev , 30 mas ; care dose ) without contrast medium was done . static emission scans , corrected for dead time , scatter , and decay were acquired from the vertex to the proximal legs requiring eight bed positions , 4 minutes each . the images were iteratively reconstructed with the osem algorithm using four iterations with eight subsets and gauss filtering to an in - plane spatial resolution of 5 mm at full - width half - maximum . the delay between both modalities ranged between 2 and 45 days ( average 14 days ) . the images were evaluated visually by two skilled nuclear medicine physicians in presence of a skilled radiologist after viewing the images in different planes . the image assessment was achieved in combination with the clinical information and close correlation with other available imaging studies . the findings concerning the bone status were assessed as positive in presence of typical findings validated with followup and/or by correlation with other modality and as negative in absence of the pathological bone uptake ( there was no need to prove the negativity ) , or false positive / negative ( if proved the contrary ) . sensitivity , specificity , npv , and ppv are the statistical indicators utilized in assessing the universal outcome . in the patients with confirmed bone metastases ( n = 18 ) , the skeleton was divided into five regions ( skull , ribs , pelvis , vertebra , and extremities ) to simplify the comparison between the matching regions in both examinations . the metastases had been drawn as black points on a separate skeleton outline ( figure 1 ) . each imaging was evaluated separately , so that the interpretation would not be influenced . the anona test was used to compare the extent of metastatic spread in both examinations in the different regions . suvs were acquired by manually drawing a volume of interest over the pathologic bone lesions that had been identified on visual analysis of static emission images . by using attenuation - corrected pet data , suvs were calculated as the ratio of regional radioactivity concentration ( becquerel / milliliter ) divided by the injected amount of radioactivity ( becquerel ) normalized to body weight in gram . ct reading was based on characteristic patterns of morphological change ; all detectable lesions on ct were categorized by a radiologist as sclerotic , lytic , or mixed metastases . the quantitative radiodensity of sclerotic lesions was measured by means of the hounsfield unit ( hu ) . for quantitative assessment of mdp uptake , we considered an uptake score based on the visual assessment and graduated from 0 till 4 ( 0 no uptake , 1 decent uptake , 2 moderate uptake , 3 high uptake , 4 extremely severe uptake ) . so the intensity of choline uptake was once compared with the radio - density represented by hu and once with mdp uptake ( evaluated visually ) . lesions detected in ct and evaluated as high suspected were encompassed in this comparison , regardless whether they showed a positive choline uptake or not . some limitations should be kept in mind such as the absence of pathological verification of the bone metastases . thus , the patients were confirmed as having bone metastases just depending on clinical followups in correlation with other diagnostic tools , for example mri . the average clinical followup duration to confirm the final diagnosis was 1 year . the characteristics of the patients are summarized in table 1 . generally , 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . since the pathological confirmation was not available , the followup and correlation with other modalities were adopted in our verification . ( followup was available in 11 patients ; in the remaining seven patients , the verification was based on a typical findings in pet , assessed by two nuclear medicine physicians matching with morphological images such as ct or mri . ) in patients with positive bone findings , the psa median was 4 ng / dl ( range 121 ) . by contrast , the patients with negative bone findings had a psa median value of 1 . 5 ng / dl ( range 0.319 ) . statistically there was a significant difference between both groups ( p = 0.02 ) ( figure 2 ) . in patients with negative bone findings the psa relapse was attributed to lymph node metastases ( n = 5 ) , local recurrence ( n = 2 ) , and lung metastases ( n = 1 ) . confirmed bone involvement cases , f - fech - pet / ct identified correctly the bone involvement in 15 cases with a sensitivity of 83.3% . no false positive was identified ; consequently its specificity was 100% , npp 86.3% , and ppv 100% . on the other hand , bone scan showed the presence of the metastases in 17 out of 18 confirmed cases with sensitivity of 94.4% . from 19 negative findings , the bone scan showed two false positive cases due to paget disease and trauma . as mentioned earlier , the skeleton had been divided into multiple regions to simplify the matching of findings . totally , the patients with positive findings have 122 lesions . f - fech - pet was capable to identify 101 lesions with a sensitivity 82.7% versus 109 identified using bs ( bone scan ) with sensitivity of 89.4% . the sensitivity of both modalities differs extremely with the anatomical region ( table 2 ) . indeed this superiority of bone scan was demonstrated in ribs ( 71% in f - fech - pet versus 94% in bs ) and cranium . in remaining regions , f - fech - pet was superior in identifying more metastases , in vertebrae ( 92% versus 85% ) and pelvis ( 91% versus 89% ) . in the extremities f - fech - pet shows also superiority over bone scan ( 90% versus 50% ) in spite of limited field of view ( fov ) figure 3 . statistically using anova test there was no significant difference between both modalities ( p > 0.1 ) in the number of detected metastases in all anatomical regions . degenerative changes : f - fech - pet / ct versus bs ( figure 4)whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . trauma : f - fech - pet / ct versus bs ( figures 5 and 6)these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . a total of 94 out of 122 with the following distribution : ( 3 in cervical spine , 10 in lumbar spine , 35 in pelvis , 7 in ribs , 4 in sternum , 15 in thoracic spine , 12 in the extremities and 7 in other regions ) were selected to make a further quantitative comparison between f - fech - pet and both ct and bone scan using the above mentioned quantitative parameters . 28 lesions were excluded from this analysis , for example , in ribs due to neighborhood to liver ( false high suv through partial volume effect ) or due to technical limitations . as previously mentioned , the quantitative assessment in bone scan was based on visual score escalating from 0 till 4 . based on the radiological portrayal in ct , lesions were divided into osteoblastic lesions ( n = 42 ) , osteolytic ( n = 19 ) , and mixed ( n = 16 ) . 16 lesions of positive choline uptake : ( 11 in the spine7 thoracic and 4 lumbar2 in the pelvis and 3 in the extremities ) were lacking morphological alterations . they were characterized with high choline uptake ( suv 5 , 95 1 , 5 ) and mostly with decent or absent mdp uptake . they were attributed to bone marrow involvement ( figure 7 ) 13 of sclerotic lesions ( 6 in pelvis , 3 in thoracic spine and 4 in ribs ) ( hu mean 739 216 ) were of negative choline uptake but mostly of intensive mdp uptake ( qmdp score 3 ) ( qmdp quantitative mdp evaluated visually ) . the decline or absence of choline uptake in sclerotic metastasis was noted in particular in patients receiving systemic therapy ( figures 8 and 11 ) . in contrast , the osteolytic metastases ( 4 in the pelvis and 1 in the humerus ) were characterized with high choline uptake ( 7 2 , nadir 5.5 ) and wide extent of mdp uptake ( 4 showed negative uptake , 4 showed intensive uptake and 2 showed moderate uptake ) . a significant negative correlation was demonstrated between tracer uptake suv(max ) and the density of sclerotic lesions assessed by hu ( r = 0.58 , p < 0.01 ) ( figure 9 ) . on the other hand , numerical score of mdp was correlated negatively with suv(max ) value ( r 0.01 ) and positively with hu ( r 0.34 ) . however , they were statistically not significant . in few mixed metastases it was demonstrated that the choline uptake was solely concentrated in the osteolytic part evading the sclerotic part ( figure 10(c ) ) . in pure sclerotic metastases it was shown that uptake concentrates at the rim of metastases , where there is a transitional region with a moderate sclerosis ( figures 10(a ) and 10(b ) ) . examples of the effects of ongoing systemic therapy on pet findingssee figures 11 and 12 . it is known that the skeleton is a favorable place for metastasis in prostate cancer . it is the second most common site of metastatic disease after lymph nodes and considered as the main cause of morbidity and mortality in prostate cancer patients and mostly related to poor prognosis . although bone scan is ranked first in assessing bone status in many malignancies including pca , it still lacks specificity . the comparison between pet modality and bone scan in detecting bone involvement was a concern of many studies , mostly involving f - fdg pet / ct [ 911 ] . cheng et al . showed , in their review of six studies , that f - fdg pet / ct has both higher sensitivity and specificity than bone scintigraphy . in patients with pca , tiwari et al . proved that f - fdg - pet / ct can play a complimentary role to the conventional skeletal scintigraphy , particularly in detection of bone marrow disease . whether or not f - fech - pet / ct is capable to assess the bone status in an adequate way or requires supplementary test is still blurred . according to our knowledge , there is lack of studies concerning the comparison of f - fech - pet / ct with standard bs . . showed that f - choline- pet / ct had lower sensitivity than f fluoride pet - ct for detection of bone metastases ( f - fluoride is bone seeking pet radiopharmaceutical ) . bone scan showed superiority over f - fech - pet / ct in patient - based results ( sensitivity 81.2% versus 93.7% ) . however , the high sensitivity of bone scan encounters a high likelihood of false positive findings . by contrast , f - fech - pet / ct appears to be less affected by such unspecific lesions . the benefit of associated diagnostic ct ( occasionally with contrast media ) should be kept in mind in minimizing the false positive cases . yet , ct is not the sole factor affecting the increase of the specificity of f - fech , mccarthy et al . suggested that f - fech - pet can separate benign conditions such as trauma and arthropathy from malignancy and may be useful as an assistant to bone scan in equivocal cases . basically , there is need to perform bone scan in case of negative f - fech - pet / ct finding if there is clinical suspicion , under suggestion that f - fech - pet is of less sensitivity . however , bone scan can be reserved and an unnecessary radiation exposure can be avoided , if proven otherwise . so whether or not bone scan can be abandoned as a complementary test to f - fech - pet / ct is of a high clinical value . we found that f - fech - pet / ct showed less sensitivity compared with bs ; however , two out of three patients demonstrating false negative pet findings were under systemic therapy , suggesting a potential impact of systemic therapy in minimizing the sensitivity . in our patient group , looking at the findings of both modalities in the various anatomical regions , we observed better lesion detection efficiency of bs in the ribs with sensitivity of 98% versus 73% . on the other hand , the f - fech - pet / ct was more effective in pelvis ( 93% versus 87% ) and in extremities in spite of the presence of few metastases outside the field of view . in vertebra the f - fech - pet / ct was also more accurate than bs . because of poor spatial resolution of bs , an uptake in the spine could be incorrectly attributed to degenerative changes that may explain the low sensitivity of bs in spine compared with f - fech - pet / ct . moreover spect ( single photon emission computed tomography ) can minimize the shortcoming of planar bs in the assessment of the spine ; however it is not routinely applied . in this regard , it should be mentioned that spinal metastases have typical locations , mostly in the posterior part of vertebrae due to the many short intraosseous arteries [ 16 , 17 ] , and they can be easily recognized and differentiated from degenerative changes in f - fech - pet / ct in the light of morphological view provided by ct component . in other anatomical regions f - fech - pet / ct showed advantage over bs ; this can be attributed to the superior spatial resolution . of course the associated ct contributed to a certain degree in increasing the sensitivity of f - fech - pet / ct through detecting the bone metastases of a negative choline uptake . in the same topic , we found that fech uptake decreases with increasing sclerosis ( negative correlation between hu and suv(max ) r = 0.58 , p < 0.01 ) . this issue emphasizes the importance of ct in compensating the deficiency of f - fech - pet in patients under aht . further benefit of ct is the validating the pet finding through excluding other benign changes that may share the same findings with the metastases . since the bone marrow is associated with minimal morphological changes and unusual to cause bone reaction , it remains undetectable or can be easily overlooked using morphological modalities or using bone seeking nuclides . kato and coworkers reported a case of bone marrow metastasis finally detected by marrow biopsy , without any abnormality on bone scintigraphy or computed tomography . generally , the bm aspiration and biopsy remain the procedures of choice to detect bm involvement . f - fdg pet is supposed to have special value in detecting bm involvement ; aydin et al . described a case of bone marrow metastasis detected by f - fdg pet and missed by bone scintigraphy in widespread melanoma . f - fech - pet / ct in prostate cancer patients shares this property , whereas there is no role of ct or bs . described a positive choline bone marrow metastasis in the thoracic spine without any ct abnormality . in our results , pet positive findings without morphological changes in spine , pelvis and proximal extremities were attributed to bone marrow involvement . bs was not of further benefit in identifying them ; they were either of negative or decent mdp uptakes . that raises the following question : what is the real clinical benefit of bs over f - fech - pet / ct , if the deficiency of choline - pet is mainly in osteoblastic metastases , which are simply to be visualized using associated ct ? in other words , if ct can adequately compensate the deficiency of f - fech - pet , still bone scan offers further diagnostic information ? indeed , in order to validate its further performance as complementary test , bone scan is required to show a superiority over f - fech - pet / ct both components pet and ct . just looking at lesion - based results , there is no real justification for additional bone scan , since the gain in detecting more lesions is not valuable in patients with multiple metastases , who should anyway undergo systemic therapy . however , in patient - based results we notice that associated ct failed in identifying three false negative cases in f - fech - pet / ct , which were ultimately detected using bs . more advantage of bone scan is emphasized in its availability and ease to repeat for followup . moreover , in multiple bone metastases if pain radiotherapy is indicated , bone scan is necessary because mdp distributes in a similar way as bone seeking therapy - tracer . in f - fech - pet / ct , choline uptake is supposed to be linked with presence of tumor cells which can transport and metabolize it . in contrast , bs displays the bone metastases indirectly through demonstrating the structural alterations . for this reason , any alteration concerning the tumor focus ( whether progress or regress ) is supposed to be first detected in choline - pet . this fact and the possibility to perform a quantitative assessing grant the f - fech - pet a unique value in therapy monitoring . subsequent to therapy onset , a drop in suv is an appropriate indicator for response . by contrast , in bone scan the therapy response may lead to transient increasing mdp uptake due to an amplified osteoblastic response in the so - called flare phenomenon . this can be mistaken with progress and typically lasts about 6 months after therapy [ 2123 ] . for this reason , the early response ct is also of negligible role in therapy monitoring , since the morphological alterations occur in delay . to understand the weakness of bs in therapy monitoring , we ought to return to the pathology of the bone metastases in prostate cancer . in this type of bone metastasis , intramembranous ossification takes place in regions of fibrous stroma and after therapy administration ; the ossification process stays for a certain time running in the manufactured stroma despite damaging the causing tumor cells and evoked mediators . in other words , the ongoing mineralization processes , thus mdp uptake , would not be inhibited after therapy onset even if the therapy is successful . compared with bs , as mentioned earlier , choline uptake in f - fech - pet / ct is supposed to reflect directly the tumor focus , and the reduction in suv early after initiation of therapy can distinguish responders from nonresponders . however , does uptake decline always indicate response ? in some cases , it was demonstrated that shortly after treatment onset , the f - fech - pet / ct turned to be negative just in osteoblastic metastases , while osteolytic metastases were preserved . this effect of aht in the sclerotic metastases was already described by beheshti et al . . it may also explain the lower total sensitivity of f - fech - pet / ct compared with bs in the area - based results ( 6 patients were under ongoing aht in our study ) . in another case of our study under aht , the followup shows a dramatic uptake decline in bone metastases therefore this uptake decline in bone metastases is likely to reflect a temporal diminishing of uptake rather to be an indicator for real response . the loss of choline uptake was also observed in osteoblastic metastases in patients undergoing no therapy . as mentioned earlier , choline uptake declines with increasing sclerosis , probably due to the drop in blood supply thus in nuclide availability . . found that a hu level above 825 is associated with an absence of metabolic activity . due to the lack of long - term followup , it is still unknown whether or not these metastases are still alive if reaching the stage of negative choline uptake . obviously , even if the metastasis center reaches this stage , the rim where there is a moderate sclerosis remains active . ultimately , the potential role of f - fech - pet / ct in therapy monitoring seems to be limited in case of bone involvement , because choline uptake is likely to be affected by local factors such as the level of sclerosis . no significant gain in sensitivity was achieved using bone scan compared with f - fech - pet / ct . in lesion - based results , the diagnostic potential of both modalities varies in the different anatomical regions and shows f - fech - pet / ct mostly of superior value . that was attributed to the higher spatial resolution and the additional benefit of accompanied ct except for its value in detecting the bone marrow involvement .

18f - fech - pet / ct has been proved to be an imaging agent for prostate carcinoma . however , its role in detecting the bone metastases is still blurred owing to the lack of related studies . the purpose of our study was to assess the efficacy of pet with 18f - ethylcholine in assessing the bone status and to compare the results with that of conventional bone scan findings . for this purpose , we selected 37 patients ( mean age 69 7 ) , who had been referred for restaging purposes due to biochemical recurrences and underwent both 18f - fech - pet / ct and bone scan in a short interval . generally 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . from 18 confirmed bone involvement cases , 18f - fech - pet / ct identified correctly the bone involvement in 15 cases with overall sensitivity of 83.3% . on the other hand , bone scan identified 17 out of 18 confirmed cases with overall sensitivity of 94.4% . the lesion - related results show that the sensitivity of each investigation differs with the anatomical regions , and by comparing both results , 18f - fech - pet / ct was mostly superior to bone scan ; however , without a statistical significance ( p > 0.1 ) . in conclusion , no significant gain in sensitivity was achieved using bone scan compared with 18f - fech - pet / ct .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

carcinoma of the prostate is the most common malignancy in men with increased incidence rates owing to the population ageing and the improvement of diagnostic procedures . the early detection of the bone metastases is of value in making decision regarding the treatment plan , which may vary extremely according to the bone status . the likelihood of the bone metastases at the first diagnosis varies with the histological score and serum level of prostate - specific antigen , and it is possible to some extent to estimate the degree of tumor spread in the light of the psa value . bone involvement is much less frequently involved with low psa level , so that the bone staging is not recommended with psa less than 10 ng / ml , except if there are known bone disorders which may later cause false positive findings [ 13 ] . however , in patients with psa relapse after primary therapy it is not easy to be guessed based on psa values whether the bone involvement or another involvement is behind a psa rising . bone scan is the widely used screening technique for assessing the bone status in the most malignancies , and it shows mostly high sensitivity in detecting the bone involvement . pet / ct with f - fluorodeoxyglucose ( fdg ) is known to have general limitation in prostate cancer . pet with radiolabeled choline such as f - fech - pet was found to be an effective method in diagnosis of primary and recurrent pca tumors as well [ 4 , 5 ] . although , f - fech - pet is being increasingly used primarily in patients with prostate cancer and for followup , its efficacy in detecting the bone involvement still needs further investigations in comparison with the established method ( bone scan ) . to our knowledge we aim in this study mainly to assess the value of f - fech - pet / ct in detecting the bone metastases and to compare the results with that of bone scan . 37 patients ( mean age 69 7 ) had been referred in our department for re - staging purposes due to biochemical recurrence , psa median 2.6 ng / dl ( range 0.321 ) . gleason 's score ranges among 3 and 9 : gs.7 ( n = 12 ) , gs.8 ( n = 8) , gs.9 ( n = 7 ) , gs . 3 ( n = 1 ) , and gs 5 ( n = 1 ) . the patients underwent a wide variety of initial therapies including radical prostatectomy ( n = 11 ) , radiotherapy ( n = 7 ) , and pure hormonotherapy in patients with locally advanced disease ( n = 3 ) . a number of patients underwent combined therapy due to previous slight psa increase such as radical prostatectomy followed by salvage radiotherapy ( n = 12 ) , radical prostatectomy followed by salvage antihormonal therapy ( n = 3 ) , or radical prostatectomy followed by hifu ( n = 1 ) . the study was approved by the ethics committee of the heidelberg university , and a written consent was obtained from all patients . bone scintigraphy was performed using a modern double - head gamma - camera ( ecam , siemens medical solution ) equipped with low - energy , high - resolution collimators . whole - body images were obtained 2 - 3 h after intravenous injection of 700 mbq ( 19 mci ) of tcm - mdp at the scan speed of 15 cm / min in the anterior and posterior projection ; additional imaging were acquired , when required . all pet studies were carried out using a biograph 6 pet / ct ( siemens / cti ) . imaging was started as dynamic imaging immediately parallel to administration of a standard of 250 mbq of f - ethylcholine for 10 min before proceeding to whole body imaging . static emission scans , corrected for dead time , scatter , and decay were acquired from the vertex to the proximal legs requiring eight bed positions , 4 minutes each . the images were iteratively reconstructed with the osem algorithm using four iterations with eight subsets and gauss filtering to an in - plane spatial resolution of 5 mm at full - width half - maximum . the image assessment was achieved in combination with the clinical information and close correlation with other available imaging studies . the findings concerning the bone status were assessed as positive in presence of typical findings validated with followup and/or by correlation with other modality and as negative in absence of the pathological bone uptake ( there was no need to prove the negativity ) , or false positive / negative ( if proved the contrary ) . sensitivity , specificity , npv , and ppv are the statistical indicators utilized in assessing the universal outcome . in the patients with confirmed bone metastases ( n = 18 ) , the skeleton was divided into five regions ( skull , ribs , pelvis , vertebra , and extremities ) to simplify the comparison between the matching regions in both examinations . the metastases had been drawn as black points on a separate skeleton outline ( figure 1 ) . each imaging was evaluated separately , so that the interpretation would not be influenced . the anona test was used to compare the extent of metastatic spread in both examinations in the different regions . suvs were acquired by manually drawing a volume of interest over the pathologic bone lesions that had been identified on visual analysis of static emission images . for quantitative assessment of mdp uptake , we considered an uptake score based on the visual assessment and graduated from 0 till 4 ( 0 no uptake , 1 decent uptake , 2 moderate uptake , 3 high uptake , 4 extremely severe uptake ) . so the intensity of choline uptake was once compared with the radio - density represented by hu and once with mdp uptake ( evaluated visually ) . lesions detected in ct and evaluated as high suspected were encompassed in this comparison , regardless whether they showed a positive choline uptake or not . some limitations should be kept in mind such as the absence of pathological verification of the bone metastases . thus , the patients were confirmed as having bone metastases just depending on clinical followups in correlation with other diagnostic tools , for example mri . generally , 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . statistically there was a significant difference between both groups ( p = 0.02 ) ( figure 2 ) . in patients with negative bone findings the psa relapse was attributed to lymph node metastases ( n = 5 ) , local recurrence ( n = 2 ) , and lung metastases ( n = 1 ) . confirmed bone involvement cases , f - fech - pet / ct identified correctly the bone involvement in 15 cases with a sensitivity of 83.3% . no false positive was identified ; consequently its specificity was 100% , npp 86.3% , and ppv 100% . on the other hand , bone scan showed the presence of the metastases in 17 out of 18 confirmed cases with sensitivity of 94.4% . from 19 negative findings , the bone scan showed two false positive cases due to paget disease and trauma . as mentioned earlier , the skeleton had been divided into multiple regions to simplify the matching of findings . f - fech - pet was capable to identify 101 lesions with a sensitivity 82.7% versus 109 identified using bs ( bone scan ) with sensitivity of 89.4% . the sensitivity of both modalities differs extremely with the anatomical region ( table 2 ) . indeed this superiority of bone scan was demonstrated in ribs ( 71% in f - fech - pet versus 94% in bs ) and cranium . in remaining regions , f - fech - pet was superior in identifying more metastases , in vertebrae ( 92% versus 85% ) and pelvis ( 91% versus 89% ) . in the extremities f - fech - pet shows also superiority over bone scan ( 90% versus 50% ) in spite of limited field of view ( fov ) figure 3 . statistically using anova test there was no significant difference between both modalities ( p > 0.1 ) in the number of detected metastases in all anatomical regions . degenerative changes : f - fech - pet / ct versus bs ( figure 4)whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . trauma : f - fech - pet / ct versus bs ( figures 5 and 6)these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . a total of 94 out of 122 with the following distribution : ( 3 in cervical spine , 10 in lumbar spine , 35 in pelvis , 7 in ribs , 4 in sternum , 15 in thoracic spine , 12 in the extremities and 7 in other regions ) were selected to make a further quantitative comparison between f - fech - pet and both ct and bone scan using the above mentioned quantitative parameters . 28 lesions were excluded from this analysis , for example , in ribs due to neighborhood to liver ( false high suv through partial volume effect ) or due to technical limitations . as previously mentioned , the quantitative assessment in bone scan was based on visual score escalating from 0 till 4 . based on the radiological portrayal in ct , lesions were divided into osteoblastic lesions ( n = 42 ) , osteolytic ( n = 19 ) , and mixed ( n = 16 ) . they were attributed to bone marrow involvement ( figure 7 ) 13 of sclerotic lesions ( 6 in pelvis , 3 in thoracic spine and 4 in ribs ) ( hu mean 739 216 ) were of negative choline uptake but mostly of intensive mdp uptake ( qmdp score 3 ) ( qmdp quantitative mdp evaluated visually ) . on the other hand , numerical score of mdp was correlated negatively with suv(max ) value ( r 0.01 ) and positively with hu ( r 0.34 ) . however , they were statistically not significant . in few mixed metastases it was demonstrated that the choline uptake was solely concentrated in the osteolytic part evading the sclerotic part ( figure 10(c ) ) . it is known that the skeleton is a favorable place for metastasis in prostate cancer . although bone scan is ranked first in assessing bone status in many malignancies including pca , it still lacks specificity . the comparison between pet modality and bone scan in detecting bone involvement was a concern of many studies , mostly involving f - fdg pet / ct [ 911 ] . showed , in their review of six studies , that f - fdg pet / ct has both higher sensitivity and specificity than bone scintigraphy . proved that f - fdg - pet / ct can play a complimentary role to the conventional skeletal scintigraphy , particularly in detection of bone marrow disease . whether or not f - fech - pet / ct is capable to assess the bone status in an adequate way or requires supplementary test is still blurred . according to our knowledge , there is lack of studies concerning the comparison of f - fech - pet / ct with standard bs . showed that f - choline- pet / ct had lower sensitivity than f fluoride pet - ct for detection of bone metastases ( f - fluoride is bone seeking pet radiopharmaceutical ) . bone scan showed superiority over f - fech - pet / ct in patient - based results ( sensitivity 81.2% versus 93.7% ) . however , the high sensitivity of bone scan encounters a high likelihood of false positive findings . by contrast , f - fech - pet / ct appears to be less affected by such unspecific lesions . yet , ct is not the sole factor affecting the increase of the specificity of f - fech , mccarthy et al . suggested that f - fech - pet can separate benign conditions such as trauma and arthropathy from malignancy and may be useful as an assistant to bone scan in equivocal cases . basically , there is need to perform bone scan in case of negative f - fech - pet / ct finding if there is clinical suspicion , under suggestion that f - fech - pet is of less sensitivity . however , bone scan can be reserved and an unnecessary radiation exposure can be avoided , if proven otherwise . so whether or not bone scan can be abandoned as a complementary test to f - fech - pet / ct is of a high clinical value . we found that f - fech - pet / ct showed less sensitivity compared with bs ; however , two out of three patients demonstrating false negative pet findings were under systemic therapy , suggesting a potential impact of systemic therapy in minimizing the sensitivity . in our patient group , looking at the findings of both modalities in the various anatomical regions , we observed better lesion detection efficiency of bs in the ribs with sensitivity of 98% versus 73% . on the other hand , the f - fech - pet / ct was more effective in pelvis ( 93% versus 87% ) and in extremities in spite of the presence of few metastases outside the field of view . in vertebra the f - fech - pet / ct was also more accurate than bs . because of poor spatial resolution of bs , an uptake in the spine could be incorrectly attributed to degenerative changes that may explain the low sensitivity of bs in spine compared with f - fech - pet / ct . moreover spect ( single photon emission computed tomography ) can minimize the shortcoming of planar bs in the assessment of the spine ; however it is not routinely applied . in this regard , it should be mentioned that spinal metastases have typical locations , mostly in the posterior part of vertebrae due to the many short intraosseous arteries [ 16 , 17 ] , and they can be easily recognized and differentiated from degenerative changes in f - fech - pet / ct in the light of morphological view provided by ct component . in other anatomical regions f - fech - pet / ct showed advantage over bs ; this can be attributed to the superior spatial resolution . of course the associated ct contributed to a certain degree in increasing the sensitivity of f - fech - pet / ct through detecting the bone metastases of a negative choline uptake . in the same topic , we found that fech uptake decreases with increasing sclerosis ( negative correlation between hu and suv(max ) r = 0.58 , p < 0.01 ) . this issue emphasizes the importance of ct in compensating the deficiency of f - fech - pet in patients under aht . further benefit of ct is the validating the pet finding through excluding other benign changes that may share the same findings with the metastases . since the bone marrow is associated with minimal morphological changes and unusual to cause bone reaction , it remains undetectable or can be easily overlooked using morphological modalities or using bone seeking nuclides . kato and coworkers reported a case of bone marrow metastasis finally detected by marrow biopsy , without any abnormality on bone scintigraphy or computed tomography . f - fdg pet is supposed to have special value in detecting bm involvement ; aydin et al . f - fech - pet / ct in prostate cancer patients shares this property , whereas there is no role of ct or bs . in our results , pet positive findings without morphological changes in spine , pelvis and proximal extremities were attributed to bone marrow involvement . that raises the following question : what is the real clinical benefit of bs over f - fech - pet / ct , if the deficiency of choline - pet is mainly in osteoblastic metastases , which are simply to be visualized using associated ct ? in other words , if ct can adequately compensate the deficiency of f - fech - pet , still bone scan offers further diagnostic information ? indeed , in order to validate its further performance as complementary test , bone scan is required to show a superiority over f - fech - pet / ct both components pet and ct . just looking at lesion - based results , there is no real justification for additional bone scan , since the gain in detecting more lesions is not valuable in patients with multiple metastases , who should anyway undergo systemic therapy . however , in patient - based results we notice that associated ct failed in identifying three false negative cases in f - fech - pet / ct , which were ultimately detected using bs . more advantage of bone scan is emphasized in its availability and ease to repeat for followup . moreover , in multiple bone metastases if pain radiotherapy is indicated , bone scan is necessary because mdp distributes in a similar way as bone seeking therapy - tracer . in f - fech - pet / ct , choline uptake is supposed to be linked with presence of tumor cells which can transport and metabolize it . in contrast , bs displays the bone metastases indirectly through demonstrating the structural alterations . for this reason , any alteration concerning the tumor focus ( whether progress or regress ) is supposed to be first detected in choline - pet . this fact and the possibility to perform a quantitative assessing grant the f - fech - pet a unique value in therapy monitoring . by contrast , in bone scan the therapy response may lead to transient increasing mdp uptake due to an amplified osteoblastic response in the so - called flare phenomenon . for this reason , the early response ct is also of negligible role in therapy monitoring , since the morphological alterations occur in delay . to understand the weakness of bs in therapy monitoring , we ought to return to the pathology of the bone metastases in prostate cancer . compared with bs , as mentioned earlier , choline uptake in f - fech - pet / ct is supposed to reflect directly the tumor focus , and the reduction in suv early after initiation of therapy can distinguish responders from nonresponders . however , does uptake decline always indicate response ? in some cases , it was demonstrated that shortly after treatment onset , the f - fech - pet / ct turned to be negative just in osteoblastic metastases , while osteolytic metastases were preserved . it may also explain the lower total sensitivity of f - fech - pet / ct compared with bs in the area - based results ( 6 patients were under ongoing aht in our study ) . in another case of our study under aht , the followup shows a dramatic uptake decline in bone metastases therefore this uptake decline in bone metastases is likely to reflect a temporal diminishing of uptake rather to be an indicator for real response . as mentioned earlier , choline uptake declines with increasing sclerosis , probably due to the drop in blood supply thus in nuclide availability . . due to the lack of long - term followup , it is still unknown whether or not these metastases are still alive if reaching the stage of negative choline uptake . ultimately , the potential role of f - fech - pet / ct in therapy monitoring seems to be limited in case of bone involvement , because choline uptake is likely to be affected by local factors such as the level of sclerosis . no significant gain in sensitivity was achieved using bone scan compared with f - fech - pet / ct . in lesion - based results , the diagnostic potential of both modalities varies in the different anatomical regions and shows f - fech - pet / ct mostly of superior value . that was attributed to the higher spatial resolution and the additional benefit of accompanied ct except for its value in detecting the bone marrow involvement .

despite the rapidity with which genomic science has enabled the identification of genes essential to bacterial homeostasis , the translation of these targets into pharmacological agents has proven to be much more difficult than anticipated . post - translational modification ( ptm ) pathways represent a unique subset of these targets since such events are scarce in prokaryotes relative to their frequency of occurrence in eukaryotes . in metabolism , bacterial ptms involve the attachment of prostheses that impart a chemical functionality distinct from that of the 20 proteogenic amino acids . thus , effector enzyme function is wholly dependent upon the installation of these groups . one such ptm is performed by 4-phosphopantetheinyl transferase ( pptase ) enzymes ( figure 1a ) , which install 4-phosphopantetheinyl ( 4-pp ) arms to carrier protein domains of synthase enzymes ( figure 1b ) . this functionality serves to store and channel reactive intermediates that would otherwise be lost to the cellular milieu by diffusion . ( a ) bacteria utilize synthase enzymatic pathways to assemble small carboxylic and amino acid monomers into complex polymeric molecules , including membrane components , virulence factors , and medicinally important compounds . a key feature of these synthases is the presence of a post - translational appendage to which the nascent polymer is tethered during extension and elaboration ; the attachment of this appendage is catalyzed by pptase enzymes . ( b ) pptase enzymes utilize coa as a 4-phosphopantetheinyl group donor and install the functionality to conserved serine residues within apo - cp domains of synthase enzymes , generating biochemically active holo - cp - containing synthases and the nucleotide pap as products . pptase = phosphopantetheinyl transferase , cp = carrier protein , and pap = 3-phosphoadenosine 5-monophosphate . pathways activated by pptases include microbial fatty acid synthase ( fas ) , a multidomain enzyme complex that has received considerable attention for both its orthogonal structural characteristics relative to the mammalian fas system and its novelty as a drug target . reduction in fas anabolic capacity is anticipated to exert pleotropic effects on cell viability by inhibiting the production of the primary membrane component palmitate and halting the assembly of virulence - determining components of the gram - negative , gram - positive , and mycobacterial cell walls ( lipid a , lipoteichoic acid , and mycolic acids , respectively ) . furthermore , fas as a drug target has been validated in vivo with chemical probes that act upon -ketoacyl - acp synthase . additionally , bacterial secondary metabolism contains polyketide and nonribosomal synthases which require 4-pp groups from pptases to produce metabolites needed for bacteria to thrive in environmental and infection settings . representative compounds from these pathways include siderophores that are secreted to scavenge iron and the phenolic glycolipids of mycobacterium spp . in many cases , the capacity to manufacture these compounds has been linked to virulence , and disruption of synthase genes precludes the establishment of infection . these avirulent phenotypes have been recapitulated in vitro with chemical probes that target synthase enzymes . since pptase enzymes represent the gatekeeper of these pathways , their inhibition stands to attenuate bacterial cell viability through direct inactivation of fas . at the same time , the concomitant disruption of secondary metabolism offers a path to mitigate numerous aspects of pathogenicity . these hypotheses have been confirmed at the genetic level , where disruption of pptase genes has been observed as either lethal or severely compromising to the fitness of both escherichia coli(14)and mycobacterium tuberculosis . thus , pptase represents an important enzyme in bacterial metabolism and is well - deserving of further study . it is noteworthy that , in most cases , bacterial genomes contain two structurally distinct pptase enzymes , the acps- and sfp - pptases . enzymes of the former class are typically responsible for activating fas , while enzymes of the latter type modify secondary metabolism synthases . given these associations and the above - mentioned therapeutic potential of targeting pptases , there have been several reports by academic and industrial researchers describing the development of small - molecule acps - pptase inhibitors , yet no reports of agents targeting sfp - pptase ( see figure 2 ) . chu and co - workers isolated sch-538415 ( figure 2 ) , a symmetrical and highly planar compound , from an unidentified bacterial extract and reported it to have moderate inhibitory activity toward acps . prior to the initiation of our current program , we had found that analogues of anthranilic acid - based leads originating from a wyeth research program targeting acps - pptase ( e.g. , ucsd-18ae , figure 2 ) possessed no activity toward sfp - pptase and had overall weak activity . an additional publication from wyeth research came out shortly thereafter detailing further medicinal chemistry efforts leading to wyeth-16 ( figure 2 ) , which was optimized for potency toward acps - pptase ( ic50 = 1.4 m ) but had limited antimicrobial activity . on the basis of the limited antibacterial activity observed for these acps - pptase - specific compounds , we have put forth a hypothesis that the presence of an sfp - pptase in the bacterial genome provides an inborn mechanism of resistance . therefore , it is our presumption that a viable antibacterial agent must simultaneously target both classes of pptase enzymes to effectively halt bacterial proliferation . previously reported inhibitors of sfp- and acps - pptase . toward this end , we developed a strategy to search for small - molecule inhibitors of sfp - pptase . these efforts identified sch-202676 as the first compound reported to have activity against sfp - pptase ( figure 2 ) . however , compounds belonging to this class are known covalent modifiers and possess promiscuous activity ( 20% hit rate in pubchem assays ) , which ultimately limits their utility as probe compounds . therefore , we conducted a high - throughput screen ( hts ) in search of additional small - molecule inhibitors of sfp - pptase . herein we describe the discovery and sar investigation of the 2-pyridinyl - n-(4-aryl)piperazine-1-carbothioamides as best in class small - molecule inhibitors of sfp - pptase . this work led to the development of ml267 ( 55 ) , a new chemical probe and potent inhibitor of this bacterial enzyme . we demonstrate that 55 possesses the required selectivity , mechanism of action , physicochemical properties , and cellular activities to interrogate the role of pptases in bacterial metabolism , and we utilize this compound to test hypotheses about the importance of pptase to the function of whole bacterial cells . as shown in scheme 1 , compound 1 was readily synthesized by 1,1-thiocarbonyldiimidazole - assisted coupling of commercially available 4-methylpyridin-2-amine and 1-(3-(trifluoromethyl)phenyl)piperazine at 40 c . the same procedure was utilized to generate a small library of compounds ( table 1 ) around the 4-methylpyridine region using differentially substituted pyridylamines , 3-toluidine , and other heterocycles ( in table 1 , analogues 111 and 1921 ) . reagents and conditions : ( a ) 1,1-thiocarbonyldiimidazole ( tcdi ) , ch2cl2 , 40 c , 1 h. ic50 values represent the half - maximal ( 50% ) inhibitory concentration as determined in the hts assay , and the experiment was performed in triplicate . the term inactive refers to compounds with ic50 114 m . through bioisosteric replacement of the thiourea , analogues 1217 were synthesized utilizing known protocols reported for similar compounds in the literature as shown in scheme 2 ( see the supporting information for details ) . phenoxycarbonyl chloride - assisted coupling of 1-(3-(trifluoromethyl)phenyl)piperazine with 4-picolin-2-amine afforded the urea analogue 12 . analogue 13 was prepared by stirring the analogue 1 with ammonium hydroxide and sodium periodate at 80 c in a dmf compounds 14 and 15 , which represent the bioisosteric replacement of the thiourea functionality , were prepared by refluxing 1-(3-(trifluoromethyl)phenyl)piperazine and 4-picolin-2-amine with diphenyl n - cyanocarbonimidate ( for 14 ) or 2-[bis(methylthio)methylene]malononitrile ( for 15 ) in acetonitrile . the thiadiazole derivative 16 was prepared utilizing xanthphos - catalyzed amination of the 2-bromo-5-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)-1,3,4-thiadiazole in the presence of pd2(dba)3 and cesium carbonate . the kindler reaction of 2-acetyl-4-picoline and 2-formyl-4-picoline with 1-(3-(trifluoromethyl)phenyl)piperazine under microwave ( mw ) conditions furnished analogues 17 and 18 , respectively . reagents and conditions : ( a ) phococl , dipea , dmap , 0 c to rt , 3 h ; ( b ) nh4oh , naio4 , dmf h2o , 80 c , 1 h ; ( c ) acetonitrile , reflux , 16 h ; ( d ) acetonitrile , 70 c , 24 h ; ( e ) xantphos , pd2(dba)3t - buona , toluene , 110 c , 3 h ; ( f ) s , dmf , mw , 130 c , 0.5 h. to explore the sar around the 3-trifluorophenyl region as shown in table 2 , a robust synthetic method was required to generate a library of arylpiperazine precursors , since many of the targeted compounds were not commercially available . given the large number of available boronic acids , we found the chan - lam reaction to be an ideal method to generate several mono- and disubstituted arylpiperazine precursors for preparation of analogues 27 , 28 , 36 , and 40 . however , the use of a large excess of copper or a constant supply of oxygen gas is necessary to regenerate the copper(ii ) catalyst , making this method less compatible with parallel library synthesis . to overcome this drawback , we used a modified version of the chan - lam reaction reported by quach et al . which requires only 10 mol % copper(ii ) acetate but still uses an oxygen atmosphere . to facilitate analogue synthesis , we further modified this method by carrying out this reaction in a sealable microwave vial . in this case , all the reactants were mixed together , the vessel was charged with oxygen gas , and then the vessel contents were stirred at 45 c for 1224 h as shown in scheme 3a . several mono- and disubstituted arylpiperazines were prepared using this method followed by boc deprotection with trifluoroacetic acid in dichloromethane . ic50 values represent the half - maximal ( 50% ) inhibitory concentration as determined in the hts assay , and the experiment was performed in triplicate . reagents and conditions : ( a ) cu(oac)2 ( 10 mol % ) , 4 molecular sieves , o2 , ch2cl2 , 45 c , 1224 h ; ( b ) tfa / ch2cl2 , rt , 1 h ; ( c ) binap ( 10 mol % ) , pd(oac)2 ( 5 mol % ) , cs2co3 ( 1.5 equiv ) , toluene , 110 c , 1224 h ; ( d ) johnphos ( 10 mol % ) , pd2(dba)3 ( 5 mol % ) , t - buona ( 1.5 equiv ) , toluene , 110 c , 28 h ; ( e ) binol ( 20 mol % ) , cubr ( 20 mol % ) , k3po4 ( 2 equiv ) , dmf , rt , 1224 h ; ( f ) 1,1-thiocarbonyldiimidazole ( tcdi ) , ch2cl2 , 40 c ; ( g ) dmf , 90 c , 1 h. despite these encouraging results , this method was insufficient for use with several di- and trisubstituted aryl and heterocyclic boronic acids . amination of the requisite aryl or heteroaryl bromides was achieved using binap or johnphos ligands in combination with pd(oac)2 or pd2(dba)3 with sodium tert - butoxide or cesium carbonate in toluene . the arylation of boc - piperazine with aryl iodides was accomplished using a binol / cubr catalytic system in the presence of potassium phosphate in dmf at room temperature ( scheme 3b ) . subsequent boc deprotection with trifluoroacetic acid ( tfa ) in dichloromethane gave the desired free piperazinamines ( see the supporting information for details ) . as shown in scheme 3c , the commercially available arylpiperazines and compounds obtained from the above methods were treated with various 4,6-substituted pyridin-2-amines in the presence of 1,1-thiocarbonyldiimidazole to furnish analogues 2256 ( table 2 ) . to explore the sar of the piperazine core itself , analogues 5967 were synthesized using 1,1-thiocarbonyldiimidazole - assisted thiourea synthesis ( table 3 ) . the acyclic analogues 57 and 58 were prepared by condensation of the 4-picoline-2-isothiocyanate with corresponding acyclic amines as shown in scheme 3d . ic50 values represent the half - maximal ( 50% ) inhibitory concentration as determined in the hts assay , and the experiment was performed in triplicate . specifically , the synthesis of analogues 5963 , 65 , and 67 was accomplished by arylation of the requisite boc - protected amine cores with 3-trifluorophenyl iodide utilizing the 2-isobutyrylcyclohexanone / cui system ( scheme 4 ) , followed by 1,1-thiocarbonyldiimidazole - assisted coupling with 4-methylpyridin-2-amine . analogues 64 and 66 were synthesized utilizing the general procedure outlined in scheme 3 using commercially available precursors 6-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline and 4-(3-(trifluoromethyl)phenyl)piperidine , respectively . reagents and conditions : ( a ) 2-isobutyrylcyclohexanone , cs2co3 , cui , dmf , 70 c , 210 h ; ( b ) tfa , dcm , rt , 1 h ; ( c ) 1,1-thiocarbonyldiimidazole ( tcdi ) , ch2cl2 , 40 c . we profiled the molecular libraries small molecule repository ( at the time totaling 311 260 compounds ) for pptase inhibitors with a previously described activity assay for sfp - pptase . the highly miniaturized format enabled the experiment to be conducted at eight concentrations ranging from 3.6 nm to 114 m following the quantitative hts methodology . data generated by this experiment are deposited in the public database pubchem under assay identifier number ( aid ) 1490 . data analysis and chemoinformatic filtering identified 388 structurally diverse compounds that were selected for follow - up in a battery of assays . this characterization began with retest of freshly prepared serial dilutions of hit compounds in the sfp - pptase screening assay ( aid 2701 ) and included further activity profiling with acps - pptase ( aid 602360 ) . in addition , this set of compounds was evaluated with sfp - pptase in a gel - based phosphopantetheinylation assay to confirm biochemical inhibition with an orthogonal detection format ( aid 2707 ) . finally , since prior work has established the essential nature of pptases to bacterial viability , we profiled these compounds for antibacterial activity with bacillus subtilis hm489 , whose viability depends solely on sfp ( aid 602366 ) . the sum of these experiments identified 1 ( figure s1a , supporting information ) as a confirmed screening hit with inhibitory activity against both acps- and sfp - pptases ( figure s1b ) and modest antibacterial activity against b. subtilis . we subsequently confirmed the identity and integrity of the chemical matter by resynthesis and reaffirmed the above findings for the resynthesized sample . to further characterize the biochemical activity of 1 , we first addressed the use of fluorescent substrates in our primary and confirmatory assays . as recently highlighted , the potential exists that the observed inhibition could be a result of compound , we developed and implemented a label - free biochemical assay that assessed pptase activity on the basis of differences in the electrophoretic mobility of the apo and holo states of the carrier protein under native page conditions . these experiments revealed that 1 elicited inhibition through a mechanism independent of the fluorescent label , with a dose - dependent inhibition being observed in the presence of the natural coa and acyl carrier protein ( acp ) substrates ( figure s1e , f , supporting information ) . to better understand the mechanism through which 1 inhibits the target , we evaluated the effects that a varying substrate concentration had on potency . in these experiments , which utilized an hplc assay and operated under an initial - rates regimen , 1 exhibited a near constant ic50 of 300 nm when coa or apo - acp acceptor substrates were increased to concentrations 50- and 100-fold above their km values , respectively ( figure s1c , d , supporting information ) . this trend is consistent with a noncompetitive mechanism of inhibition and indicates that the compound binds to the free enzyme , enzyme substrate complex , or ternary complex with similar affinities at a location other than the active site : a form of allosteric modulation . finally , we probed the reversibility of inhibition with a rescue - by - dilution experiment that assessed the recovery of enzymatic activity after a large ( 100-fold ) dilution from conditions producing 90% inhibition of enzymatic activity . these experiments included control compounds pap and sch-202676 , the latter of which was discovered during assay optimization as an active compound from lopac . inhibition by the former proved to be reversible , as anticipated by the fact that it is one of the reaction products ( figure s2a , supporting information ) , while the latter was found be an irreversible inhibitor , a result that is consistent with other reports of the compound s reactivity ( figure s2b ) . parallel evaluation of our primary hit 1 ( figure s1 g , supporting information ) in the same manner revealed completely reversible inhibition of sfp , with enzymatic activity restored upon dilution of the enzyme1 solution in a compound - free buffer ( orange data symbols , figure s1 g ) , relative to a buffer containing a constant concentration of the compound ( green data symbols , figure s1 g ) . after confirming the activity of 1 , we planned systematic structural modifications to the various regions of the molecule in an effort to establish tractable sar . our first area of exploration was the western 2-aminopyridine moiety as shown in table 1 . removal of the 4-me group ( 2 ) resulted in only a slight drop in potency , whereas addition of electron - withdrawing groups such as 4-cf3 ( 4 ) and 4-c(o)ome ( 7 ) resulted in a significant loss of activity . moreover , removal of the pyridine nitrogen ( 5 ) or changing the position of the pyridine ( 6 ) also led to inactive compounds . thus , it appears that the pyridine nitrogen needs to be adjacent to the thiourea motif to maintain sfp - pptase inhibitory activity . conversely , modification with electron - donating groups , such as addition of an extra methyl group adjacent to the nitrogen ( 3 ) , 4-ome ( 8) , and other heterocycles ( 10 and 11 ) , led to compounds with activity comparable to that of 1 . with initial sar explorations of the western region of the molecule complete , we then focused our attention on the thiourea moiety . as shown in table 1 , replacement of the thiourea was not well tolerated with the urea ( 12 ) , guanidine ( 13 ) , and thiadiazole ( 16 ) all being inactive . attempts to replace the thiourea structural motif with bioisosteres such as cyanoguanidine ( 14 ) or a 1,1-dicyanoketene group ( 15 ) also led to complete loss of activity . methylation of the nitrogen ( 19 ) or changing the nitrogen from being a part of a ring to being exocyclic ( 20 ) resulted in loss of potency . interestingly , replacement of the 2-amino group with a methylene spacer ( 17 ) retained some activity toward sfp - pptase albeit much reduced , with an ic50 value of 5.8 m . as such , these data suggested that the thiourea moiety was clearly the most optimal group for the desired activity . however , the thiourea functionality is often considered an undesirable structural feature , primarily due to its potential in forming toxic metabolites via oxidation to the s - oxide or sulfinic acid ( typically p450 or fmo - catalyzed ) , followed by gsh - trapping . these covalent gsh adducts could ultimately lead to oxidative - stress - induced cell death . therefore , upon completion of the sar efforts , we planned to investigate the propensity of this undesired reaction to occur with our lead compound(s ) ( vide infra ) . given that the 4,6-dimethyl substitution in 3 displayed potency comparable to that of the 4-methylpyridine moiety of 1 , we decided to further investigate both pyridine derivatives when exploring the sar of the eastern phenyl region . in these efforts , a large number of analogues were synthesized , and representative compounds are shown in table 2 . overall , substitution of the pendant phenyl group was very well tolerated , with most of the analogues 2244 having comparable activity . the general trend suggested that electron - withdrawing groups are preferred , with most of these analogues exhibiting submicromolar potency . the analogues which showed the most potent activity included 29 ( r = 3,5-cf3 ) , 40 ( r = 3-cf3 - 4-cl ) , 41 ( r = 2-cf3 - 4-cl ) , and 43 ( r = 3,4,5-cl ) . in contrast , compounds in which electron - donating substituents were incorporated ( e.g. , 24 ( r = 3-nme2 ) and 34 ( r = 3,5-ome ) ) had weaker potency with ic50 values of 5.8 and 2.0 m , respectively . these data suggest that this region of the molecule may provide a handle for modulating the adme properties ( e.g. , solubility ) in future sar efforts given the general tolerance for structural modifications . with analogues 4556 ( table 2 ) , we aimed to investigate more significant structural changes to this region , including removal of the aryl group ( 45 ) or replacing it with smaller groups such as a methyl group ( 46 ) , and both changes significantly diminished the potency ( ic50 = 14.5 and 6.5 m respectively ) . addition of a methylene spacer between the aryl group and piperazine ( 48 ) was not well tolerated ( ic50 = 11.5 m ) . however , incorporation of a sulfonamide moiety ( 49 ) resulted in potency comparable to that of 1 . replacement of the aryl group with a thiadiazole ( 50 ) resulted in a 2-fold drop in potency . in an effort to improve the overall solubility of the molecule gratifyingly , this change was tolerated and led to the discovery of analogue 55 , which was ultimately determined to be our probe molecule ( ml267 ) after all compound attributes such as potency , in vitro adme properties , and antibacterial activity were taken into consideration ( vide infra ) . notably , compound 55 possesses a 4-ome group in contrast to the 4,6-dimethyl derivative , which led to a 23-fold improvement in potency ( ic50 = 0.81 m ( 54 ) vs ic50 = 0.29 m ( 55 ) ) but also contributed to improvement in other attributes mentioned above . we had synthesized and tested a small set of analogues containing the 4-methoxypyridine combined with the best arylpiperazines in the right - hand side of the molecule . these analogues showed similar or less potency compared to compound 55 and exhibited a consistent sar trend ( data not shown ) . our final area of sar exploration was modification of the piperazine linker , as shown in table 3 . a literature search uncovered troviridine [ 1-(5-bromopyridin-2-yl)-3-(2-(pyridin-2-yl)ethyl)thiourea ] ( also known as ly300045-hcl ) , a thiourea - containing small molecule that was originally developed by eli lilly as a non - nucleoside reverse transcriptase inhibitor but later was found to have antimicrobial properties . similar to our lead scaffold , troviridine contains a 2-aminopyridine attached to the thiourea moiety ; however , instead of a piperazine , it contains an acyclic linker . therefore , we replaced the piperazine core with two acyclic linkers ( 57 and 58 ) ; however , these compounds lost all activity . despite this , we were encouraged that a compound with a structure comparable to that of our lead had advanced into human trials . moreover , the inactivity of troviridine - like analogues 57 and 58 assured us that our compound has a divergent mechanisms of action , particularly in reference to the antimicrobial activity . additional sar explorations of the piperazine motif included varying the ring size to smaller , larger , and fused ring systems . the smaller rings showed similar activity ( 60 ) , whereas larger rings ( 67 ) or the substituted piperazine ( 62 ) had reduced potency . the fused pyrrolidine motif ( 65 ) had comparable potency ( ic50 = 0.73 m ) , but the solubility was markedly reduced . interestingly , when the nitrogen linked to thiourea is exocyclic ( e.g. , 59 ) , all activity is lost , whereas the piperazine nitrogen attached to an aryl ring is not essential ( e.g. , 66 , ic50 = 0.81 m ) . initial sar explorations around compound 1 led to a thorough understanding of the structural features that are required for activity . thus , we then sought to more thoroughly characterize the best sfp - pptase inhibitors for their activity against acps - pptase and antibacterial activity . additionally , we profiled this panel for activity with the human pptase , an important antitarget , and cytotoxicity with human hepg2 cells ( table 4 ) . as mentioned above , our primary target of interest was sfp - pptase , yet activity against acps - pptase is desirable for maximal antimicrobial activity . as shown in table 4 , most compounds display activity toward acps - pptase , but the ic50 values were generally 510-fold less potent . to assess acute cytotoxicity , we chose to profile the top compounds using hepg2 ( hepatocellular carcinoma ) cells since hepatotoxicity has been reported for some thiourea - containing compounds , and this profiling not only reveals potential toxicity of the parent compound , but also its metabolites . while this method is certainly not to be considered a thorough investigation of toxicity , it provided us a means to quickly profile numerous compounds . notably , despite the generally favorable activity of the original compound 1 , it demonstrated moderate toxicity toward this cell line , whereas most other compounds were inactive . finally , as part of our initial biological activity profile , we sought to further characterize the compounds for antibacterial activity using the b. subtilis hm489 strain . this strain contains sfp as the only locus encoding a functional pptase gene product , making the allele essential to viability of this organism . these experiments revealed that we had modest inhibitors of bacterial growth that generally tracked with sar in the biochemical assay . this important finding is exemplified by urea derivative 12 , which was inactive against sfp / acps - pptase and lacked activity in the antibacterial assays ( table 4 ) . while the antibacterial activity of these compounds was modest in these high - throughput assays , in subsequent antibacterial studies using more traditional methods of minimum inhibitory concentration ( mic ) determination , we found the compounds to be generally more potent ( vide infra ) . after careful analysis of the data in table 4 , and profiling of select compounds for their in vitro adme properties , 55 emerged as the compound with the best balance of properties . in these profiling studies , 55 demonstrated dual activity toward the bacterial sfp- and acps - pptase targets ( figure s4 , supporting information ) , presenting ic50 values of 290 nm and 8.1 m , respectively . furthermore , we profiled top compounds for activity with the human pptase , an important antitarget . while we observed inhibition of this enzyme with pap and sch202676 , 55 exhibited no inhibition at concentrations up to 125 m ( figure s4b , lower panel ) . comparison of these data to the inhibition observed in the same biochemical assay for sfp - pptase ( figure s4b , upper panel ) indicated the selectivity index , the ratio of inhibition observed for the bacterial target relative to the human enzyme , to be greater than 500-fold . sfp - pptase and acps - pptase ic50 values were determined using the hts assay . compound toxicity toward hepg2 cells was assessed by measuring the cellular atp content using a luciferase - coupled atp quantitation assay ( celltiter - glo , promega corp . , antibacterial activity against b. subtilis ( hm489 ) was accessed by measuring the cellular atp content using a luciferase - coupled atp quantitation assay ( bac - titer glo , promega corp . ) . to evaluate the antibacterial spectrum of activity possessed by 55 , we assembled a panel of microorganisms which included b. subtilis strains hm489 , 168 , and okb105 , which possess all possible pptase genotype combinations sfp / acps , sfp / acps , and sfp / acps , respectively , in a strain 168 isogenic background , as well as e. coli k12 and the laboratory strain bw25113 . with respect to human pathogens , the panel contained pseudomonas aeruginosa atcc9028 , a gram - negative organism of clinical importance , as well as three strains of methicillin - sensitive and community - acquired methicillin - resistant staphylococcus aureus ( atcc6538 , usa300 , and usa500 ) . finally , the panel also contained fluconazole - sensitive and -resistant strains of candida albicans as archetypical fungal pathogens ( atcc90028 and atcc96901 , respectively ) . compound 55 displayed a spectrum of activity that targets gram - positive organisms , where , in addition to inhibiting the growth of b. subtilis at a concentration of 1.7 g / ml , it also thwarted the growth of both methicillin - sensitive and -resistant strains of s. aureus in a similar concentration range [ figure 3 , where the growth indicator resazurin ( purple ) is reduced to resorufin ( pink ) by metabolically active microorganisms]. no growth inhibition was observed for the gram - negative organisms e. coli and p. aeruginosa , a result that was investigated further ( vide infra ) , or for fungal organisms . the finding of null activity with eukaryotic microbes was not discouraging , as total coverage of the panel would have been taken as nonspecific inhibition of growth and could be considered suspicious for an antibacterial candidate . broth dilution experiments were performed to assess the microbicidal activity of 55 with different organisms . in these experiments , growth is detected with the indicator resazurin , a purple dye which is reduced to the pink compound resorufin by metabolically active organisms . the antibacterial activity of 55 was restricted to gram - positive organisms , represented here by strains of b. subtilis and s. aureus , with no growth inhibition observed for gram - negative bacteria or fungi . we followed up on this finding with a study of bacterial cell membrane integrity , as gram - positive whole - cell experiments can be convoluted by nonspecific damage to the lipid bilayer . we employed the differential nucleic acid staining of b. subtilis 168 bacterial cells by syto9 and propidium iodide in the baclight live / dead microscopy assay , which utilizes green and red staining to differentiate healthy and compromised cells , respectively . chloramphenicol ( cam ) and cetyltrimethylammonium bromide ( ctab ) , two compounds that possess minimum inhibitory concentration ( mic ) values similar to that of 55 ( 2 g / ml ) , were included as controls to serve as known specific and nonspecific agents , respectively . testing of these agents indicated a rapid compromise of bacterial cell integrity for ctab after 15 min of incubation , where similar testing of chloramphenicol and 55 revealed continued cellular viability over the short incubation period at concentrations that exceeded mic by 10-fold ( figure 4a ) . therefore , we concluded that compound 55 does not elicit antibacterial activity through the nonspecific disruption of the gram - positive membrane and likely acts on an intracellular target . compound 55 does not disturb bacterial membranes and is expelled by efflux pumps in gram - negative bacteria . ( a ) microscopy experiments using the exclusion of propidium iodide ( pi ) were conducted to evaluate the effects of 55 on the integrity of the membrane in b. subtilis . in these , syto9 stains dna of all bacteria green , while pi can only penetrate compromised bacterial cells . cam , ctab , and 55 were evaluated at 1 , 4 , and 10 their mic values , and the number of observed cells was counted . chloramphenicol ( cam ) , a non - membrane - active antibiotic , and cetyltrimethylammonium bromide ( ctab ) , a known membrane - disrupting agent , were included as controls for the experiment . ( b ) disruption of tolc , the primary efflux pump of e. coli , induced a 55-sensitive phenotype in broth dilution experiments , where concentrations as low as 12 m inhibited bacterial growth ( red - outlined well ) . ( c ) building upon the results of ( b ) , checkerboard synergy experiments were conducted with the acrab - tolc inhibitor phenylarginine--napthamide ( pan ) and demonstrate an effective chemical knockout strategy to induce a 55-sensitive phenotype in wild - type e. coli . wells containing no metabolically active bacteria are observed as purple in this experiment , and wells corresponding to the lowest concentration of pan capable of synergizing with 55 to halt bacterial proliferation at each concentration of 55 are outlined in red . with this information , we further characterized the antibacterial activity of 55 to discern whether the compound merely halted proliferation of bacteria or possessed bactericidal activity . the results of these experiments , summarized in table s1 ( supporting information ) , demonstrated that the minimum bactericidal concentration ( mbc ) of 55 fell within a range of 14 the mic in s. aureus and b. subtilis . the generally accepted criteria of defining antibacterial activity as cidal is a ratio of mbc to mic less than or equal to 4 . by this definition , the limited spectrum of activity possessed by 55 was surprising , since our biochemical testing indicated that the compound was active with acps in the direct enzymatic assay . this led us to hypothesize that the gram - negative outer membrane was the source of this complication , as it presents a formidable barrier possessing both low penetrability and an active efflux mechanism . acrb gene products constitutes the primary efflux pump and source of resistance through this mechanism . thus , we tested e. coli bw25113 and jw0451 , the wild - type strain and an efflux - defective mutant that contains a lesion in the acrb locus , respectively , for susceptibility to 55 . tolc system would manifest as an observed hypersusceptibility of the acrb strain , while insensitivity would indicate cellular penetration or another mechanism as the source of resistance . for these experiments , we observed a clear differential susceptibility between the two strains ( figure 4b ) , where disruption of the acrb locus rendered the organism susceptible to 55 at concentrations as low as 12.5 g / ml . these findings indicated that efflux is the primary mechanism of resistance in this organism and that 55 is capable of penetrating the gram - negative bacterial cell . to further validate the above findings using a chemical genetic model , we tested 55 for synergistic activity with the broad - spectrum efflux pump inhibitor phenylarginine--naphthamide ( pan ) . in these experiments , neither compound alone was found to possess antibacterial activity against the wild - type strain bw25113 at the top concentrations tested , but offered pronounced growth inhibition in combination ( figure 4c ) . the fractional inhibition concentration index , a ratio of the potency of the combined test articles relative to that of the singular components , was estimated to be less than 0.25 , a finding strongly indicative of synergism , although we were not able to precisely determine this parameter since neither compound elicited growth inhibition at the top concentrations tested . to further understand the on - target engagement of pptase enzymes within the bacterial cell , we exploited the association of sfp - pptase with secondary metabolism in b. subtilis . the lipopeptide surfactin 68 is produced by a nonribosomal peptide synthetase pathway , diagrammed in figure 5a . this canonical megasynthase complex contains 24 functional domains , 7 of which are 4-pp - accepting carrier protein domains . a singly missed 4-pp transfer event will disrupt processivity in the megasynthase and render the complex incapable of producing the metabolite . as such , this system represents a strategy to study target engagement by 55 in the bacterial cell through monitoring of the fermentative yield of 68 . toward this end , we conducted time course experiments for cultures of b. subtilis in the presence of increasing concentrations of 55 , where we periodically measured both bacterial growth and surfactin titer over 28 h ( figure 5b , c ) . given the potent antibacterial activity of 55 against this organism , delicate selection of test conditions was required to supply high enough concentrations of compound to elicit a response but also provide for normal bacterial growth . in these experiments , cultures treated with 55 at concentrations of 0.53 or 1.1 g / ml resulted in 33% and 41% reductions in surfactin titer relative to a vehicle control after 28 h of culture , respectively ( figure 5b ) . these reductions in titer were accompanied by modest extensions in the culture lag phase , although continued culture revealed that 55 had a null effect on both the doubling time in the log phase and final cellular densities ( figure 5c ) . this dose - dependent attenuation of surfactin titer in cultures which accumulated similar quantities of biomass is consistent with engagement of sfp - pptase by 55 inside living bacteria . ( a ) surfactin 68 is produced by a nonribosomal peptide synthetase pathway in b. subtilis that contains 24 distinct functional domains , represented as cubes . seven of these are cp domains that require post - translational activation by sfp - pptase , and the monitoring of the fermentative yield provides a means to assess the blockade of pptase - dependent processes in the bacterial cell . ( b ) culture time course experiments demonstrate that sublethal concentrations of 55 attenuate surfactin production by b. subtilis okb105 . ( c ) culture density data are plotted for the culture time course experiment in ( b ) and demonstrate a modest effect of 55 on the growth of the cultures , indicating that the compound did not impede their ability to reach a terminal density . while our preliminary studies involved some characterization of the in vitro adme properties , after selection of 55 as a lead compound , a more detailed analysis was obtained . as shown in table 5 , 55 has a modest solubility in pbs buffer ( ph 7.4 ) of 20 m , which is approximately 60 times higher than the recorded ic50 value ( 290 nm ) . moreover , 55 displayed stability to mouse and rat liver microsomes with t1/2 values of 49.5 and > 30 min , respectively . the compound was completely stable ( measured for 1 h ) in the absence of the nadph cofactor , suggesting a cyp - mediated oxidation event . passive permeability was assessed using the pampa assay , and very favorable permeability was observed ( 1122 10 cm / s ) . next , we wanted to examine whether the probe compound inhibits specific cytochrome p450 enzymes 2d6 and 3a4 as these two isoforms account for the metabolism of approximately 80% of drugs . to assess the potential for cyp inhibition and drug drug interactions ( ddis ) , we looked at the effect of coincubation of our compound(s ) at 3 m with known cyp substrates ( 2d6 with dextromethorphan and 3a4 with 6-hydroxytestosterone ) . we found that 55 exhibits modest cyp inhibition of 25% and 20% for 2d6 and 3a4 , respectively . to further elucidate the potential for cyp liabilities , follow - up ic50 values and testing of a more expansive representation of cyp isoforms will be required . however , we also investigated cyp inhibition for other analogues , and inhibition was inconsistent across the series , so we are confident that these liabilities could be addressed through targeted structural modifications . given the potential instability of the thiourea moiety in aqueous and biological media , we were encouraged that 55 showed no signs of degradation in aqueous media at a wide ph range ( 29 ) , buffers ( pbs and hepes ) ( figure s7 , supporting information ) , and mouse plasma ( table 5 ) . in addition , the thiourea functionality is a known structural alert that can cause toxicity due to formation of reactive metabolites under physiological conditions ( vide supra ) . however , our probe compound 55 and analogue 41 are not susceptible to bioactivation and did not show any gsh adducts in follow - up studies ( table s3 , supporting information ) . aqueous solubility ( pbs buffer ) , mouse liver microsome ( mlm ) stability , cyp2d6/3a4 inhibition , and plasma stability were determined at pharmaron inc . dextromethorphan and 6-hydroxytestosterone were the substrates used for the cyp2d6 and cyp3a4 inhibition studies , respectively . having demonstrated a favorable in vitro adme profile of 55 , we next sought to investigate the in vivo pk profile in support of testing this compound in proof of concept ( poc ) antibacterial animal models ( vide infra ) . as shown in table 6 , 55 was administered to cd1 mice via both iv and ip routes at 3 and 30 mg / kg doses , respectively , using a solution formation consisting of 5/10/85 ( w / w / w ) dmso , solutol , and water . compound 55 exhibits favorable systemic exposure levels ( aucinf = 68860 hng / ml ) that are 4050-fold higher than the mic values against s. aureus strains at 30 mg / kg dosing . moreover , the compound has a reasonable t1/2 ( 2.0 h ) , and low clearance ( 7.2 ml / min / kg ) results in an exposure ( total drug concentration , not free drug concentration ) that exceeds the in vitro antibacterial mic value for over 8 h. the compound also efficiently crosses the bbb and thus could be potentially used for bacterial infections which reside in the brain . furthermore , dosing of 55 at these concentrations did not result in any adverse clinical observations over the 24 h period , which seems to indicate that no acute toxicity is to be expected at the doses used for in vivo poc studies . data were collected in triplicate at eight time points over a 24 h period . 55 was formulated as a solution ( 5% dmso and 10% solutol in h2o ) . for iv , mrt = mean residence time ( the time for elimination of 63.2% of the iv dose ) . as discussed above , 55 exhibits antibacterial activity against mrsa and has a desirable adme / pk profile , and thus , it was poised to be tested in vivo . for these studies , we profiled a panel of clinical isolates to assess the susceptibility of various genotypes ( table s2 , supporting information ) , and this revealed similar potencies ( within 3-fold ) for all strains tested . with this information , we pursued an in vivo poc study that utilized the mrsa - usa500 strain ( bk2395 ) since our compound had demonstrated favorable in vitro activity against it and it has demonstrated suitable virulence in mouse models of sepsis . unfortunately , in this experiment , 55 did not increase survival compared to the vehicle control , while vancomycin treatment completely protected animals from morbidity ( figure s6 , supporting information ) . these findings led us to investigate the source of discrepancy between our in vitro and in vivo results . to rule out the rapid development of resistance in vivo , bacteria were harvested from the kidneys of mice treated with the vehicle or 55 . for each crude we then determined mic values for these strains in parallel along with the parental mrsa - usa500 strain and observed identical susceptibility of all strains to 55 . these results indicated that neither passage of the organism in mice nor challenge with 55 in vivo led to reduced susceptibility to the compound . realizing that the antibacterial activity of compounds can be attenuated in the presence of whole blood and/or serum , we repeated the mic determinations of 55 in the presence of calf serum ( 20% , v / v ) in cation - adjusted mueller hinton ii broth and observed a significant shift in potency ( > 57 m vs 3.4 m ) . these data , along with subsequent analysis via equilibrium dialysis , revealed that 55 displays a high degree ( 98.6% ) of plasma protein binding ( ppb ) , and thus , the lack of activity is likely a consequence of the lipophilic nature of this compound . while disappointing , other groups have shown the ability to successfully modulate the lipophilicity of the lead compound through the strategic placement of polar moieties to improve the antibacterial activity in vivo . in fact , a group of researchers at pfizer recently reported improved in vivo efficacy of their lead antibacterial candidate by lowering ppb via lowering clogp . as described above , the eastern region of the molecule is highly tolerant to structural modifications ( table 2 ) , which could be exploited to modulate the clogp and thus hopefully improve activity in vivo . in addition , we plan on conducting more extensive pk studies to look at tissue distribution and free drug concentration , which may guide not only a future medicinal chemistry effort , but also aid the design of our in vivo proof of concept models . over the course of this work , a heated debate has emerged as to the suitability of the fas as a drug target . it appears that , in some gram - positive pathogens , fas is dispensable when bacteria are propagated in the presence of serum , which is a nutrient source rich in fatty acids . further study has found that the results may not be accurately predictive for even closely related bacteria , and further investigation of this phenomenon is warranted to tease apart the details with finer resolution . nonetheless , this discrepancy provides another mechanism through which bacterial tolerance to 55 may be occurring in vivo , and such a mechanism may confound the advancement of in vitro pptase - targeting inhibitors through in vivo poc studies of staphylococcal septicemia . however , this does not speak to the role that pptase plays in the production of virulence factors and elaborate cell wall components , especially in the case of mycobacterium spp . , where recent findings have confirmed the essential nature of both acps- and sfp - pptases . these organisms depend on the concerted effort of fas and pks systems for the assembly of mycolic acids , and these pptase dependencies have been substantiated in rodent models of infection . thus , pptase inhibitors remain a target worthy of pursuit and may find a more immediate applicability as tools to pharmacologically probe the role of pptases in mycobacterial pathogenesis . pptase enzymes catalyze an essential ptm that activates the assembly of fatty acid , polyketide , and nonribosomal peptides . metabolites from all three of these classes are necessary for bacterial cell viability and virulence . as a result , inhibition of these pathways has received attention as an attractive approach to the development of new antimicrobial compounds . rather than targeting the machinery involved in the production of a single metabolite , we have chosen to investigate the inhibition of this ptm as it represents a unifying feature between these pathways . pptase inhibitors may leverage this overlap to downregulate the production of key cellular components and virulence - determining factors . the critical role of this ptm in bacterial metabolism has been studied by others , but to date these efforts have not produced compounds with appreciable antibacterial activity . this limited success can likely be attributed to the inability of these programs to consider the presence of an sfp - pptase in addition to their primary target , acps - pptase . this secondary enzyme is capable of compensating for loss of the acps locus and thus provides a direct mechanism through which bacterial organisms may develop resistance through simple transcriptional / translational upregulation . to address this issue , we have pursued the development of a chemical probe with dual pptase inhibitory activity . here we have detailed the development and characterization of 55 , a novel small molecule that possesses these characteristics . we have demonstrated the qualities of this chemical probe and evaluated it according to the benchmarks suggested by the community . the features of 55 can be summarized according to the five principles of a quality chemical probe as follows : ( i ) we have performed detailed molecular profiling with relevant molecular targets , with observed potencies of 290 nm and 8.1 m against the primary development target sfp - pptase and bacterial orthologue acps , respectively , which is contrasted by null activity with the human enzyme . in the context of an antibacterial discovery campaign , where the end goal is the inhibition of bacterial growth in man , this indicates a selectivity index of > 500-fold with respect to the human orthologue . ( ii ) with respect to the mechanism of action , we demonstrate that the chemotype inhibits sfp - pptase independent of a fluorescent label through an allosteric mechanism that is noncompetitive with substrates and is rapidly reversible . ( iii ) regarding the identity of the active species , synthetic / medicinal chemistry efforts verified the structure , purity , stability , and chemical tractability of the chemotype . in addition , a structurally similar but inactive control was identified which was devoid of acps / sfp - pptase inhibitory activity as well as antibacterial activity . ( iv ) to demonstrate the utility of 55 as a probe , we confirmed the ability of a dual - specific pptase inhibitor to thwart the growth of bacteria in the absence of a rapid cytotoxic response . additionally , 55 demonstrated activity against clinically relevant microorganisms , where it stifled the growth of methicillin - resistant s. aureus . expanding on these findings , we also identified the primary mechanism of resistance in e. coli to be efflux by the acrab tolc system and demonstrated a chemical genetic approach to mitigate this issue and extend the spectrum of antibacterial activity to include gram - negative organisms . we recognize that optimizing a chemotype to circumvent efflux is a formidable task and is exacerbated in clinically relevant gram - negative pathogens where efflux pathways are more complex than in the model organism e. coli . beyond these assessments of antimicrobial activity , the ability of 55 to attenuate the production of surfactin , a metabolite dependent on the biochemical functionality of sfp - pptase ( figure 5b ) , strongly indicates that 55 is acting on - target inside the bacterial cell . ( v ) finally , in conjunction with the spirit and principles of the scientific community and the probe characteristic of availability , we will provide samples of 55 freely upon request . all air- or moisture - sensitive reactions were performed under positive pressure of nitrogen with oven - dried glassware . anhydrous solvents such as dichloromethane , n , n - dimethylformamide ( dmf ) , acetonitrile , methanol , and triethylamine were purchased from sigma - aldrich . the column used was a phenomenex luna c18 ( 5 m , 30 75 mm ) at a flow rate of 45 ml / min . the mobile phase consisted of acetonitrile and water ( each containing 0.1% trifluoroacetic acid ) . analytical analysis was performed on an agilent lc / ms system ( agilent technologies , santa clara , ca ) . method 1 : a 7 min gradient of 4100% acetonitrile ( containing 0.025% trifluoroacetic acid ) in water ( containing 0.05% trifluoroacetic acid ) was used with an 8 min run time at a flow rate of 1 ml / min . a phenomenex luna c18 column ( 3 m , 3 75 mm ) method 2 : a 3 min gradient of 4100% acetonitrile ( containing 0.025% trifluoroacetic acid ) in water ( containing 0.05% trifluoroacetic acid ) was used with a 4.5 min run time at a flow rate of 1 ml / min . a phenomenex gemini phenyl column ( 3 m , 3 100 mm ) purity determination was performed using an agilent diode array detector for both method 1 and method 2 . mass determination was performed using an agilent 6130 mass spectrometer with electrospray ionization in the positive mode . chemical shifts are reported in parts per million with undeuterated solvent ( dmso - d6 at 2.49 ppm ) as the internal standard for dmso - d6 solutions . all of the analogues tested in the biological assays have purity greater than 95% on the basis of both analytical methods . high - resolution mass spectrometry was recorded on an agilent 6210 time - of - flight lc / ms system . confirmation of the molecular formula was accomplished using electrospray ionization in the positive mode with the agilent masshunter software ( version b.02 ) . a mixture of substituted pyridin-2-amine ( 1 equiv ) and 1,1-thiocarbonyldiimidazole ( tcdi ) ( 1.05 equiv ) in dichloromethane ( 2 ml / mmol ) was stirred for 15 min at room temperature . to the clear yellow solution was added arylpiperazine ( 1.1 equiv ) , and the reaction mixture was stirred at 40 c for 1 h. the solvent was evaporated , and the crude product was taken up in 2 ml of dmso and purified via reversed - phase chromatography to give the products as tfa salts ( the details and characterization for all the compounds is depicted in the supporting information ) . this compound was prepared following the above general procedure starting from commerically available 4-methylpyridin-2-amine and 1-(3-(trifluoromethyl)phenyl)piperazine : lc / ms retention time t1 ( method 1 , 7 min ) = 4.91 min and t2 ( method 2 , 3 min ) = 3.13 min ; h nmr ( 400 mhz , dmso - d6 ) 10.09 ( s , 1h ) , 8.22 ( d , j = 5.4 hz , 1h ) , 7.537.42 ( m , 2h ) , 7.337.18 ( m , 2h ) , 7.167.07 ( m , 1h ) , 7.03 ( d , j = 5.4 hz , 1h ) , 4.08 ( t , j = 5.2 hz , 4h ) , 3.40 ( dd , j = 6.5 , 3.9 hz , 4h ) , 2.36 ( s , 3h ) ; c nmr ( 101 mhz , dmso - d6 ) 181.16 , 158.49 , 153.14 , 150.95 , 130.53 , 130.49 , 130.22 , 126.22 , 120.70 , 119.17 , 118.96 , 115.15 , 111.29 , 111.25 , 48.49 , 47.41 , 21.38 ; hrms ( esi ) m / z ( m + h ) calcd for c18h20f3n4s 381.1355 , found 381.1341 . this compound was prepared following the above general procedure starting from commerically available 4-methylpyridin-2-amine and 1-(2-(trifluoromethyl)phenyl)piperazine : lc / ms retention time t1 ( method 1 , 7 min ) = 4.993 min and t2 ( method 2 , 3 min ) = 3.215 min ; h nmr ( 400 mhz , dmso - d6 ) 8.18 ( d , j = 5.3 hz , 1h ) , 7.767.57 ( m , 3h ) , 7.467.34 ( m , 2h ) , 6.96 ( d , j = 5.1 hz , 1h ) , 4.093.95 ( m , 4h ) , 2.93 ( m , 4h ) , 2.32 ( s , 3h ) ; hrms ( esi ) m / z ( m + h ) calcd for c18h20f3n4s 381.1355 , found 381.1361 . this compound was prepared following the above general procedure starting from intermediate 1-(4-chloro-2-(trifluoromethyl)phenyl)piperazine ( see the supporting information for the synthesis ) and commerically available 2,4-dimethylpyridin-2-amine : lc / ms retention time t1 ( method 1 , 7 min ) = 5.384 min and t2 ( method 2 , 3 min ) = 3.247 min ; h nmr ( 400 mhz , dmso - d6 ) 7.75 ( dd , j = 7.1 , 2.5 hz , 2h ) , 7.687.59 ( m , 1h ) , 7.23 ( s , 1h ) , 6.85 ( s , 1h ) , 4.01 ( d , j = 5.6 hz , 4h ) , 2.93 ( q , j = 4.6 , 3.7 hz , 4h ) , 2.40 ( s , 3h ) , 2.29 ( s , 3h ) ; hrms ( esi ) m / z ( m + h ) calcd for c19h21clf3n4s 429.1122 , found 429.1133 . this compound was prepared following the above general procedure starting from commerically available precursors 2,4-dimethylpyridin-2-amine and 1-(5-(trifluoromethyl)pyridin-2-yl)piperazine : lc / ms retention time t1 ( method 1 , 7 min ) = 4.615 min and t2 ( method 2 , 3 min ) = 3.114 min ; h nmr ( 400 mhz , dmso - d6 ) 8.45 ( d , j = 2.5 hz , 1h ) , 7.85 ( dd , j = 9.2 , 2.6 hz , 1h ) , 7.29 ( s , 1h ) , 6.96 ( d , j = 9.5 hz , 2h ) , 4.103.99 ( m , 4h ) , 3.79 ( dd , j = 6.5 , 4.0 hz , 4h ) , 2.44 ( s , 3h ) , 2.33 ( s , 3h ) ; hrms ( esi ) m / z ( m + h ) calcd for c18h21f3n5s 396.1464 , found 396.1469 . this compound was prepared following the above general procedure starting from commerically available precursors 2,4-dimethylpyridin-2-amine and 1-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine : lc / ms retention time t1 ( method 1 , 7 min ) = 5.113 min and t2 ( method 2 , 3 min ) = 3.287 min ; h nmr ( 400 mhz , dmso - d6 ) 8.58 ( d , j = 2.1 hz , 1h ) , 8.23 ( d , j = 2.1 hz , 1h ) , 7.25 ( s , 1h ) , 6.93 ( s , 1h ) , 4.06 ( dd , j = 6.5 , 3.6 hz , 4h ) , 3.61 ( dd , j = 6.6 , 3.7 hz , 4h ) , 2.43 ( s , 3h ) , 2.31 ( s , 3h ) ; hrms ( esi ) m / z ( m + h ) calcd for c18h20clf3n5s 430.1075 , found 430.1089 . this compound was prepared following the above general procedure starting from commerically available precursors 4-methoxypyridin-2-amine and 1-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine : lc / ms retention time t1 ( method 1 , 7 min ) = 4.88 min and t2 ( method 2 , 3 min ) = 3.04 min ; h nmr ( 400 mhz , dmso - d6 ) 8.56 8.61 ( m , 1h ) , 8.168.26 ( m , 2h ) , 7.15 ( s , 1h ) , 6.83 ( s , 1h ) , 4.054.13 ( m , 4h ) , 3.88 ( s , 3h ) , 3.583.67 ( m , 4 h ) ; c nmr ( 101 mhz , dmso - d6 ) 180.45 , 168.50 , 159.01 , 153.62 , 143.08 , 136.44 , 136.41 , 124.78 , 122.09 , 119.61 , 118.77 , 118.44 , 107.22 , 102.47 , 102.37 , 56.43 , 56.29 , 48.06 , 47.56 ; hrms ( esi ) m / z ( m + h ) calcd for c17h18clf3n5os 432.0867 , found 432.0856 . a mixture of 4-methylpyridin-2-amine ( 0.1 g , 0.925 mmol , 1 equiv ) and tcdi ( 0.165 g , 0.925 mmol , 1.0 equiv ) in dichloromethane ( 3 ml ) was stirred for 15 min at room temperature . to the clear yellow solution was added commercially available n-(3-(trifluoromethyl)phenyl)piperidin-4-amine ( 0.226 g , 0.925 mmol , 1 equiv ) , and the resulting solution was stirred at 40 c for 1 h. the solvent was evaporated , and the crude product was taken up in 2 ml of dmso and purified via reversed - phase chromatography to give the product as a tfa salt : lc / ms retention time t1 ( method 1 , 7 min ) = 4.954 min and t2 ( method 2 , 3 min ) = 3.025 min ; h nmr ( 400 mhz , dmso - d6 ) 8.21 ( d , j = 5.4 hz , 1h ) , 7.39 ( d , j = 1.6 hz , 1h ) , 7.317.22 ( m , 1h ) , 7.057.00 ( m , 1h ) , 6.916.85 ( m , 2h ) , 6.836.77 ( m , 1h ) , 5.50 ( br s , 1h ) , 4.54 ( d , j = 13.2 hz , 2h ) , 3.68 ( tt , j = 9.4 , 3.9 hz , 1h ) , 3.43 ( ddd , j = 13.7 , 11.1 , 2.8 hz , 2h ) , 2.35 ( s , 3h ) , 2.071.89 ( m , 2h ) , 1.43 ( dtd , j = 13.6 , 10.2 , 3.8 hz , 2h ) ; hrms ( esi ) m / z ( m + h ) calcd for c19h22f3n4s 395.1512 , found 395.1510 . to a mixture of 4-methylpyridin-2-amine ( 0.5 g , 4.62 mmol , 1 equiv ) , ( i - pr)2net ( 0.970 ml , 5.55 mmol , 1.2 equiv ) , and dmap ( 0.113 g , 0.925 mmol , 0.2 equiv ) in dichloromethane ( 25 ml ) was added phenyl carbonochloridate ( 0.664 ml , 5.09 mmol , 1.1 equiv ) at 0 c , and the reaction was allowed to stir at room temperature for 1 h. to the resulting clear solution was added 1-(3-(trifluoromethyl)phenyl)piperazine ( 1.13 ml , 6.01 mmol , 1.3 equiv ) , and the reaction mixture was stirred for an additional 2 h at room temperature . volatiles were removed by forced air , and the residue was dissolved in dmf and purified via reversed - phase chromatography to give 12 as a tfa salt : lc / ms retention time t1 ( method 1 , 7 min ) = 4.43 min and t2 ( method 2 , 3 min ) = 3.095 min ; h nmr ( 400 mhz , dmso - d6 ) 10.21 ( s , 1h ) , 8.17 ( d , j = 5.9 hz , 1h ) , 7.547.39 ( m , 2h ) , 7.297.18 ( m , 2h ) , 7.177.05 ( m , 2h ) , 3.713.63 ( m , 4h ) , 3.313.29 ( m , 4h ) , 2.40 ( s , 3h ) ; c nmr ( 101 mhz , dmso - d6 ) 158.27 , 153.81 , 150.90 , 150.53 , 140.63 , 130.07 , 130.04 , 129.76 , 119.59 , 119.03 , 115.02 , 114.70 , 111.33 , 111.29 , 47.41 , 43.50 , 21.45 ; hrms ( esi ) m / z ( m + h ) calcd for c18h20f3n4o 365.1584 , found 365.1590 . the assay was performed in 50 mm hepesna ( ph 7.6 ) , 10 mm mgcl2 , 0.01% nonidet p-40 , and 0.01% bsa . a 3 l volume of each reagent , consisting of buffer ( in columns 3 and 4 as a negative control ) and sfp- or acps - pptase ( in columns 1 , 2 , and 548 , final concentration of 15 or 100 nm , respectively ) were dispensed into a 1536-well greiner black solid - bottom plate . compounds ( 23 nl ) were transferred via a kalypsys pintool equipped with a 1536-pin array . the plate was incubated for 15 min at room temperature , followed by the addition of 1 l of substrate ( final concentrations for rhodamine coa and bhq-2-ybbr were 5 and 12.5 m , respectively ) to start the reaction . the plate was then centrifuged at 1000 rpm for 15 s , and the fluorescence intensity was recorded on a viewlux high - throughput charge - coupled device ( ccd ) imager ( perkin - elmer ) using standard bodipy optics ( 525 nm excitation and 598 nm emission ) . the plate was then incubated for 30 or 60 min ( sfp or acps , respectively ) , and a second read on the viewlux was performed . the fluorescence intensity difference over the 30 or 60 min period ( sfp or acps , respectively ) was used to calculate the respective reaction rate for each well . all screening operations were performed on a fully integrated robotic system ( kalypsys inc . , san diego , ca ) as described elsewhere . plates containing dmso only ( instead of compound solutions ) were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or a decrease in enzyme specific activity . a dmso solution of confirmed hits ( 0.5 l ) was added to a 1.33 enzyme solution ( 15 l , containing 26.6 nm sfp , 66 mm hepesna , 13.3 mm mgcl2 , 0.0133% np-40 , and 0.133% bsa , ph 7.6 ) . after a 10 min incubation , the enzymatic reaction was initiated by the addition of a 4 substrate solution ( 4 l , containing 50 m rhodamine coa and 50 m apo - actinorhodin acp ) . the reactions were terminated after a 30 min incubation at room temperature by the addition of a 2 quench solution ( 20 l , containing 4 m urea , 25 mm edta , and 0.004% phenol red , ph 8.0 ) . samples were separated under native conditions on a 20% polyacrylamide gel using standard laemmli conditions . following the run , the gels were imaged with a chemi - doc plus imager ( bio - rad , hercules , ca ) , and the band intensity was quantified using the imagej software package . pixel density values were normalized to control wells and fit with the four - parameter hill equation using in - house tools . methods for mic determination were made in accordance with standards put forth by the national clinical laboratory standards institute detailed in documents m07-a8 and m27-a2 for bacterial and fungal species , respectively . innoculum was prepared from overnight liquid cultures of bacteria or by suspending 2 day old colonies in rpmi 1640 medium . test articles dissolved in dmso ( 1 l ) were added to sterile medium [ 50 l , cation - adjusted mueller hinton ii broth for bacteria ( bd bbl , franklin lakes , nj ) or rmpi 1640 for fungi ( invitrogen corp . , carlsbad , ca ) ] followed by innoculum prepared in the same medium ( 50 l , containing 1 10 cfu ) and incubated at 3037 c for 1620 h ( 48 h for fungi ) . the plates were visually inspected for microbial growth , and the first well containing no visible microbial growth was scored as the mic . coli k12 , p. aeruginosa atcc 9028 , s. aureus atcc 6538 ( methicillin - sensitive ) , s. aureus atcc baa-1717 ( community - acquired methicillin - resistant strain usa300-hou - mr ) , c. albicans atcc 90028 ( fluconazole - sensitive ) , and c. albicans atcc 96901 ( fluconazole - resistant ) . for the acquisition of images for figures 3d and 4c , resazurin was employed as an indicator to assist in visualization in a method similar to that of sarker et al . a 10 l volume of sterile aqueous resazurin solution ( 0.7% w / v ) was added to the wells of test plates resulting from the protocol above . after further incubation 4 h at 37 c ,

4-phosphopantetheinyl transferases ( pptases ) catalyze a post - translational modification essential to bacterial cell viability and virulence . we present the discovery and medicinal chemistry optimization of 2-pyridinyl - n-(4-aryl)piperazine-1-carbothioamides , which exhibit submicromolar inhibition of bacterial sfp - pptase with no activity toward the human orthologue . moreover , compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells . an advanced analogue of this series , ml267 ( 55 ) , was found to attenuate production of an sfp - pptase - dependent metabolite when applied to bacillus subtilis at sublethal doses . additional testing revealed antibacterial activity against methicillin - resistant staphylococcus aureus , and chemical genetic studies implicated efflux as a mechanism for resistance in escherichia coli . additionally , we highlight the in vitro absorption , distribution , metabolism , and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small - molecule inhibitor .

Introduction Chemistry Results and Discussion Conclusion Experimental Procedures

a key feature of these synthases is the presence of a post - translational appendage to which the nascent polymer is tethered during extension and elaboration ; the attachment of this appendage is catalyzed by pptase enzymes . reduction in fas anabolic capacity is anticipated to exert pleotropic effects on cell viability by inhibiting the production of the primary membrane component palmitate and halting the assembly of virulence - determining components of the gram - negative , gram - positive , and mycobacterial cell walls ( lipid a , lipoteichoic acid , and mycolic acids , respectively ) . since pptase enzymes represent the gatekeeper of these pathways , their inhibition stands to attenuate bacterial cell viability through direct inactivation of fas . given these associations and the above - mentioned therapeutic potential of targeting pptases , there have been several reports by academic and industrial researchers describing the development of small - molecule acps - pptase inhibitors , yet no reports of agents targeting sfp - pptase ( see figure 2 ) . , ucsd-18ae , figure 2 ) possessed no activity toward sfp - pptase and had overall weak activity . an additional publication from wyeth research came out shortly thereafter detailing further medicinal chemistry efforts leading to wyeth-16 ( figure 2 ) , which was optimized for potency toward acps - pptase ( ic50 = 1.4 m ) but had limited antimicrobial activity . on the basis of the limited antibacterial activity observed for these acps - pptase - specific compounds , we have put forth a hypothesis that the presence of an sfp - pptase in the bacterial genome provides an inborn mechanism of resistance . toward this end , we developed a strategy to search for small - molecule inhibitors of sfp - pptase . these efforts identified sch-202676 as the first compound reported to have activity against sfp - pptase ( figure 2 ) . however , compounds belonging to this class are known covalent modifiers and possess promiscuous activity ( 20% hit rate in pubchem assays ) , which ultimately limits their utility as probe compounds . therefore , we conducted a high - throughput screen ( hts ) in search of additional small - molecule inhibitors of sfp - pptase . herein we describe the discovery and sar investigation of the 2-pyridinyl - n-(4-aryl)piperazine-1-carbothioamides as best in class small - molecule inhibitors of sfp - pptase . this work led to the development of ml267 ( 55 ) , a new chemical probe and potent inhibitor of this bacterial enzyme . this characterization began with retest of freshly prepared serial dilutions of hit compounds in the sfp - pptase screening assay ( aid 2701 ) and included further activity profiling with acps - pptase ( aid 602360 ) . finally , since prior work has established the essential nature of pptases to bacterial viability , we profiled these compounds for antibacterial activity with bacillus subtilis hm489 , whose viability depends solely on sfp ( aid 602366 ) . the sum of these experiments identified 1 ( figure s1a , supporting information ) as a confirmed screening hit with inhibitory activity against both acps- and sfp - pptases ( figure s1b ) and modest antibacterial activity against b. subtilis . as recently highlighted , the potential exists that the observed inhibition could be a result of compound , we developed and implemented a label - free biochemical assay that assessed pptase activity on the basis of differences in the electrophoretic mobility of the apo and holo states of the carrier protein under native page conditions . with analogues 4556 ( table 2 ) , we aimed to investigate more significant structural changes to this region , including removal of the aryl group ( 45 ) or replacing it with smaller groups such as a methyl group ( 46 ) , and both changes significantly diminished the potency ( ic50 = 14.5 and 6.5 m respectively ) . in an effort to improve the overall solubility of the molecule gratifyingly , this change was tolerated and led to the discovery of analogue 55 , which was ultimately determined to be our probe molecule ( ml267 ) after all compound attributes such as potency , in vitro adme properties , and antibacterial activity were taken into consideration ( vide infra ) . notably , compound 55 possesses a 4-ome group in contrast to the 4,6-dimethyl derivative , which led to a 23-fold improvement in potency ( ic50 = 0.81 m ( 54 ) vs ic50 = 0.29 m ( 55 ) ) but also contributed to improvement in other attributes mentioned above . a literature search uncovered troviridine [ 1-(5-bromopyridin-2-yl)-3-(2-(pyridin-2-yl)ethyl)thiourea ] ( also known as ly300045-hcl ) , a thiourea - containing small molecule that was originally developed by eli lilly as a non - nucleoside reverse transcriptase inhibitor but later was found to have antimicrobial properties . thus , we then sought to more thoroughly characterize the best sfp - pptase inhibitors for their activity against acps - pptase and antibacterial activity . additionally , we profiled this panel for activity with the human pptase , an important antitarget , and cytotoxicity with human hepg2 cells ( table 4 ) . finally , as part of our initial biological activity profile , we sought to further characterize the compounds for antibacterial activity using the b. subtilis hm489 strain . this important finding is exemplified by urea derivative 12 , which was inactive against sfp / acps - pptase and lacked activity in the antibacterial assays ( table 4 ) . in these profiling studies , 55 demonstrated dual activity toward the bacterial sfp- and acps - pptase targets ( figure s4 , supporting information ) , presenting ic50 values of 290 nm and 8.1 m , respectively . comparison of these data to the inhibition observed in the same biochemical assay for sfp - pptase ( figure s4b , upper panel ) indicated the selectivity index , the ratio of inhibition observed for the bacterial target relative to the human enzyme , to be greater than 500-fold . to evaluate the antibacterial spectrum of activity possessed by 55 , we assembled a panel of microorganisms which included b. subtilis strains hm489 , 168 , and okb105 , which possess all possible pptase genotype combinations sfp / acps , sfp / acps , and sfp / acps , respectively , in a strain 168 isogenic background , as well as e. coli k12 and the laboratory strain bw25113 . with respect to human pathogens , the panel contained pseudomonas aeruginosa atcc9028 , a gram - negative organism of clinical importance , as well as three strains of methicillin - sensitive and community - acquired methicillin - resistant staphylococcus aureus ( atcc6538 , usa300 , and usa500 ) . compound 55 displayed a spectrum of activity that targets gram - positive organisms , where , in addition to inhibiting the growth of b. subtilis at a concentration of 1.7 g / ml , it also thwarted the growth of both methicillin - sensitive and -resistant strains of s. aureus in a similar concentration range [ figure 3 , where the growth indicator resazurin ( purple ) is reduced to resorufin ( pink ) by metabolically active microorganisms]. the antibacterial activity of 55 was restricted to gram - positive organisms , represented here by strains of b. subtilis and s. aureus , with no growth inhibition observed for gram - negative bacteria or fungi . therefore , we concluded that compound 55 does not elicit antibacterial activity through the nonspecific disruption of the gram - positive membrane and likely acts on an intracellular target . with this information , we further characterized the antibacterial activity of 55 to discern whether the compound merely halted proliferation of bacteria or possessed bactericidal activity . to further validate the above findings using a chemical genetic model , we tested 55 for synergistic activity with the broad - spectrum efflux pump inhibitor phenylarginine--naphthamide ( pan ) . in these experiments , neither compound alone was found to possess antibacterial activity against the wild - type strain bw25113 at the top concentrations tested , but offered pronounced growth inhibition in combination ( figure 4c ) . to further understand the on - target engagement of pptase enzymes within the bacterial cell , we exploited the association of sfp - pptase with secondary metabolism in b. subtilis . this dose - dependent attenuation of surfactin titer in cultures which accumulated similar quantities of biomass is consistent with engagement of sfp - pptase by 55 inside living bacteria . seven of these are cp domains that require post - translational activation by sfp - pptase , and the monitoring of the fermentative yield provides a means to assess the blockade of pptase - dependent processes in the bacterial cell . given the potential instability of the thiourea moiety in aqueous and biological media , we were encouraged that 55 showed no signs of degradation in aqueous media at a wide ph range ( 29 ) , buffers ( pbs and hepes ) ( figure s7 , supporting information ) , and mouse plasma ( table 5 ) . having demonstrated a favorable in vitro adme profile of 55 , we next sought to investigate the in vivo pk profile in support of testing this compound in proof of concept ( poc ) antibacterial animal models ( vide infra ) . moreover , the compound has a reasonable t1/2 ( 2.0 h ) , and low clearance ( 7.2 ml / min / kg ) results in an exposure ( total drug concentration , not free drug concentration ) that exceeds the in vitro antibacterial mic value for over 8 h. the compound also efficiently crosses the bbb and thus could be potentially used for bacterial infections which reside in the brain . as discussed above , 55 exhibits antibacterial activity against mrsa and has a desirable adme / pk profile , and thus , it was poised to be tested in vivo . for these studies , we profiled a panel of clinical isolates to assess the susceptibility of various genotypes ( table s2 , supporting information ) , and this revealed similar potencies ( within 3-fold ) for all strains tested . with this information , we pursued an in vivo poc study that utilized the mrsa - usa500 strain ( bk2395 ) since our compound had demonstrated favorable in vitro activity against it and it has demonstrated suitable virulence in mouse models of sepsis . these findings led us to investigate the source of discrepancy between our in vitro and in vivo results . as described above , the eastern region of the molecule is highly tolerant to structural modifications ( table 2 ) , which could be exploited to modulate the clogp and thus hopefully improve activity in vivo . in addition , we plan on conducting more extensive pk studies to look at tissue distribution and free drug concentration , which may guide not only a future medicinal chemistry effort , but also aid the design of our in vivo proof of concept models . nonetheless , this discrepancy provides another mechanism through which bacterial tolerance to 55 may be occurring in vivo , and such a mechanism may confound the advancement of in vitro pptase - targeting inhibitors through in vivo poc studies of staphylococcal septicemia . metabolites from all three of these classes are necessary for bacterial cell viability and virulence . rather than targeting the machinery involved in the production of a single metabolite , we have chosen to investigate the inhibition of this ptm as it represents a unifying feature between these pathways . this limited success can likely be attributed to the inability of these programs to consider the presence of an sfp - pptase in addition to their primary target , acps - pptase . the features of 55 can be summarized according to the five principles of a quality chemical probe as follows : ( i ) we have performed detailed molecular profiling with relevant molecular targets , with observed potencies of 290 nm and 8.1 m against the primary development target sfp - pptase and bacterial orthologue acps , respectively , which is contrasted by null activity with the human enzyme . in the context of an antibacterial discovery campaign , where the end goal is the inhibition of bacterial growth in man , this indicates a selectivity index of > 500-fold with respect to the human orthologue . ( ii ) with respect to the mechanism of action , we demonstrate that the chemotype inhibits sfp - pptase independent of a fluorescent label through an allosteric mechanism that is noncompetitive with substrates and is rapidly reversible . ( iv ) to demonstrate the utility of 55 as a probe , we confirmed the ability of a dual - specific pptase inhibitor to thwart the growth of bacteria in the absence of a rapid cytotoxic response . additionally , 55 demonstrated activity against clinically relevant microorganisms , where it stifled the growth of methicillin - resistant s. aureus . expanding on these findings , we also identified the primary mechanism of resistance in e. coli to be efflux by the acrab tolc system and demonstrated a chemical genetic approach to mitigate this issue and extend the spectrum of antibacterial activity to include gram - negative organisms . beyond these assessments of antimicrobial activity , the ability of 55 to attenuate the production of surfactin , a metabolite dependent on the biochemical functionality of sfp - pptase ( figure 5b ) , strongly indicates that 55 is acting on - target inside the bacterial cell . to the clear yellow solution was added commercially available n-(3-(trifluoromethyl)phenyl)piperidin-4-amine ( 0.226 g , 0.925 mmol , 1 equiv ) , and the resulting solution was stirred at 40 c for 1 h. the solvent was evaporated , and the crude product was taken up in 2 ml of dmso and purified via reversed - phase chromatography to give the product as a tfa salt : lc / ms retention time t1 ( method 1 , 7 min ) = 4.954 min and t2 ( method 2 , 3 min ) = 3.025 min ; h nmr ( 400 mhz , dmso - d6 ) 8.21 ( d , j = 5.4 hz , 1h ) , 7.39 ( d , j = 1.6 hz , 1h ) , 7.317.22 ( m , 1h ) , 7.057.00 ( m , 1h ) , 6.916.85 ( m , 2h ) , 6.836.77 ( m , 1h ) , 5.50 ( br s , 1h ) , 4.54 ( d , j = 13.2 hz , 2h ) , 3.68 ( tt , j = 9.4 , 3.9 hz , 1h ) , 3.43 ( ddd , j = 13.7 , 11.1 , 2.8 hz , 2h ) , 2.35 ( s , 3h ) , 2.071.89 ( m , 2h ) , 1.43 ( dtd , j = 13.6 , 10.2 , 3.8 hz , 2h ) ; hrms ( esi ) m / z ( m + h ) calcd for c19h22f3n4s 395.1512 , found 395.1510 . volatiles were removed by forced air , and the residue was dissolved in dmf and purified via reversed - phase chromatography to give 12 as a tfa salt : lc / ms retention time t1 ( method 1 , 7 min ) = 4.43 min and t2 ( method 2 , 3 min ) = 3.095 min ; h nmr ( 400 mhz , dmso - d6 ) 10.21 ( s , 1h ) , 8.17 ( d , j = 5.9 hz , 1h ) , 7.547.39 ( m , 2h ) , 7.297.18 ( m , 2h ) , 7.177.05 ( m , 2h ) , 3.713.63 ( m , 4h ) , 3.313.29 ( m , 4h ) , 2.40 ( s , 3h ) ; c nmr ( 101 mhz , dmso - d6 ) 158.27 , 153.81 , 150.90 , 150.53 , 140.63 , 130.07 , 130.04 , 129.76 , 119.59 , 119.03 , 115.02 , 114.70 , 111.33 , 111.29 , 47.41 , 43.50 , 21.45 ; hrms ( esi ) m / z ( m + h ) calcd for c18h20f3n4o 365.1584 , found 365.1590 . a 3 l volume of each reagent , consisting of buffer ( in columns 3 and 4 as a negative control ) and sfp- or acps - pptase ( in columns 1 , 2 , and 548 , final concentration of 15 or 100 nm , respectively ) were dispensed into a 1536-well greiner black solid - bottom plate . coli k12 , p. aeruginosa atcc 9028 , s. aureus atcc 6538 ( methicillin - sensitive ) , s. aureus atcc baa-1717 ( community - acquired methicillin - resistant strain usa300-hou - mr ) , c. albicans atcc 90028 ( fluconazole - sensitive ) , and c. albicans atcc 96901 ( fluconazole - resistant ) .

there is much controversy regarding the use and timing of enteral feeding support in patients with dysphagia and aspiration risk . the decision process becomes far more complex in those patients with advanced dementia , due to ethical and moral issues associated with the continuing comfort care per os ( po ) feeds with the knowledge that the person is aspirating , and the use of percutaneous endoscopic gastrostomy tube ( peg ) placement , and associated mortality rates . dementia is a leading cause of death in the us , with mortality affected by aspiration , hydration , and nutritional status . data in the year 2000 show there were approximately 4.5 million people in north america with a diagnosis of dementia , and more than half progressed to the moderate to severe stages of their disease . in 2001 there were 24.3 million people in the world with dementia , and by 2040 , the number is estimated to increase to over 81 million . the prevalence of dementia is estimated to double every 5 years after 65 years old , and at age 85 years , the prevalence is approximately 50%.1,2 dementia is a terminal diagnosis . alzheimer s disease and other related illnesses causing dementia are progressive , incurable , and lead to a complete loss of cognitive function , and subsequent death . a characteristic feature of the final phase of dementia , which can last from 6 months to 2 years , is loss of interest in eating , dysphagia , or both.3 an estimated 60% of nursing home residents have dementia , with approximately half ( 480,000 ) being in the last stages of their disease . the prognosis and progressive clinical course affects the important decision about peg insertion.4 mean survival after dementia diagnosis varies between 1 and 16 years , but one - third of demented individuals live to advanced stages.4 the ability to perform activities of daily living are typically lost in a hierarchical fashion , with eating and bed mobility lost last . the most severe phase of dementia is characterized by loss of capacity to provide self - care in basic activities of daily living , such as eating , bathing , and walking independently . in this stage there are also many behavioral symptoms that compromise quality of life for both patients and caregivers , and are sources of great stress to the latter , with institutionalization being the ultimate consequence . many patients and their families have limited understanding of the terminal nature of a dementia diagnosis . due to the complexity that characterizes advanced dementia , many moral , ethical , religious , and medical decisions arise ; these involve appreciating the risks , benefits , and alternatives to adjunctive enteral ( peg ) feeding , non per os ( npo ) status , and the role of comfort care or compassionate po which is defined as continuing to feed a patient by mouth for quality of life and or comfort , in spite of and with information that the patient is aspirating and at risk for aspiration pneumonia or worse . surprisingly , there is little discussion of these issues in the literature and in routine medical care . in this paper , we reviewed the existing literature on peg placement in patients with dementia , in terms of subsequent mortality rates , beginning with the hypothesis that peg does not prolong life . we also explored the impact of peg in specific dementia groups to identify refined prognostic indicators that may be further used to develop comprehensive guidelines for the placement of pegs in this population . a systematic literature search was performed using pubmed , to identify studies and their levels of evidence . key words were identified as search terms : aspiration , dysphagia , swallowing difficulty , dementia , alzheimer , peg , and enteral and feeding tube . three searches gleaned the most results . the search combining dementia and ( dysphagia or aspiration ) and ( endoscopic gastrostomy or enteral ) yielded 97 articles . the search combining ( dementia or alzheimer ) and ( dysphagia or aspiration ) and ( percutaneous endoscopic gastrostomy or enteral or feeding tube ) yielded 99 articles , and the search gleaning the most results , 100 articles , and therefore used for this study combined the following terms ( dementia or alzheimer ) and ( dysphagia or aspiration or swallowing difficulty ) and ( percutaneous endoscopic gastrostomy or enteral or feeding tube ) . our inclusion criteria required that the article 1 ) must have been a scientific research paper ; 2 ) must have addressed dementia , dysphagia or aspiration risk , or peg tube placement ; and 3 ) must have been originally written in or translated into the english language . the exclusion criteria comprised the following : 1 ) studies focused solely on the pediatric population ; 2 ) studies focused on peg placements in other diagnoses only ( studies including cohorts with dementia patients among other neurological diagnoses were not excluded if the dementia patients were separated out in the results ) ; 3 ) abstracts only ; and 4 ) nonscientific research , editorial , text book , or opinion - based papers . once the articles were selected , the reference sections were scanned for other relevant studies . it was then determined whether the study was derived from or based on a systematic review of the literature . next , we looked at the size of the study , the number of patients with dementia within the study , and specific outcomes , most importantly survival , comparing those patients with dementia who received pegs to those who did not . ten articles were highlighted ( the review process is summarized in the preferred reporting items for systematic reviews and meta - analyses [ prisma ] diagram as figure 1 ) . overall results are summarized in table 1 , stratified by year , study design , number of subjects , level of evidence , and study findings . the retrospective case series , which included a dementia subgroup containing 103 people with a peg , revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years , which contained 8,688 demented patients who received pegs , broke down mortality rates between males and females.2 according to this study , at 1 year , mortality rates for males and females with dementia status post - peg insertion were 61% and 50% , respectively . at 3 years , the mortality for males and females with dementia status post - peg was 78% and 84% , respectively . a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old . the participants were classified into seven diagnoses : stroke ; dementia ; parkinson s ; other neurological diseases ( mainly amyotrophic lateral sclerosis ) ; malignancies with dysphagia ; malignancies without dysphagia ; and miscellaneous . results based on time frames were as follows : at 30 days , demented patients had 25% mortality ; at 3 months , 37% mortality ; and at 1 year , 58% mortality . another retrospective study , of 90 patients with dementia and dysphagia and of average age ~86 years , revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members , 23 received pegs and 18 did not , secondary to family member refusal after discussion . the median survival in the group status post - peg was 59 days compared with the group without the peg , who survived 60 days . a retrospective cohort study with 311 patients with pegs , 143 with dementia , compared demented patient survival with peg to that of patients with other diagnoses ( patients without dementia).9 the 12-month survival was 51% in patients with dementia , and 49% in the group without dementia . more than 20% of patients with dementia lived more than 3 years after peg . two predictors of poor survival after peg ( among other medical issues ) were patients who were male and older than 80 years of age . in this study , there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact , unless the patients are older than 80 years of age , male , or have the other poor prognostic indicators mentioned in this study , including hypoalbuminemia . one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs , 135 ( 18% ) had dementia , 82% were 70 years of age or older , and 50% were greater than 80 years of age . of note , there was a particularly low rate ( 29% ) of written consent for the procedure among dementia patients . after their review , acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable , the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status feeding tube was 195 days , and in the group without the feeding tube , this was 189 days . overall , the peg and non - peg median mortality at 6 months was no different , at 50% . a prospective study , which included a dementia cohort of 55 patients , compared tube - fed ( tf ) versus ( vs ) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls . the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient . this study specifically focused on patients with inadequate oral intake secondary to dementia , in order to determine whether the nutritional parameters at the time of peg placement would predict survival . it also recommended that prior to initiating peg feeding , the limited benefits that would be achieved should be taken into consideration . a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years , with an age distribution similar to that of patients undergoing peg placements nationally in the united states . the median survival was 171 days in patients over age 80 with dementia , which was worse when compared with other subgroups . the median survival reached 423 days in demented patients from nursing homes and 467 days in demented patients younger than 80 years . results from five studies reported mortality for the patients with dementia after peg placement.57,10 , the studies reviewed reported on varied mortality time frames . the specific results were : 13%18%,6 14.4% , 18%,10 25%,7 and 54%.5 one study reported 3-month mortality at 37%.7 six month mortality was revealed in one study as 44%.12 the mortality at 1 year was reported in five of the studies reviewed . those specific results were as follows : 45.6% , 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows . one study revealed dementia patient median survival with a feeding tube as 195 days ( 6.5 months ) compared with median survival without tube feeding as 189 days ( 6.3 months ) , with no obvious difference in survival when comparing both groups.11 the following are results from individual studies . one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality . this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days , a low number . yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days , whereas dementia patients with peg and stroke median survival was at 181 days , a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years , which included 8,688 demented patients who received pegs , broke down the mortality rates between males and females.2 this study revealed females fared slightly better , though mortality rates were still high after peg . the female dementia subgroup had 50% mortality at 1 year and 78% at 3 years . the male dementia subgroup had 61% mortality at 1 year and 84% mortality at 3 years . as previously reported in retrospective studies , an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement , and survival after peg.7 the mean age of participants was 79 years old . patients were classified into seven diagnoses : stroke , dementia , parkinson s , other neurological diseases ( mainly amyotrophic lateral sclerosis ) , malignancies with or without dysphagia , and miscellaneous . mortality rates were broken down farther into specific time frames : 30 days , 3 months and 1 year . regarding consideration of age prior to placement of peg in patients with dementia , only 33% of patients over age 80 were still alive after 1 year , as compared with 73% under age 80 . in this particular study , dementia was not found to be a negative prognostic factor for survival in elderly patients after peg , specifically those younger than 80 years old . another retrospective study of 90 patients with dementia and dysphagia , whose average age was 86 years , revealed 14.4% mortality within 30 days , and 1-year survival of 54.4% . the retrospective case series , which included a dementia subgroup containing 103 people with a peg , revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years , which contained 8,688 demented patients who received pegs , broke down mortality rates between males and females.2 according to this study , at 1 year , mortality rates for males and females with dementia status post - peg insertion were 61% and 50% , respectively . at 3 years , the mortality for males and females with dementia status post - peg was 78% and 84% , respectively . a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old . the participants were classified into seven diagnoses : stroke ; dementia ; parkinson s ; other neurological diseases ( mainly amyotrophic lateral sclerosis ) ; malignancies with dysphagia ; malignancies without dysphagia ; and miscellaneous . results based on time frames were as follows : at 30 days , demented patients had 25% mortality ; at 3 months , 37% mortality ; and at 1 year , 58% mortality . another retrospective study , of 90 patients with dementia and dysphagia and of average age ~86 years , revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members , 23 received pegs and 18 did not , secondary to family member refusal after discussion . the median survival in the group status post - peg was 59 days compared with the group without the peg , who survived 60 days . a retrospective cohort study with 311 patients with pegs , 143 with dementia , compared demented patient survival with peg to that of patients with other diagnoses ( patients without dementia).9 the 12-month survival was 51% in patients with dementia , and 49% in the group without dementia . two predictors of poor survival after peg ( among other medical issues ) were patients who were male and older than 80 years of age . in this study , there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact , unless the patients are older than 80 years of age , male , or have the other poor prognostic indicators mentioned in this study , including hypoalbuminemia . one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs , 135 ( 18% ) had dementia , 82% were 70 years of age or older , and 50% were greater than 80 years of age . of note , there was a particularly low rate ( 29% ) of written consent for the procedure among dementia patients . after their review , acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable , the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status post feeding tube was 195 days , and in the group without the feeding tube , this was 189 days . overall , the peg and non - peg median mortality at 6 months was no different , at 50% . a prospective study , which included a dementia cohort of 55 patients , compared tube - fed ( tf ) versus ( vs ) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls . the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient . this study specifically focused on patients with inadequate oral intake secondary to dementia , in order to determine whether the nutritional parameters at the time of peg placement would predict survival . it also recommended that prior to initiating peg feeding , the limited benefits that would be achieved should be taken into consideration . a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years , with an age distribution similar to that of patients undergoing peg placements nationally in the united states . the median survival was 171 days in patients over age 80 with dementia , which was worse when compared with other subgroups . the median survival reached 423 days in demented patients from nursing homes and 467 days in demented patients younger than 80 years . results from five studies reported mortality for the patients with dementia after peg placement.57,10 , the studies reviewed reported on varied mortality time frames . the specific results were : 13%18%,6 14.4% , 18%,10 25%,7 and 54%.5 one study reported 3-month mortality at 37%.7 six month mortality was revealed in one study as 44%.12 the mortality at 1 year was reported in five of the studies reviewed . those specific results were as follows : 45.6% , 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows . one study revealed dementia patient median survival with a feeding tube as 195 days ( 6.5 months ) compared with median survival without tube feeding as 189 days ( 6.3 months ) , with no obvious difference in survival when comparing both groups.11 the following are results from individual studies . one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality . this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days , a low number . yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days , whereas dementia patients with peg and stroke median survival was at 181 days , a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years , which included 8,688 demented patients who received pegs , broke down the mortality rates between males and females.2 this study revealed females fared slightly better , though mortality rates were still high after peg . the female dementia subgroup had 50% mortality at 1 year and 78% at 3 years . the male dementia subgroup had 61% mortality at 1 year and 84% mortality at 3 years . as previously reported in retrospective studies , an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement , and survival after peg.7 the mean age of participants was 79 years old . patients were classified into seven diagnoses : stroke , dementia , parkinson s , other neurological diseases ( mainly amyotrophic lateral sclerosis ) , malignancies with or without dysphagia , and miscellaneous . mortality rates were broken down farther into specific time frames : 30 days , 3 months and 1 year . regarding consideration of age prior to placement of peg in patients with dementia , only 33% of patients over age 80 were still alive after 1 year , as compared with 73% under age 80 . in this particular study , dementia was not found to be a negative prognostic factor for survival in elderly patients after peg , specifically those younger than 80 years old . another retrospective study of 90 patients with dementia and dysphagia , whose average age was 86 years , revealed 14.4% mortality within 30 days , and 1-year survival of 54.4% . there are many controversies regarding whether or not to place a peg in a patient with dementia who is aspirating . quality of life must be considered , not only for the patient but for the caregiver as well . while an in - depth discussion of these issues goes well beyond the scope of this paper , it is important to recognize some of the major problems that health care professionals , caregivers , and the patients themselves encounter . stages 6 ( moderate severe dementia ) and 7 ( severe dementia ) are listed in table 2 , with some of the hallmark signs of disease progression . voice and speech deterioration , as well as dysphagia with aspiration risk , demonstrate the cognitive effects on laryngeal function and are included in stage 6e.3 discussions regarding peg insertion do not appear to be happening during the early stages of dementia diagnosis , and many patients who receive a peg do not have health care proxies . patients diagnosed with dementia could be seen when presenting with initial signs of difficulty eating or swallowing , and could have regular follow - up so that aspiration risk can be assessed and potentially , intervention undertaken . at are they being inserted too late ? would there be a better outcome if the pegs were inserted earlier ? when is too late ? the research has shown an increased survival rate with demented patients under age 80 , nearly double that of patients over age 80 . other considerations are : age of onset of dysphagia , age of onset of dementia , severity of dementia , and disease duration , course , and comorbidities . every patient must still be regarded as an individual . medications should be assessed for their effects on swallowing and perhaps , medication schedules adjusted to facilitate eating , to prolong safe eating by mouth . caregivers , and often health care professionals as well , do not have all the information prior to making a decision . the data should be reviewed by medical staff and family members when deciding whether and when to insert a peg . when the discussion and or recommendation to not insert a peg is brought up with caregivers , this is often wrongly interpreted as a recommendation that will mean no food or no care for their loved one . caregivers need to be educated regarding quality of life po feeding , which may be interpreted as continuing to feed patients by mouth , with the safest diet and strict aspiration precautions in place , so they may still enjoy the taste of food . future studies that divide cohorts of retrospective studies into levels of severity of dementia would be informative . the study of the specific divisions and levels of dementia could assist in formulating future recommendations based on age , sex , nutritional status and the home environment . some benefits of peg placement that were not addressed in our review include : provision of a route for reliably administering medications ; supplementation of hydration and nutritional status while still allowing for compassionate po or comfort care ; as well as ensuring the bridging of nutritional needs during interval hospitalizations for conditions that may cause a transient decline in mental status . also , while this review does not document that peg prolongs life in this population , it is possible that a prospective study has yet to be constructed that accurately measures prolongation as well as quality of life for the patients , and their families , during their declining years . there is presently no evidence in the existing literature to suggest long - term survival rates improved in patients with advanced dementia who underwent peg placement for dysphagia and aspiration risk . however , the relevance for quality of life , need for nutrition and hydration , and ethical consideration are complex and controversial issues that warrant further study prior to achieving consensus and formalizing clinical care guidelines .

purposeover 4.5 million people in north america had a diagnosis of dementia in the year 2000 , and more than half had advanced disease with potential aspiration risk . there is much controversy regarding the use and timing of enteral feeding support in these patients with dysphagia . the management of dysphagia is far more complex when considering quality of life , comfort care hand feeding , the use of percutaneous endoscopic gastrostomy tube ( peg ) , and associated mortality rates . this study seeks to critically review the literature that evaluates peg placement in this population.methodsa systematic literature review of pubmed , from 19952012 , was conducted to identify studies relating to peg placement in dementia patients with dysphagia . the principal outcomes and related survival rates for this population were compared.resultsin total , 100 articles were identified in the search . of these , ten met the search criteria and were analyzed . there was one study with a 2b level of evidence , one with 3b , and the remainder had level 4 . all studies discussed long - term survival in the peg versus non - peg populations . no studies showed definitive evidence to suggest long - term survival rates improved in patients who underwent peg placement as compared to those who did not . two studies documented median survival worse in patients over age 80 with dementia and peg placement.conclusionthere is presently no evidence to suggest long - term survival rates improved in patients with advanced dementia who underwent peg placement for dysphagia . relevance to quality of life , need for nutrition and hydration , and ethical considerations in the decision process are discussed .

Introduction Methods Results Retrospective studies Prospective studies Summary of results Male vs female mortality subgroup Age-based mortality subgroup Discussion Conclusion

there is much controversy regarding the use and timing of enteral feeding support in patients with dysphagia and aspiration risk . the decision process becomes far more complex in those patients with advanced dementia , due to ethical and moral issues associated with the continuing comfort care per os ( po ) feeds with the knowledge that the person is aspirating , and the use of percutaneous endoscopic gastrostomy tube ( peg ) placement , and associated mortality rates . dementia is a leading cause of death in the us , with mortality affected by aspiration , hydration , and nutritional status . data in the year 2000 show there were approximately 4.5 million people in north america with a diagnosis of dementia , and more than half progressed to the moderate to severe stages of their disease . in 2001 there were 24.3 million people in the world with dementia , and by 2040 , the number is estimated to increase to over 81 million . the prevalence of dementia is estimated to double every 5 years after 65 years old , and at age 85 years , the prevalence is approximately 50%.1,2 dementia is a terminal diagnosis . a characteristic feature of the final phase of dementia , which can last from 6 months to 2 years , is loss of interest in eating , dysphagia , or both.3 an estimated 60% of nursing home residents have dementia , with approximately half ( 480,000 ) being in the last stages of their disease . the most severe phase of dementia is characterized by loss of capacity to provide self - care in basic activities of daily living , such as eating , bathing , and walking independently . in this stage there are also many behavioral symptoms that compromise quality of life for both patients and caregivers , and are sources of great stress to the latter , with institutionalization being the ultimate consequence . due to the complexity that characterizes advanced dementia , many moral , ethical , religious , and medical decisions arise ; these involve appreciating the risks , benefits , and alternatives to adjunctive enteral ( peg ) feeding , non per os ( npo ) status , and the role of comfort care or compassionate po which is defined as continuing to feed a patient by mouth for quality of life and or comfort , in spite of and with information that the patient is aspirating and at risk for aspiration pneumonia or worse . surprisingly , there is little discussion of these issues in the literature and in routine medical care . in this paper , we reviewed the existing literature on peg placement in patients with dementia , in terms of subsequent mortality rates , beginning with the hypothesis that peg does not prolong life . we also explored the impact of peg in specific dementia groups to identify refined prognostic indicators that may be further used to develop comprehensive guidelines for the placement of pegs in this population . a systematic literature search was performed using pubmed , to identify studies and their levels of evidence . key words were identified as search terms : aspiration , dysphagia , swallowing difficulty , dementia , alzheimer , peg , and enteral and feeding tube . the search combining dementia and ( dysphagia or aspiration ) and ( endoscopic gastrostomy or enteral ) yielded 97 articles . the search combining ( dementia or alzheimer ) and ( dysphagia or aspiration ) and ( percutaneous endoscopic gastrostomy or enteral or feeding tube ) yielded 99 articles , and the search gleaning the most results , 100 articles , and therefore used for this study combined the following terms ( dementia or alzheimer ) and ( dysphagia or aspiration or swallowing difficulty ) and ( percutaneous endoscopic gastrostomy or enteral or feeding tube ) . the exclusion criteria comprised the following : 1 ) studies focused solely on the pediatric population ; 2 ) studies focused on peg placements in other diagnoses only ( studies including cohorts with dementia patients among other neurological diagnoses were not excluded if the dementia patients were separated out in the results ) ; 3 ) abstracts only ; and 4 ) nonscientific research , editorial , text book , or opinion - based papers . once the articles were selected , the reference sections were scanned for other relevant studies . it was then determined whether the study was derived from or based on a systematic review of the literature . next , we looked at the size of the study , the number of patients with dementia within the study , and specific outcomes , most importantly survival , comparing those patients with dementia who received pegs to those who did not . ten articles were highlighted ( the review process is summarized in the preferred reporting items for systematic reviews and meta - analyses [ prisma ] diagram as figure 1 ) . overall results are summarized in table 1 , stratified by year , study design , number of subjects , level of evidence , and study findings . the retrospective case series , which included a dementia subgroup containing 103 people with a peg , revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years , which contained 8,688 demented patients who received pegs , broke down mortality rates between males and females.2 according to this study , at 1 year , mortality rates for males and females with dementia status post - peg insertion were 61% and 50% , respectively . at 3 years , the mortality for males and females with dementia status post - peg was 78% and 84% , respectively . a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old . another retrospective study , of 90 patients with dementia and dysphagia and of average age ~86 years , revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members , 23 received pegs and 18 did not , secondary to family member refusal after discussion . the median survival in the group status post - peg was 59 days compared with the group without the peg , who survived 60 days . a retrospective cohort study with 311 patients with pegs , 143 with dementia , compared demented patient survival with peg to that of patients with other diagnoses ( patients without dementia).9 the 12-month survival was 51% in patients with dementia , and 49% in the group without dementia . more than 20% of patients with dementia lived more than 3 years after peg . in this study , there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact , unless the patients are older than 80 years of age , male , or have the other poor prognostic indicators mentioned in this study , including hypoalbuminemia . one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs , 135 ( 18% ) had dementia , 82% were 70 years of age or older , and 50% were greater than 80 years of age . of note , there was a particularly low rate ( 29% ) of written consent for the procedure among dementia patients . after their review , acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable , the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status feeding tube was 195 days , and in the group without the feeding tube , this was 189 days . overall , the peg and non - peg median mortality at 6 months was no different , at 50% . a prospective study , which included a dementia cohort of 55 patients , compared tube - fed ( tf ) versus ( vs ) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls . the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient . this study specifically focused on patients with inadequate oral intake secondary to dementia , in order to determine whether the nutritional parameters at the time of peg placement would predict survival . it also recommended that prior to initiating peg feeding , the limited benefits that would be achieved should be taken into consideration . a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years , with an age distribution similar to that of patients undergoing peg placements nationally in the united states . the median survival was 171 days in patients over age 80 with dementia , which was worse when compared with other subgroups . results from five studies reported mortality for the patients with dementia after peg placement.57,10 , the studies reviewed reported on varied mortality time frames . those specific results were as follows : 45.6% , 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows . one study revealed dementia patient median survival with a feeding tube as 195 days ( 6.5 months ) compared with median survival without tube feeding as 189 days ( 6.3 months ) , with no obvious difference in survival when comparing both groups.11 the following are results from individual studies . one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality . this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days , a low number . yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days , whereas dementia patients with peg and stroke median survival was at 181 days , a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years , which included 8,688 demented patients who received pegs , broke down the mortality rates between males and females.2 this study revealed females fared slightly better , though mortality rates were still high after peg . as previously reported in retrospective studies , an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement , and survival after peg.7 the mean age of participants was 79 years old . patients were classified into seven diagnoses : stroke , dementia , parkinson s , other neurological diseases ( mainly amyotrophic lateral sclerosis ) , malignancies with or without dysphagia , and miscellaneous . regarding consideration of age prior to placement of peg in patients with dementia , only 33% of patients over age 80 were still alive after 1 year , as compared with 73% under age 80 . in this particular study , dementia was not found to be a negative prognostic factor for survival in elderly patients after peg , specifically those younger than 80 years old . another retrospective study of 90 patients with dementia and dysphagia , whose average age was 86 years , revealed 14.4% mortality within 30 days , and 1-year survival of 54.4% . the retrospective case series , which included a dementia subgroup containing 103 people with a peg , revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years , which contained 8,688 demented patients who received pegs , broke down mortality rates between males and females.2 according to this study , at 1 year , mortality rates for males and females with dementia status post - peg insertion were 61% and 50% , respectively . at 3 years , the mortality for males and females with dementia status post - peg was 78% and 84% , respectively . a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old . another retrospective study , of 90 patients with dementia and dysphagia and of average age ~86 years , revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members , 23 received pegs and 18 did not , secondary to family member refusal after discussion . the median survival in the group status post - peg was 59 days compared with the group without the peg , who survived 60 days . a retrospective cohort study with 311 patients with pegs , 143 with dementia , compared demented patient survival with peg to that of patients with other diagnoses ( patients without dementia).9 the 12-month survival was 51% in patients with dementia , and 49% in the group without dementia . in this study , there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact , unless the patients are older than 80 years of age , male , or have the other poor prognostic indicators mentioned in this study , including hypoalbuminemia . one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs , 135 ( 18% ) had dementia , 82% were 70 years of age or older , and 50% were greater than 80 years of age . of note , there was a particularly low rate ( 29% ) of written consent for the procedure among dementia patients . after their review , acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable , the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status post feeding tube was 195 days , and in the group without the feeding tube , this was 189 days . overall , the peg and non - peg median mortality at 6 months was no different , at 50% . a prospective study , which included a dementia cohort of 55 patients , compared tube - fed ( tf ) versus ( vs ) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls . the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient . this study specifically focused on patients with inadequate oral intake secondary to dementia , in order to determine whether the nutritional parameters at the time of peg placement would predict survival . it also recommended that prior to initiating peg feeding , the limited benefits that would be achieved should be taken into consideration . a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years , with an age distribution similar to that of patients undergoing peg placements nationally in the united states . the median survival was 171 days in patients over age 80 with dementia , which was worse when compared with other subgroups . results from five studies reported mortality for the patients with dementia after peg placement.57,10 , the studies reviewed reported on varied mortality time frames . those specific results were as follows : 45.6% , 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows . one study revealed dementia patient median survival with a feeding tube as 195 days ( 6.5 months ) compared with median survival without tube feeding as 189 days ( 6.3 months ) , with no obvious difference in survival when comparing both groups.11 the following are results from individual studies . one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality . this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days , a low number . yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days , whereas dementia patients with peg and stroke median survival was at 181 days , a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years , which included 8,688 demented patients who received pegs , broke down the mortality rates between males and females.2 this study revealed females fared slightly better , though mortality rates were still high after peg . as previously reported in retrospective studies , an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement , and survival after peg.7 the mean age of participants was 79 years old . patients were classified into seven diagnoses : stroke , dementia , parkinson s , other neurological diseases ( mainly amyotrophic lateral sclerosis ) , malignancies with or without dysphagia , and miscellaneous . regarding consideration of age prior to placement of peg in patients with dementia , only 33% of patients over age 80 were still alive after 1 year , as compared with 73% under age 80 . in this particular study , dementia was not found to be a negative prognostic factor for survival in elderly patients after peg , specifically those younger than 80 years old . another retrospective study of 90 patients with dementia and dysphagia , whose average age was 86 years , revealed 14.4% mortality within 30 days , and 1-year survival of 54.4% . there are many controversies regarding whether or not to place a peg in a patient with dementia who is aspirating . quality of life must be considered , not only for the patient but for the caregiver as well . while an in - depth discussion of these issues goes well beyond the scope of this paper , it is important to recognize some of the major problems that health care professionals , caregivers , and the patients themselves encounter . voice and speech deterioration , as well as dysphagia with aspiration risk , demonstrate the cognitive effects on laryngeal function and are included in stage 6e.3 discussions regarding peg insertion do not appear to be happening during the early stages of dementia diagnosis , and many patients who receive a peg do not have health care proxies . patients diagnosed with dementia could be seen when presenting with initial signs of difficulty eating or swallowing , and could have regular follow - up so that aspiration risk can be assessed and potentially , intervention undertaken . the research has shown an increased survival rate with demented patients under age 80 , nearly double that of patients over age 80 . other considerations are : age of onset of dysphagia , age of onset of dementia , severity of dementia , and disease duration , course , and comorbidities . caregivers need to be educated regarding quality of life po feeding , which may be interpreted as continuing to feed patients by mouth , with the safest diet and strict aspiration precautions in place , so they may still enjoy the taste of food . the study of the specific divisions and levels of dementia could assist in formulating future recommendations based on age , sex , nutritional status and the home environment . some benefits of peg placement that were not addressed in our review include : provision of a route for reliably administering medications ; supplementation of hydration and nutritional status while still allowing for compassionate po or comfort care ; as well as ensuring the bridging of nutritional needs during interval hospitalizations for conditions that may cause a transient decline in mental status . also , while this review does not document that peg prolongs life in this population , it is possible that a prospective study has yet to be constructed that accurately measures prolongation as well as quality of life for the patients , and their families , during their declining years . there is presently no evidence in the existing literature to suggest long - term survival rates improved in patients with advanced dementia who underwent peg placement for dysphagia and aspiration risk . however , the relevance for quality of life , need for nutrition and hydration , and ethical consideration are complex and controversial issues that warrant further study prior to achieving consensus and formalizing clinical care guidelines .

the endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis via the regulation of a multitude of vasoactive processes . disruption to these processes may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease ( cvd ) . furthermore , measures of peripheral endothelial function have been shown to reflect coronary endothelial function , and as such are regarded as good predictors of cardiovascular disease . this is perhaps hardly surprising given that atherosclerosis is now broadly appreciated to be a systemic disorder . assessments of peripheral endothelial function typically quantify the vasodilatory response of the vessel to a specific stimulus , with an attenuation of the dilatory response indicative of endothelial dysfunction , and can be measured in different vascular beds . assessments of advanced structural changes in the vessel can be characterised by ultrasound examination of the intima - media thickness . in the microcirculation , laser doppler flowmetry ( ldf ) and laser doppler imaging ( ldi ) with iontophoresis of vasodilator agonists can provide useful information on microvascular perfusion . both techniques measure the doppler shift created by scattered light from moving red blood cells . perfusion is represented as blood flux rather than blood flow ( ml / min ) , with blood flux reflecting average red blood cell velocity and concentration . the assessment of ldi offers considerable advantages over ldf , because unlike ldf , ldi can scan over a vast area thus accounting for heterogeneity in skin blood flow and increasing the reproducibility of the technique . the stimulus for increasing blood flux during ldi are provided by iontophoresis of vasodilator agonists acetylcholine ( ach ) and sodium nitroprusside ( snp ) , which assess endothelium - dependent and endothelium - independent function respectively , into the skin using a weak electrical current . once through the skin , ach binds to endothelial cell muscarinic receptors releasing the vasodilator nitric oxide ( no ) . the use of snp directly activates smooth muscle cell receptors to allow for maximum vasodilatation of the vessel and examination of smooth muscle integrity . there is some uncertainty on whether ach - mediated dilatation involves no at all , as ach may stimulate non - no pathways such as cycloxygenase - mediated pathways . nevertheless , we have previously reported that ach and snp responses are impaired in patient populations at increased risk of cvd and that exercise interventions known to improve no bioactivity also improve ach - mediated blood flux using ldi . the vehicle for transporting the agents into the skin microvessels often include sodium chloride or deionised water . microvascular endothelial function can be quantified using different approaches , with cutaneous vascular conductance a product of flux divided by arterial pressure , used in studies where blood pressure may change over the study duration ( i.e. , during exercise or anti - hypertensive treatment ) . another commonly used quantification is to calculate the area under the curve for blood flux or express the percentage increase in flux from baseline . it is important to note that there are no established guidelines for presenting data , but investigators should utilise an approach which shows good reproducibility . in the large vessels , flow - mediated dilatation ( fmd ) and glyceryl - trinitrate mediated dilatation ( gtn ) are performed to assess endothelium - dependent and endothelium - independent function respectively . fmd is typically carried out in the brachial artery where a cuff is used to occlude arterial blood flow for 5 min ; release of the cuff causes a sudden increase in blood flow ( reactive hyperaemia ) through the brachial artery resulting in shear - stress mediated dilatation of the vessel . the baseline and post - cuff release diameter are quantified by ultrasound imaging of the vessel with subsequent assessments of the vessel diameter performed manually or using automated edge detection software . the use of gtn helps to determine if abnormalities in vasodilatation are due to a loss in smooth muscle cell integrity , or impaired release of no from the endothelial cells . fmd and gtn are expressed as the percentage increase in post - stimulus vessel diameter relative to the baseline diameter . the correct assessment of fmd requires a number of important considerations in the study protocol . the duration of cuff occlusion must be carefully timed ; 5 min of cuff occlusion is sufficient for no - mediated dilatation while longer cuff occlusion results in non - no mediated dilatation . similarly , placement of the occluding cuff around the wrist and distal from the ultrasound probe predominantly invokes no - mediated dilatation , whereas cuff placement on the upper arm and proximal to the probe only partially stimulates no . it is also important to measure peak dilatation following cuff deflation over a protracted period of time , as measurement of peak diameter within the first 60 sec following cuff deflation can underestimate fmd by 25 40% . indeed , a period of 180 sec is likely to be sufficient in capturing true peak diameter , with most peak values occurring within the first 120 sec . the stimulus for fmd involves the production of shear stress , which activates specific endothelial receptors to release no . however , shear stress may also activate several other vasoactive factors ( some of which may cause vasoconstriction ) , making it essential that the evoked shear stress stimulus reflects vasodilation from no pathways . it is also important to account for the shear stress stimulus during fmd , with calculation of shear rate ( velocity / diameter ) serving as an adequate measure of shear stress , but not necessarily reflecting peak flow . recent physiological recommendations suggest that the shear stress profile should always be characterised when ultrasound systems allow simultaneous measurement of pulse wave velocity and active b - mode imaging in duplex mode . assessment of the carotid arteries using b - mode ultrasound can provide information on the carotid intima - media thickness ( cimt ) , and was first described in 1986 by pignoli and colleagues . assessment of cimt reflects proliferation of smooth muscle cells into the intima of the vessel and is a useful predictor of clinical events in early atherosclerosis . carotid ultrasound can often predict arterial structure better than similar techniques ( such as magnetic resonance imaging or radiographic assessments ) . in addition , cimt associates with a number of classical cvd risk factors including ageing , hypertension , and dyslipidemia . changes to the walls of the carotid artery are usually initiated by a reduction in no bioavailability which promotes inflammation within the vessel . the common carotid artery , internal carotid artery and carotid bifurcation points can be used to determine cimt , as each site can similarly predict cardiovascular events . in the present manuscript , we provide detailed methodology on assessment of microvascular endothelial function ( ldi with iontophoresis ) , large vessel endothelial function ( fmd and gtn ) and vascular morphology ( cimt ) . atherosclerosis is a multi - stage process which begins with endothelial dysfunction and ends with focal atherosclerotic lesions in the large arteries . the rationale for choosing the above assessments is that they reflect the different stages of atherosclerosis and help to account for the heterogeneous nature of the vasculature . furthermore , we have previously shown that in a population of patients at increased risk for cvd , microvascular endothelial function was independent from large vessel endothelial function , and functional assessments were independent from structural assessments of the vasculature . therefore , global assessments of the vasculature can help to decipher the different stages of atherosclerosis . note : the protocol follows guidelines from dudley group nhs foundation trust s human research ethics committee . perform all described techniques in a temperature controlled laboratory ( 21 22 c ) , with stable lighting and absence of noise . ask individuals undergoing assessments to refrain from food , drink , smoking and exercise 12 hr prior to the test . switch on laser doppler imager ( ldi ) and allow the scanner to automatically stabilise for approximately 30 min measurement ( the software s home screen will then be shown ) . on the home screen , manually enter the protocol ( the protocol used in our laboratory involves a total of 13 scans , with electrical current for iontophoretic drug delivery set from scan 2 to scan 11 at a voltage of 30 a ) . set scan 1 as a baseline scan with no electric current , and scan 12 & 13 as recovery scans also with no electrical current . click ok to confirm settings and return to home screen . ask participant to relax in a semi - recumbent chair with their forearm resting 90 degrees on a comfortable , firm pillow , and place a black mat under the forearm . note : it is important that the participants arm is strapped firmly to the pillow so that there is no movement and associated artefacts . connect the wired plugs at the opposite end of each perspex chamber to the iontophoresis controller . connect the chamber containing a 2.5 ml dose of 1% acetylcholine ( ach ) to the anodal connection of the iontophoresis controller , and connect the second chamber containing a 2.5 ml dose of 1% sodium - nitroprusside ( snp ) to the cathodal connection . connect the two chambers to the volar aspect of the participant s right forearm using double sided adhesive pads . cover the chambers by 32 mm coverslips to prevent leakage of fluid . before starting the scan , open the scanner setup window located on the top left of the home screen . select the video and distance tab and select the auto distance function to measure the distance of the scanner head from the participants forearm . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the mark button in the bottom right corner of the window . if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the assessment , open the data file using ldi image analysis software to perform measurements of perfusion . click image review on the main software window , and open the image file that is to be analysed.use the software to mark out a region of interest around the outer diameters of each chamber . adjust the region of interest so that it fits correctly on the area where the chambers were present . statistics icon and a column containing the median perfusion units for each chamber will be displayed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : this method of analysis is specific to our laboratory ; however , other methods can be used to express data obtained from the ldi scan . for a comprehensive review image review on the main software window , and open the image file that is to be analysed . use the software to mark out a region of interest around the outer diameters of each chamber . adjust the region of interest statistics icon and a column containing the median perfusion units for each chamber will be displayed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : this method of analysis is specific to our laboratory ; however , other methods can be used to express data obtained from the ldi scan . for a comprehensive review please refer to guidelines from roustit and cracowski . to calculate percentage change in perfusion in response to ach and snp , subtract baseline perfusion from the peak perfusion , divide by baseline perfusion and then multiply by 100 . note : in our lab , changes in perfusion relative to baseline have shown good intra - observer coefficient of variation for ach ( 7% ) and snp ( 6% ) . switch on the doppler ultrasound machine and networked pc containing vascular image analysis ( via ) software . note : the via software captures a live image ( at 25 frames per sec ) and provides information on the vessel diameter as well as the quality of the vascular borders being detected by the ultrasound machine . ask participant to relax in a semi - recumbent armchair and place their arm on a comfortable pillow out to their side but level with the heart . place a blood pressure cuff around the participant s wrist . note : the patient should be asked to keep their arm as still as possible to prevent movement artefacts during the measurement . secure the linear array transducer from the ultrasound machine into a stereotactic clamp , and tighten the clamp using the wingnuts so that the ultrasound transducer remains in a fixed position . note : the clamp will ensure that the ultrasound transducer will remain stable once the blood vessel is located . on the ultrasound machine , scroll into the menu and set the scanning frequency at 5 mhz and optimize the depth ( the recommended depth setting is 3.5 cm ) and gain settings on the ultrasound machine . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . using the linear array transducer , locate the brachial artery which is usually found 2 - 10 cm above the antecubital fossa in the longitudinal scanning plane . make any adjustments to clarify the image quality at this stage . to help identify the artery , turn on the colour doppler to help show pulsatile arterial blood flow and distinguish it from continuous venous blood flow . view the brachial artery horizontally across the screen ; it should appear as two solid parallel lines , separated by a clear area in between the lines which represents the lumen of the vessel . to allow the via software to automatically record vessel diameter , use the cursor to mark a predetermined region of interest to detect and track the anterior and posterior walls of the artery . note : the size of the region of interest can be increased or decreased using the x and y buttons located on the main software screen . start on the via software and image the artery for 2 min . following this , inflate on the via software and simultaneously inflate the blood pressure cuff placed around the wrist to suprasystolic pressures ( usually above 220 mmhg ) for 5 min . note : the purpose of the wrist cuff is to occlude blood flow to the hand . after 5 min deflate the blood pressure cuff to induce reactive hyperaemia which , in a healthy vessel , will stimulate no - mediated vasodilatation . note : peak dilatation can occur up to 180 sec following cuff deflation , so it is advisable to continue recording vascular diameters for 3 min after cuff release . following a 10 min rest period , re - locate the brachial artery using the linear array transducer and record a 2 min baseline diameter reading in the same manner as step 2.7 . then ask the participant to place a 500 g sublingual glyceryl - trinitrate ( gtn ) tablet under their tongue and continue to measure the brachial artery diameter for a further 5 min . after this period , ask the participant to remove the gtn tablet and monitor the participant to make sure they do not experience any adverse effects to the drug . carry out all analysis of data offline . twenty - five data points are available for each second of the assessment ; collapse this data into one - second epochs in microsoft excel . export the data to a digital signal analysis package and filter with a 3 sec moving average filter . establish the baseline diameter from the 120 sec of data prior to the cuff - inflation . visually inspect the baseline region and exclude artefacts . for the flow - mediated dilatation ( fmd ) analysis , use the software to automatically scan the post cuff - deflation region for peak dilation and use the cursor to mark this peak for visual inspection . if the peak has been misidentified , use the cursor to select a more confined region within which the peak could then be identified . adopt an identical procedure to that used with fmd , except search for peak dilation in the region following the 5 min of drug administration . to calculate fmd % and gtn % , subtract baseline diameter from the peak diameter , divide by baseline diameter and then multiply by 100 . note : in our laboratory , the intra - observer coefficient of variation is 11% for fmd , and 12% for gtn . ask participant to lie comfortably on a bed , and place a pillow under the head to offer support to the neck . connect the electrocardiogram ( ecg ) leads to the doppler ultrasound and then attach them onto the patient limbs . only a basic ecg trace is required , so place the appropriate leads on the left and right arms , and on the left ankle . menu and setting the scanning frequency at 10 mhz and optimizing depth ( the recommended depth setting is 3 4 cm ) and gain settings . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . ask participant to tilt their head slightly to the left , and using the linear array transducer , scan the right carotid artery along all its sections ( common , internal and external carotid artery ) using the longitudinal scanning plane to identify the presence of any plaques . save images that display any evidence of plaque . to help identify the artery , look for a bifurcation point in the vessel , as this shows the common carotid artery bifurcating into the internal and external carotid arteries . for measurement of carotid intima - media thickness ( cimt ) , attain at least 3 images of a section of the common carotid artery that is free of plaque , and is 1 cm proximal to the carotid bulb . attain all images at the peak of the r wave on the ecg as this corresponds to ventricular diastole and the point at which the vessel is under the least amount of shear stress . ask the participant to tilt their head slightly to the right for this measurement . to assist in attaining clear images of the near and far walls , carefully manipulate the ultrasound probe during the assessment to ensure the vessel is perpendicular to the ultrasound beam . achieve this by subtly changing the tilt and rotation of the transducer along with minor adjustments to the pressure applied to the proximal - to - distal angle ( heel - toe movement ) of the probe . carry out analysis of images offline using artery measurement software ( ams ) to detect the vascular boundary according to the lines of pignoli . load up the image to be analyzed , and then using the cursor , create a region of interest in a section of the vessel that is free from plaque . click detect on the software and record the values displayed on the screen for cimt and lumen diameter . note : accurate readings can only be obtained from the far wall , so ignore readings from the near wall . take three measurements for each side , and then average these to give the mean cimt for the right and left carotid arteries separately . note : the intra - observer coefficient of variation for this technique in our laboratory is 9% . perform measurement of any plaque using the same software by manually marking out the plaque using the cursor . click classify on ams to automatically calculate the echogenicity of the plaque and grade according to its susceptibility for rupture . switch on laser doppler imager ( ldi ) and allow the scanner to automatically stabilise for approximately 30 min . start the ldi software and click measurement ( the software s home screen will then be shown ) . on the home screen , manually enter the protocol ( the protocol used in our laboratory involves a total of 13 scans , with electrical current for iontophoretic drug delivery set from scan 2 to scan 11 at a voltage of 30 a ) . set scan 1 as a baseline scan with no electric current , and scan 12 & 13 as recovery scans also with no electrical current . click ok to confirm settings and return to home screen . ask participant to relax in a semi - recumbent chair with their forearm resting 90 degrees on a comfortable , firm pillow , and place a black mat under the forearm . note : the mat helps to limit artefact measurements generated by background surfaces surrounding the tissue . it is important that the participants arm is strapped firmly to the pillow so that there is no movement and associated artefacts . connect the wired plugs at the opposite end of each perspex chamber to the iontophoresis controller . connect the chamber containing a 2.5 ml dose of 1% acetylcholine ( ach ) to the anodal connection of the iontophoresis controller , and connect the second chamber containing a 2.5 ml dose of 1% sodium - nitroprusside ( snp ) to the cathodal connection . connect the two chambers to the volar aspect of the participant s right forearm using double sided adhesive pads . cover the chambers by 32 mm coverslips to prevent leakage of fluid . before starting the scan , open the scanner setup window located on the top left of the home screen . select the video and distance tab and select the auto distance function to measure the distance of the scanner head from the participants forearm . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . open the data file using ldi image analysis software to perform measurements of perfusion . click image review on the main software window , and open the image file that is to be analysed.use the software to mark out a region of interest around the outer diameters of each chamber . adjust the region of interest so that it fits correctly on the area where the chambers were present . then click the statistics icon and a column containing the median perfusion units for each chamber will be displayed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : this method of analysis is specific to our laboratory ; however , other methods can be used to express data obtained from the ldi scan . for image review on the main software window , and open the image file that is to be analysed . use the software to mark out a region of interest around the outer diameters of each chamber . adjust the region of interest so that it fits correctly on the area where the chambers were present statistics icon and a column containing the median perfusion units for each chamber will be displayed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : this method of analysis is specific to our laboratory ; however , other methods can be used to express data obtained from the ldi scan . for a comprehensive review please refer to guidelines from roustit and cracowski . to calculate percentage change in perfusion in response to ach and snp , subtract baseline perfusion from the peak perfusion , divide by baseline perfusion and then multiply by 100 . note : in our lab , changes in perfusion relative to baseline have shown good intra - observer coefficient of variation for ach ( 7% ) and snp ( 6% ) . switch on the doppler ultrasound machine and networked pc containing vascular image analysis ( via ) software . note : the via software captures a live image ( at 25 frames per sec ) and provides information on the vessel diameter as well as the quality of the vascular borders being detected by the ultrasound machine . ask participant to relax in a semi - recumbent armchair and place their arm on a comfortable pillow out to their side but level with the heart . place a blood pressure cuff around the participant s wrist . note : the patient should be asked to keep their arm as still as possible to prevent movement artefacts during the measurement . secure the linear array transducer from the ultrasound machine into a stereotactic clamp , and tighten the clamp using the wingnuts so that the ultrasound transducer remains in a fixed position . note : the clamp will ensure that the ultrasound transducer will remain stable once the blood vessel is located . on the ultrasound machine , scroll into the menu and set the scanning frequency at 5 mhz and optimize the depth ( the recommended depth setting is 3.5 cm ) and gain settings on the ultrasound machine . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . using the linear array transducer , locate the brachial artery which is usually found 2 - 10 cm above the antecubital fossa in the longitudinal scanning plane . make any adjustments to clarify the image quality at this stage . to help identify the artery , turn on the colour doppler to help show pulsatile arterial blood flow and distinguish it from continuous venous blood flow . view the brachial artery horizontally across the screen ; it should appear as two solid parallel lines , separated by a clear area in between the lines which represents the lumen of the vessel . to allow the via software to automatically record vessel diameter , use the cursor to mark a predetermined region of interest to detect and track the anterior and posterior walls of the artery . note : the size of the region of interest can be increased or decreased using the x and y buttons located on the main software screen . start on the via software and image the artery for 2 min . following this , inflate on the via software and simultaneously inflate the blood pressure cuff placed around the wrist to suprasystolic pressures ( usually above 220 mmhg ) for 5 min . note : the purpose of the wrist cuff is to occlude blood flow to the hand . after 5 min deflate the blood pressure cuff to induce reactive hyperaemia which , in a healthy vessel , will stimulate no - mediated vasodilatation . note : peak dilatation can occur up to 180 sec following cuff deflation , so it is advisable to continue recording vascular diameters for 3 min after cuff release . following a 10 min rest period , re - locate the brachial artery using the linear array transducer and record a 2 min baseline diameter reading in the same manner as step 2.7 . then ask the participant to place a 500 g sublingual glyceryl - trinitrate ( gtn ) tablet under their tongue and continue to measure the brachial artery diameter for a further 5 min . after this period , ask the participant to remove the gtn tablet and monitor the participant to make sure they do not experience any adverse effects to the drug . carry out all analysis of data offline . twenty - five data points are available for each second of the assessment ; collapse this data into one - second epochs in microsoft excel . export the data to a digital signal analysis package and filter with a 3 sec moving average filter . establish the baseline diameter from the 120 sec of data prior to the cuff - inflation . visually inspect the baseline region and exclude artefacts . for the flow - mediated dilatation ( fmd ) analysis , use the software to automatically scan the post cuff - deflation region for peak dilation and use the cursor to mark this peak for visual inspection . if the peak has been misidentified , use the cursor to select a more confined region within which the peak could then be identified . for the gtn data , adopt an identical procedure to that used with fmd , except search for peak dilation in the region following the 5 min of drug administration . to calculate fmd % and gtn % , subtract baseline diameter from the peak diameter , divide by baseline diameter and then multiply by 100 . note : in our laboratory , the intra - observer coefficient of variation is 11% for fmd , and 12% for gtn . ask participant to lie comfortably on a bed , and place a pillow under the head to offer support to the neck . connect the electrocardiogram ( ecg ) leads to the doppler ultrasound and then attach them onto the patient limbs . only a basic ecg trace is required , so place the appropriate leads on the left and right arms , and on the left ankle . menu and setting the scanning frequency at 10 mhz and optimizing depth ( the recommended depth setting is 3 4 cm ) and gain settings . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . ask participant to tilt their head slightly to the left , and using the linear array transducer , scan the right carotid artery along all its sections ( common , internal and external carotid artery ) using the longitudinal scanning plane to identify the presence of any plaques . save images that display any evidence of plaque . to help identify the artery , look for a bifurcation point in the vessel , as this shows the common carotid artery bifurcating into the internal and external carotid arteries . for measurement of carotid intima - media thickness ( cimt ) , attain at least 3 images of a section of the common carotid artery that is free of plaque , and is 1 cm proximal to the carotid bulb . attain all images at the peak of the r wave on the ecg as this corresponds to ventricular diastole and the point at which the vessel is under the least amount of shear stress . ask the participant to tilt their head slightly to the right for this measurement . to assist in attaining clear images of the near and far walls , carefully manipulate the ultrasound probe during the assessment to ensure the vessel is perpendicular to the ultrasound beam . achieve this by subtly changing the tilt and rotation of the transducer along with minor adjustments to the pressure applied to the proximal - to - distal angle ( heel - toe movement ) of the probe . carry out analysis of images offline using artery measurement software ( ams ) to detect the vascular boundary according to the lines of pignoli . load up the image to be analyzed , and then using the cursor , create a region of interest in a section of the vessel that is free from plaque . click detect on the software and record the values displayed on the screen for cimt and lumen diameter . note : accurate readings can only be obtained from the far wall , so ignore readings from the near wall . take three measurements for each side , and then average these to give the mean cimt for the right and left carotid arteries separately . note : the intra - observer coefficient of variation for this technique in our laboratory is 9% . perform measurement of any plaque using the same software by manually marking out the plaque using the cursor . click classify on ams to automatically calculate the echogenicity of the plaque and grade according to its susceptibility for rupture . laser doppler imaging with iontophoresis the median blood flux units following the laser doppler imaging scans from a healthy middle aged female free from cvd are shown in figure 1 . there was a marked increase in median blood flux for both ach and snp . baseline blood flux was 48 perfusion units for ach , and 67 perfusion units for snp . peak blood flux in response to ach was 455 perfusion units , and for snp 446 perfusion units . this yielded an 831% and 566% increase in perfusion ( relative to baseline ) for ach and snp respectively . the values that are provided are highly - dependent on the equipment used to examine skin blood flux along with environmental factors . flow - mediated dilatation and glyceryl trinitrate - mediated dilation figure 2 displays the baseline and peak diameters for fmd and gtn assessments from a healthy young male free from cvd . the baseline diameter of the brachial artery was 3.0 mm for the fmd and gtn assessments . the peak diameter in the fmd test was 3.3 mm , while for the gtn assessment it was 3.9 mm , which corresponds to a 10 and 30% increase in blood flow respectively , relative to baseline . carotid intima - media thickness figure 3 shows the left carotid artery of a healthy individual . the cimt in the far wall was 0.83 mm and the lumen diameter of the vessel was 7.71 mm . the results for the right carotid artery in the same individual were 0.87 mm for cimt , and 7.80 mm for the lumen diameter . when averaging the reading from both sides , cimt was 0.85 mm , and lumen diameter was 7.76 mm . after completion of a baseline scan to measure baseline blood flux , 10 scans ( scan 1 to 10 ) with iontophoresis of ach and snp using a 30 a electrical current were performed . the graph displays the baseline diameter and a clear increase in peak diameters following application of the flow - mediated and glyceryl trinitrate - mediated dilatation stimuli . an ultrasound scan of the left carotid artery is shown with a region of interest placed 1 cm from the carotid bulb ( point of bifurcation ) . the present manuscript details the methodology of several distinct assessments of vascular function and morphology which can be performed in the peripheral vasculature . each assessment provides information on the distinct stages of atherosclerosis , and help to characterize the vascular profile of different vascular territories . we have previously reported that microvascular endothelial function is independent from large vessel endothelial function in a population of rheumatoid arthritis patients at increased risk of cvd . moreover , assessments of vascular function and morphology were also independent from each other in the same group of patients and in patients with cvd . these findings can be explained by the heterogeneity of function and structure of endothelial cells in different vascular territories , as well as a possible time lag for progression of functional alterations to morphological abnormalities in the vessel . a study by hashimoto and colleagues revealed that several participants with atherosclerosis had decreased fmd values but normal cimt values . these findings suggest that examination of subclinical atherosclerosis using a variety of methods is important to decipher the global effects of cvd . the importance of the microvasculature in health and disease is gaining increasing attention in the medical literature . the microvessels form a much larger surface area than large vessels making them significant targets for damage from injurious stimuli . it has been hypothesized that microvessels might be the primary source of inflammatory mediators which infiltrate the endothelium of the larger vessels leading to lesion formation . in type ii diabetics , microvascular disease often precedes large vessel disease , and in other populations with increased risk for cvd such as rheumatoid arthritis , interventions which reduce the cvd risk improve microvascular , but not large vessel , endothelial function . collectively , these findings suggest that examination of microvascular function may help in understanding the complex mechanisms which initiate atherosclerosis . in the present work , several other assessments can be used to assess microvascular function including nailfold capillaroscopy and venous occlusion plethysmography . however , the former assessment provides information on microvascular morphology only , while the latter is time consuming and in some protocols invasive due to administration of intra - brachial vasoactive agents . in contrast , ldi offers a simple , time - efficient approach to measure microvascular perfusion of skin blood vessels in response to vasoactive agents which are administered non - invasively . the measurement of skin blood flow has gained widespread acceptance in the literature due to its ease of accessibility and strong correlation with established cvd . moreover , the advantage of ldi over other doppler techniques such as laser doppler flowmetry , is that it can simultaneously scan multiple points in a given area and can therefore account for cellular movement artefacts and spatial differences of skin blood flow , both of which can affect the perfusion of the vessel . despite the obvious advantages of iontophoresis , it is important to note that current induced vasodilatation ( civ ) from iontophoresis may confound the effects of the vasoactive agents especially at the cathode . the choice of vehicle for drug delivery could help to reduce this effect , with 0.5% sodium chloride ( as used in the current protocol ) effective in limiting civ . furthermore , use of larger diameter chambers and low electrical currents ( as used in the current protocol ) all help to reduce civ . for example , circadian variation and smoking have been shown to influence microvascular endothelial function . strict recording conditions must be adhered to in order to obtain accurate results and established guidelines should be followed when designing protocols . measurement of fmd and gtn - mediated dilatation provides information on endothelial dysfunction in the large blood vessels , and is used widely in non - invasive vascular research . the fmd technique provides surrogate information on no bioavailability and is a useful prognostic marker of cardiac events in different clinical populations . in the present work , the protocol presented accounts for many of the factors that are necessary for adequate stimulation of no - mediated vasodilatation . for example , the occluding cuff was placed distal to the ultrasound probe and around the wrist , the duration of ischemia was 5 min and adequate time was allowed to record the unfortunately , the protocol did not include characterization of the shear stress profile as the automatic edge detection software did not allow simultaneous recording of vessel diameter and pulse wave velocity signal . the calculation of shear stress is integral to accurate measurement of fmd and we recommended that , where possible , vascular research groups use software which allows such measurements to be performed . assessments of fmd and gtn - mediated dilatation are also susceptible to environmental and biological variations , as small changes in vascular diameter can elicit large fmd / gtn responses . for example , typical fmd values for healthy participants range from 5 - 10% , which corresponds to a 0.25 0.5 mm change in arterial diameter for an artery with a diameter of 5 mm . given such small changes to the arterial diameter , careful attention must be paid to technical and biological factors that may influence the measurement . indeed , fmd can be affected by a variety of biologic and behavioural factors such as sympathetic activation , sleep deprivation , caffeine consumption , smoking , antioxidant therapy and time of day . accordingly , it is important to control for these factors by utilising information from established guidelines . assessment of advanced but subclinical atherosclerosis was done using cimt . the technique has been utilised in several clinical populations and provides great detail on arterial structure when compared to more sophisticated techniques such as magnetic resonance imaging . as with the other vascular techniques , measurement of cimt generally , cimt should be performed in areas free from plaque , in the far wall of the common carotid artery . similar to fmd , measurement of cimt is performed using high - resolution ultrasonography and so is highly user - dependent . reported coefficient of variation ( cofv ) range from 2.4 18.3% , while for fmd it is 1 84% . however , even when both techniques are performed by competent ultrasonographers with external factors well - controlled , there remains a high cofv . one reason for this could be that analysis of vascular borders are carried out using manual methods . such analysis can reduce reliability as imaging artefacts such as false borders , noise from the ultrasound signal , and distortion of vessels can affect interpretation of the image . recent developments in continuous automated edge - detection software have greatly improved the detection of vascular wall boundaries . in the present study , via software was used to measure the brachial artery diameter , while ams was used to detect cimt . the use of these software greatly reduces operator dependence , yet in the case of ams , some degree of operator control is still available in situations where image quality may be poor . laboratories which use automated edge detection software generally tend to have low cofv , thus it should be the aim of all vascular research laboratories to incorporate automated measurement of vascular boundaries in order to ensure accuracy of the results . it is also good practice to report results of reproducibility studies for specific protocols when publishing study outcomes . although assessments of vascular function are routinely used in clinical research , a limitation of the techniques is that normative values for ldi with iontophoresis and fmd do not exist . it is therefore important that healthy age- and sex - matched control groups are examined to compare findings with the experimental group . even though these techniques associate with poor prognosis in a variety of populations with evidence of cvd , there is still a dearth of studies which have examined the relationship between poor endothelial function and adverse cardiovascular outcomes such as myocardial infarction and stroke . another limitation is the use of human operators to perform the assessments and carry out the analysis . this introduces a potential source of bias ; however , this can be limited by blinding the operator to the results or ensuring that the reader is different from the operator . it is also important to ensure that the reader follows a standardised protocol for data analysis , so that all data is analysed consistently . in summary , the present manuscript provides detailed information on the methodological steps necessary to successfully perform assessments of microvessel and large vessel endothelial function as well as vascular morphology of the peripheral circulation . when used together , the assessments provide global information on the different stages of atherosclerosis .

the endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis . damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease . assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function . the present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser doppler imaging with iontophoresis , large vessel endothelial function using flow - mediated dilatation , and carotid atherosclerosis using carotid artery ultrasound . a discussion on the methodological considerations for each of the techniques is also presented , and recommendations are made for future research .

Introduction Protocol 1. Laser Doppler Imaging with Iontophoresis 2. Flow-mediated Dilatation and Glyceryl trinitrate-mediated Dilatation 3. Carotid Intima-media Thickness Representative Results Discussion Disclosures

the endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis via the regulation of a multitude of vasoactive processes . disruption to these processes may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease ( cvd ) . furthermore , measures of peripheral endothelial function have been shown to reflect coronary endothelial function , and as such are regarded as good predictors of cardiovascular disease . assessments of peripheral endothelial function typically quantify the vasodilatory response of the vessel to a specific stimulus , with an attenuation of the dilatory response indicative of endothelial dysfunction , and can be measured in different vascular beds . assessments of advanced structural changes in the vessel can be characterised by ultrasound examination of the intima - media thickness . in the microcirculation , laser doppler flowmetry ( ldf ) and laser doppler imaging ( ldi ) with iontophoresis of vasodilator agonists can provide useful information on microvascular perfusion . the assessment of ldi offers considerable advantages over ldf , because unlike ldf , ldi can scan over a vast area thus accounting for heterogeneity in skin blood flow and increasing the reproducibility of the technique . the use of snp directly activates smooth muscle cell receptors to allow for maximum vasodilatation of the vessel and examination of smooth muscle integrity . there is some uncertainty on whether ach - mediated dilatation involves no at all , as ach may stimulate non - no pathways such as cycloxygenase - mediated pathways . microvascular endothelial function can be quantified using different approaches , with cutaneous vascular conductance a product of flux divided by arterial pressure , used in studies where blood pressure may change over the study duration ( i.e. in the large vessels , flow - mediated dilatation ( fmd ) and glyceryl - trinitrate mediated dilatation ( gtn ) are performed to assess endothelium - dependent and endothelium - independent function respectively . fmd is typically carried out in the brachial artery where a cuff is used to occlude arterial blood flow for 5 min ; release of the cuff causes a sudden increase in blood flow ( reactive hyperaemia ) through the brachial artery resulting in shear - stress mediated dilatation of the vessel . the baseline and post - cuff release diameter are quantified by ultrasound imaging of the vessel with subsequent assessments of the vessel diameter performed manually or using automated edge detection software . the correct assessment of fmd requires a number of important considerations in the study protocol . the duration of cuff occlusion must be carefully timed ; 5 min of cuff occlusion is sufficient for no - mediated dilatation while longer cuff occlusion results in non - no mediated dilatation . similarly , placement of the occluding cuff around the wrist and distal from the ultrasound probe predominantly invokes no - mediated dilatation , whereas cuff placement on the upper arm and proximal to the probe only partially stimulates no . it is also important to account for the shear stress stimulus during fmd , with calculation of shear rate ( velocity / diameter ) serving as an adequate measure of shear stress , but not necessarily reflecting peak flow . assessment of the carotid arteries using b - mode ultrasound can provide information on the carotid intima - media thickness ( cimt ) , and was first described in 1986 by pignoli and colleagues . assessment of cimt reflects proliferation of smooth muscle cells into the intima of the vessel and is a useful predictor of clinical events in early atherosclerosis . changes to the walls of the carotid artery are usually initiated by a reduction in no bioavailability which promotes inflammation within the vessel . the common carotid artery , internal carotid artery and carotid bifurcation points can be used to determine cimt , as each site can similarly predict cardiovascular events . in the present manuscript , we provide detailed methodology on assessment of microvascular endothelial function ( ldi with iontophoresis ) , large vessel endothelial function ( fmd and gtn ) and vascular morphology ( cimt ) . the rationale for choosing the above assessments is that they reflect the different stages of atherosclerosis and help to account for the heterogeneous nature of the vasculature . furthermore , we have previously shown that in a population of patients at increased risk for cvd , microvascular endothelial function was independent from large vessel endothelial function , and functional assessments were independent from structural assessments of the vasculature . therefore , global assessments of the vasculature can help to decipher the different stages of atherosclerosis . connect the chamber containing a 2.5 ml dose of 1% acetylcholine ( ach ) to the anodal connection of the iontophoresis controller , and connect the second chamber containing a 2.5 ml dose of 1% sodium - nitroprusside ( snp ) to the cathodal connection . connect the two chambers to the volar aspect of the participant s right forearm using double sided adhesive pads . before starting the scan , open the scanner setup window located on the top left of the home screen . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the mark button in the bottom right corner of the window . if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the assessment , open the data file using ldi image analysis software to perform measurements of perfusion . click image review on the main software window , and open the image file that is to be analysed.use the software to mark out a region of interest around the outer diameters of each chamber . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . for a comprehensive review image review on the main software window , and open the image file that is to be analysed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : the via software captures a live image ( at 25 frames per sec ) and provides information on the vessel diameter as well as the quality of the vascular borders being detected by the ultrasound machine . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . view the brachial artery horizontally across the screen ; it should appear as two solid parallel lines , separated by a clear area in between the lines which represents the lumen of the vessel . note : the size of the region of interest can be increased or decreased using the x and y buttons located on the main software screen . note : the purpose of the wrist cuff is to occlude blood flow to the hand . twenty - five data points are available for each second of the assessment ; collapse this data into one - second epochs in microsoft excel . for the flow - mediated dilatation ( fmd ) analysis , use the software to automatically scan the post cuff - deflation region for peak dilation and use the cursor to mark this peak for visual inspection . ask participant to lie comfortably on a bed , and place a pillow under the head to offer support to the neck . only a basic ecg trace is required , so place the appropriate leads on the left and right arms , and on the left ankle . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . ask participant to tilt their head slightly to the left , and using the linear array transducer , scan the right carotid artery along all its sections ( common , internal and external carotid artery ) using the longitudinal scanning plane to identify the presence of any plaques . to help identify the artery , look for a bifurcation point in the vessel , as this shows the common carotid artery bifurcating into the internal and external carotid arteries . for measurement of carotid intima - media thickness ( cimt ) , attain at least 3 images of a section of the common carotid artery that is free of plaque , and is 1 cm proximal to the carotid bulb . attain all images at the peak of the r wave on the ecg as this corresponds to ventricular diastole and the point at which the vessel is under the least amount of shear stress . to assist in attaining clear images of the near and far walls , carefully manipulate the ultrasound probe during the assessment to ensure the vessel is perpendicular to the ultrasound beam . achieve this by subtly changing the tilt and rotation of the transducer along with minor adjustments to the pressure applied to the proximal - to - distal angle ( heel - toe movement ) of the probe . carry out analysis of images offline using artery measurement software ( ams ) to detect the vascular boundary according to the lines of pignoli . load up the image to be analyzed , and then using the cursor , create a region of interest in a section of the vessel that is free from plaque . take three measurements for each side , and then average these to give the mean cimt for the right and left carotid arteries separately . connect the chamber containing a 2.5 ml dose of 1% acetylcholine ( ach ) to the anodal connection of the iontophoresis controller , and connect the second chamber containing a 2.5 ml dose of 1% sodium - nitroprusside ( snp ) to the cathodal connection . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . following completion of the auto distance measurement , select the image scan tab and determine the area that is to be scanned by clicking the if needed , change the size of the region of interest by manually entering in the size of the scanning area into the scan area section near the top of the window . ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . for image review on the main software window , and open the image file that is to be analysed . adjust the region of interest so that it fits correctly on the area where the chambers were present statistics icon and a column containing the median perfusion units for each chamber will be displayed . note the baseline perfusion unit , as well as the highest perfusion unit from each of the preceding 12 scans for each chamber . note : the via software captures a live image ( at 25 frames per sec ) and provides information on the vessel diameter as well as the quality of the vascular borders being detected by the ultrasound machine . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . view the brachial artery horizontally across the screen ; it should appear as two solid parallel lines , separated by a clear area in between the lines which represents the lumen of the vessel . note : the size of the region of interest can be increased or decreased using the x and y buttons located on the main software screen . twenty - five data points are available for each second of the assessment ; collapse this data into one - second epochs in microsoft excel . for the flow - mediated dilatation ( fmd ) analysis , use the software to automatically scan the post cuff - deflation region for peak dilation and use the cursor to mark this peak for visual inspection . ask participant to lie comfortably on a bed , and place a pillow under the head to offer support to the neck . adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel . ask participant to tilt their head slightly to the left , and using the linear array transducer , scan the right carotid artery along all its sections ( common , internal and external carotid artery ) using the longitudinal scanning plane to identify the presence of any plaques . to help identify the artery , look for a bifurcation point in the vessel , as this shows the common carotid artery bifurcating into the internal and external carotid arteries . for measurement of carotid intima - media thickness ( cimt ) , attain at least 3 images of a section of the common carotid artery that is free of plaque , and is 1 cm proximal to the carotid bulb . attain all images at the peak of the r wave on the ecg as this corresponds to ventricular diastole and the point at which the vessel is under the least amount of shear stress . to assist in attaining clear images of the near and far walls , carefully manipulate the ultrasound probe during the assessment to ensure the vessel is perpendicular to the ultrasound beam . carry out analysis of images offline using artery measurement software ( ams ) to detect the vascular boundary according to the lines of pignoli . load up the image to be analyzed , and then using the cursor , create a region of interest in a section of the vessel that is free from plaque . take three measurements for each side , and then average these to give the mean cimt for the right and left carotid arteries separately . laser doppler imaging with iontophoresis the median blood flux units following the laser doppler imaging scans from a healthy middle aged female free from cvd are shown in figure 1 . flow - mediated dilatation and glyceryl trinitrate - mediated dilation figure 2 displays the baseline and peak diameters for fmd and gtn assessments from a healthy young male free from cvd . the baseline diameter of the brachial artery was 3.0 mm for the fmd and gtn assessments . the peak diameter in the fmd test was 3.3 mm , while for the gtn assessment it was 3.9 mm , which corresponds to a 10 and 30% increase in blood flow respectively , relative to baseline . the cimt in the far wall was 0.83 mm and the lumen diameter of the vessel was 7.71 mm . the results for the right carotid artery in the same individual were 0.87 mm for cimt , and 7.80 mm for the lumen diameter . the graph displays the baseline diameter and a clear increase in peak diameters following application of the flow - mediated and glyceryl trinitrate - mediated dilatation stimuli . an ultrasound scan of the left carotid artery is shown with a region of interest placed 1 cm from the carotid bulb ( point of bifurcation ) . the present manuscript details the methodology of several distinct assessments of vascular function and morphology which can be performed in the peripheral vasculature . each assessment provides information on the distinct stages of atherosclerosis , and help to characterize the vascular profile of different vascular territories . we have previously reported that microvascular endothelial function is independent from large vessel endothelial function in a population of rheumatoid arthritis patients at increased risk of cvd . moreover , assessments of vascular function and morphology were also independent from each other in the same group of patients and in patients with cvd . these findings can be explained by the heterogeneity of function and structure of endothelial cells in different vascular territories , as well as a possible time lag for progression of functional alterations to morphological abnormalities in the vessel . the importance of the microvasculature in health and disease is gaining increasing attention in the medical literature . it has been hypothesized that microvessels might be the primary source of inflammatory mediators which infiltrate the endothelium of the larger vessels leading to lesion formation . in type ii diabetics , microvascular disease often precedes large vessel disease , and in other populations with increased risk for cvd such as rheumatoid arthritis , interventions which reduce the cvd risk improve microvascular , but not large vessel , endothelial function . in the present work , several other assessments can be used to assess microvascular function including nailfold capillaroscopy and venous occlusion plethysmography . moreover , the advantage of ldi over other doppler techniques such as laser doppler flowmetry , is that it can simultaneously scan multiple points in a given area and can therefore account for cellular movement artefacts and spatial differences of skin blood flow , both of which can affect the perfusion of the vessel . for example , circadian variation and smoking have been shown to influence microvascular endothelial function . measurement of fmd and gtn - mediated dilatation provides information on endothelial dysfunction in the large blood vessels , and is used widely in non - invasive vascular research . in the present work , the protocol presented accounts for many of the factors that are necessary for adequate stimulation of no - mediated vasodilatation . for example , the occluding cuff was placed distal to the ultrasound probe and around the wrist , the duration of ischemia was 5 min and adequate time was allowed to record the unfortunately , the protocol did not include characterization of the shear stress profile as the automatic edge detection software did not allow simultaneous recording of vessel diameter and pulse wave velocity signal . assessments of fmd and gtn - mediated dilatation are also susceptible to environmental and biological variations , as small changes in vascular diameter can elicit large fmd / gtn responses . as with the other vascular techniques , measurement of cimt generally , cimt should be performed in areas free from plaque , in the far wall of the common carotid artery . such analysis can reduce reliability as imaging artefacts such as false borders , noise from the ultrasound signal , and distortion of vessels can affect interpretation of the image . recent developments in continuous automated edge - detection software have greatly improved the detection of vascular wall boundaries . in the present study , via software was used to measure the brachial artery diameter , while ams was used to detect cimt . laboratories which use automated edge detection software generally tend to have low cofv , thus it should be the aim of all vascular research laboratories to incorporate automated measurement of vascular boundaries in order to ensure accuracy of the results . although assessments of vascular function are routinely used in clinical research , a limitation of the techniques is that normative values for ldi with iontophoresis and fmd do not exist . in summary , the present manuscript provides detailed information on the methodological steps necessary to successfully perform assessments of microvessel and large vessel endothelial function as well as vascular morphology of the peripheral circulation .

body size , one of the most important quantitative traits under evolutionary scrutiny , varies considerably at both the inter- and intraspecific levels ( blanckenhorn et al . 1999 ; kraushaar and blanckenhorn 2002 ) . understanding the genetic foundation of body size variation is particularly important , especially in humans for studies on the genetic mechanisms underlying associated diseases and in domesticated animals for understanding the patterns of artificial selection and for improving production traits . compared with populations that only experience natural selection , tremendous variation in body size is observed in domesticated species , such as dogs and chickens , which have experienced strong artificial selective pressures aimed at this trait for improvements or for exaggeration . numerous genes that have small phenotypic effect on overall human body size have been identified , whereas the reverse pattern ( few genes with large effect ) has been described in domesticated animals , with a subset of genes convergently identified from both lists ( sutter et al . for example , igf1 together with almost 700 other genes explains only approximately 16% of the height variation observed in humans ( lango allen et al . 2010 ) , but a single mutation within igf1 is the dominant explanation for small body size in several different breeds of dogs ( sutter et al . 2007 ) , and the majority of the variation in the average body mass of dogs from different breeds can be explained by only six loci ( boyko et al . the official definition of a pony is a horse that measures less than 14.2 hands ( 58 inches , 147 cm ) at the withers ( mattern 2010 ) . guangxi mountains of southern china is of particular interest due to its extremely small size ( withers height less than 106 cm ) ( china national commission of animal genetic resources 2011 ) ( fig . it is thought that this breed was selected for its small size to facilitate work in mountainous regions . similar small stature horse breeds ( e.g. , shetland and caspian ponies ) are found in other location in the world , where they serve a variety of functions ( e.g. , transport in mines and carrying children ) . given the restricted geographic distributions of these different breeds , it is possible that small stature was independently selected across these distinct horse breed lineages . although previous studies have identified quantitative trait loci on equine chromosomes ( e.g. , horse chromosomes 3 , 6 , 9 , and 11 ) that contribute to size variation in breeds developed in europe , america and the middle east , where frequent migration occurred during the breeding process , it remains unknown if these loci account for the size variation observed in all horse breeds ( makvandi - nejad et al . ( a ) height of the three breeds : db , mg , and yl . data were retrieved from china national commission of animal genetic resources ( 2011 ) . ( b ) phylogenetic tree constructed by treemix with bootstrap analysis with the przewalski s horse as the outgroup . names labeled in blue are short stature horses / pony , and green are tall horses . a , akhal ; b , andalusian ; c , arabian ; d , belgian ; e , caspian ; f , clydesdale ; g , debao ; h , exmoor ; i , fell pony ; j , finnhorse ; k , franches - montagnes ; l , french trotter ; m , hanoverian ; n , icelandic ; o , inner mongolian ; p , mangalargapaulista ; q , miniature ; r , mongolian ; s , morgan ; t , new forest pony ; u , north swedish horse ; v , norwegian fjord ; w , paint ; x , percheron ; y , peruvian paso ; z , puerto rican paso fino ; a , quarter horse ; b , saddlebred ; d : shetland ; e , shire ; f , standardbred ; g , swiss warmblood ; h , thoroughbred ; i , tuva ; j , yili . ( a ) height of the three breeds : db , mg , and yl . data were retrieved from china national commission of animal genetic resources ( 2011 ) . ( b ) phylogenetic tree constructed by treemix with bootstrap analysis with the przewalski s horse as the outgroup . names labeled in blue are short stature horses / pony , and green are tall horses . a , akhal ; b , andalusian ; c , arabian ; d , belgian ; e , caspian ; f , clydesdale ; g , debao ; h , exmoor ; i , fell pony ; j , finnhorse ; k , franches - montagnes ; l , french trotter ; m , hanoverian ; n , icelandic ; o , inner mongolian ; p , mangalargapaulista ; q , miniature ; r , mongolian ; s , morgan ; t , new forest pony ; u , north swedish horse ; v , norwegian fjord ; w , paint ; x , percheron ; y , peruvian paso ; z , puerto rican paso fino ; a , quarter horse ; b , saddlebred ; d : shetland ; e , shire ; f , standardbred ; g , swiss warmblood ; h , thoroughbred ; i , tuva ; j , yili . , we conducted a genomic scan to search for signatures of positive selection for diminutive body size in the debao pony ( db ) and selection for the relatively higher stature of the yili ( yl ) and inner mongolian ( mg ) horse breeds from northern china ( fig . 1a ) , using a comparative approach with a world - wide sample of 730 horses . our objectives were to 1 ) investigate the phylogenic relationship between the three chinese horses and previously studied horses to estimate the origin of the db , 2 ) compare the population genomic structure of the db with those of other horses to identify genetic mechanisms underlying the small size of the db , and 3 ) identify if introgression of size - related genes occurred among different horse breeds to determine whether small stature had a single or multiple origins . a total of 96 horse individuals from three horse breeds ( db [ n = 32 ] , yl [ n = 32 ] , and mg [ n = 32 ] ) were genotyped using the geneseek equine snp 65 bead chip panel . genotyped snps data from 32 other breeds of horses ( n = 729 ) from previous studies ( petersen , mickelson , cothran , et al . snps that failed in exact tests for hardy weinberg equilibrium at p < 0.001 or had more than 10% missing genotype data or had minor allele frequency less than 5% were excluded using plink ( purcell et al . individuals with more than 10% missing genotyped data were also removed . in total , 816 individuals with 21,740 snps were retrieved for the phylogenetic analysis and multidimensional clustering ( purcell et al . snp data from a przewalski s horse were downloaded from a study ( orlando et al . 2013 ) and merged with the data from the domesticated horses to construct a maximum - likelihood tree using treemix ( pickrell and pritchard 2012 ) , based on the genome - wide allele frequency data . with a pruned data set , which discarded snps in linkage disequilibrium ( ld ) across breeds ( plink indep 50 5 2 ) , we implemented a bayesian model - based approach to assess the population relationship of the world - wide horses using the program structure ( pritchard et al . a new small data set including only the three chinese breeds was created and the population relationships were analyzed after ld - based snp pruning . to compare the diversity of the chinese population with reported diversity of other horse breeds ( petersen , mickelson , cothran , et al . 2013 ) , we pruned the data set for pairwise r < 0.1 considering 100 snp windows with a step size of 25 snps . to compare ld decay among breeds with different sample sizes , we selected a random subset of 14 horses from each of the 35 breeds . all breeds combined ld decay were calculated with each 14 individuals for a breed ( 14 35 = 490 ) . we used a total of 52,244 snps to calculate the population differentiation among the db , yl , and mg horses as described in akey et al . ( 2002 ) . to retrieve candidate snps under selection in the db , di values xp - ehh values of each snp in the debao compared with the yl and the mg were calculated by the xpehh program ( http://hgdp.uchicago.edu/software/ , last accessed december 16 , 2015 ) . the two values of xp - ehh ( db vs. yl ) and xp - ehh ( db vs. mg ) were merged using mxpehh = pdb - yili/(1pdb - yili)pdb - mg/(1pdb - mg ) , where p is the percentile rank value of the xp - ehh values of each snp . all snps within this small data set were also considered in scans for association ( pickrell and pritchard 2012 ) . a potential difference in association with stature variation between db and yl although the influence of sex was not significant in the multiple regression analysis , we still considered sex when performing the association analysis . we also performed fdr adjustment and adaptive model - based permutation using plink ( pickrell and pritchard 2012 ) . candidate genes were identified whether they are located within 150 kb of an snp of interest . gene ontology ( go ) enrichment analysis was performed on g : profiler ( http://biit.cs.ut.ee/gprofiler ) . the geneseek equine snp ( single nucleotide polymorphism ) 65 bead chip panel was used to genotype 32 individuals each from the db , yl , and mg breeds of horse . to assess the phylogenetic relationships of the chinese horses to other horse breeds , we obtained snp genotype data from 32 different breeds from a previous study ( petersen , mickelson , rendahl , et al . 2013 ) , which yielded a total of 21,740 snps that passed our quality filtration ( see materials and methods ) . after merging with the snp data from a przewalski s horse ( orlando et al . 2013 ) , a total of 14,959 snps were used to infer a maximum - likelihood tree of the horse populations using the genome - wide allele frequency from the przewalski s horse as the outgroup ( fig . as expected , all three chinese breeds genotyped here clustered with the mongolian horses ( fig . 1b ) , consistent with their close geographic distribution . the relationships of the ponies , scandinavian breeds , heavy draft horses , recent breeds derived from the thoroughbred , modern us breeds , and the trotting breeds ( fig . 1b ) were in agreement with that described in a previous study ( petersen , mickelson , cothran , et al . landrace breeds , which originated geographically , ranged freely and experience lesser degrees of management , located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . considering the fact that most modern breeds were recently created by infusing arabian , and/or thoroughbred , which is also inherited majorly from arabian , we simply grouped these breeds into an arab group and the remaining into a non - arab group ( materials and methods ) . ancient breeds located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . 1b ) . breeds with short stature were located at disperse locations in the phylogenetic trees , suggesting independent domestication of these short stature breeds . however , when the treemix program ( pickrell and pritchard 2012 ) was used to infer migration events , the tree topology frequently changed with changes in the defined number of migration events ( supplementary fig . s1 , supplementary material online ) , suggesting a complex history for horse domestication with genetic admixtures occurring among breeds . the data also suggest that the exact phylogenetic relationship might be difficult to reveal based only on allele frequency data , particularly here , since genetic admixture occurred and only one wild przewalski s horse was available . ( 2013 ) found that the mangalarga paulista was located basally in a phylogenetic tree constructed by the parsimony method , whereas in contrast , mccue et al . ( 2012 ) identified the norwegian fjord and icelandic as being basal in their phylogenetic tree , and here , we have the norwegian fjord , n swedish , and shetland horses appearing close to the przewalski s horse ( fig . 1b ) ; however , we also detected migrations from the przewalski s horse to these three breeds ( supplementary fig . s1 , supplementary material online).to better analyze the phylogenetic relationships , we next implemented a bayesian model - based approach to assess the individual assignment using the program structure ( pritchard et al . a gradient from non - arab group to arab group was found whether we assumed that there were two ancestral populations ( k = 2 ) . some european draft ( clydesdale , shire ) and pony breeds ( icelandic , miniature , shetland ) separated when four ancestral populations ( k = 4 ) were assumed . horses from mongolia , china and siberia grouped into a distinct cluster when k = 12 , suggesting a much closer relationship among these horses from the middle part of the eurasian continent . when k = 12 , przewalski s horse showed closer relationship and clustered with the chinese breeds . consistently , the neighbor - joining tree based on all individuals showed that the chinese breeds had the closest relationship to the przewalski s horse ( supplementary fig . s2 , supplementary material online ) , which contrasts with the phylogenetic tree inferred by whole population allele frequency data ( fig . these results also suggest that the phylogenetic relationships are difficult to reveal based only on allele frequency data . genetic migrations between the przewalski s horse and the chinese horse breeds were not detected by our treemix analysis , which potentially suggests that there is a true closer relationship between the przewalski s horse and the chinese breeds ( supplementary fig . s1 , supplementary material online ) . to exclude the possibility that the estimated population structures were influenced by the specific algorithmic strategy , we also used principal component analysis ( pca ) to examine the population relationships ( supplementary fig . similar to the above analysis , the first dimension moderately distinguished the arab group and non - arab group ( fig . s4 , supplementary material online ) , whereas the second dimension moderately separated horses with differing statures ( fig . horses from the middle east and asia region clustered together in the central part of the multidimensional scaling plot ( fig . consistent with the above , the przewalski s horse is close to the ancient breeds and non - arab breeds ( fig . s4b , supplementary material online ) , but in two dimensions we could not distinguish clearly the przewalski s horse from the domestic breeds . the individual closest to the przewalski s horse in the pca was the exmoor , but the treemix analysis found strong evidence of admixture between the exmoor and the przewalski s horse ( supplementary fig . consistent with the phylogenetic tree and the structural analysis , the chinese breeds were located within the ancient breeds in the pca analysis ( supplementary fig . s4b , supplementary material online ) , and closer than most breeds to the przewalski s horse ( supplementary fig . s7 , supplementary material online ) , which were comparable to the expected level of heterozygosity ( petersen , mickelson , cothran , et al . in addition , ld , which can be informative for population demography , found a similar decay in each of the three chinese breeds , with their low levels of ld similar to those seen within other landrace breeds ( fig . there were two exceptions : clydesdale and exmoor , which have small population sizes compared with other landrace breeds ( petersen , mickelson , cothran , et al . thus , the higher levels of genetic diversity , the closer relationship to the ancient breeds , and the lower levels of ld in the db than in other recent breeds suggest an ancient origin for the db , consistent with its antiquity record dating to at least 2,000 years ago ( hendricks 2007 ) . the three chinese breeds showed similar roh means , with the peaks of the roh distributions near the same lengths ( supplementary fig . s8a and b , supplementary material online ) , indicative of similar contemporary population sizes and mating systems . taken together , these results indicate that the three chinese breeds have an ancient origin , which is consistent with their anecdotal records of breed history ( wentong 1990 ; china national commission of animal genetic resources 2011 ) . perhaps surprisingly , ld across whole breeds did not rapidly decay , a pattern that is very different from that observed in dogs ( boyko et al . 1c ) , which may indicate that numerous ibd ( identity by descent ) segments are shared across multiple breeds and/or the shared segments are quite large . this pattern is consistent with horse breeding history as many modern breeds originated , at least in part , from the arabian horses , in a relatively short period of time . as the db individuals could not be assigned to a single cluster using the snps detected by all horses , we next assessed the populations of the three chinese breeds as a group using the 52,244 snps passed after quality filtration . the results from both structure and pca clearly distinguished the db from the other two populations ( supplementary fig . s8c and d , supplementary material online ) , which may indicate a differentiation between south china and north china horse populations . given that the db possesses a significantly low stature and their population characteristics imply an ancient history , we sought to identify candidate loci that regulate this phenotype and estimate the origin of this phenotype . population structure analysis implied a much closer relationship among the db , yl , and mg breeds ; therefore , we first evaluated the population differentiation of db from the other two chinese breeds to search for loci that show evidence of positive selection specific to the db , using the statistic di , as described in akey et al . we first extracted the top snps using a cutoff of 0.5% , and found 262 snps , which mapped to 153 genes ( within 150 kb of the snps ) . gene enrichment analysis found some categories associated with development of the skeletal system , such as abnormal morphology of forearm bone , abnormality of the ischium , dumbbell - shaped long bone , and aplasia / hypoplasia involving bones of the feet , but each had very few genes ( supplementary table s1 , supplementary material online ) . when a cutoff of 1% was used , a total of 523 snps ( di > 6.34 ) were identified ( fig . . gene enrichment analysis of the 338 genes closest to these snp found a significant overrepresentation in developmental process , for example , 87 genes involved in anatomical structure development ( go:0048856 ) , 17 genes involved in skeletal system development ( go:0001501 ) , 10 genes in cartilage development ( go:0051216 ) , 11 genes ( hand2 , lef1 , alx3 , col2a1 , zbtb16 , tbx3 , sulf1 , asph , en1 , pcsk5 , chst11 ) involved in limb development ( go:0060173 ) , and limb morphogenesis ( go:0035108 ) ( table 1 ) . we also extracted the top snps using cutoffs of 0.1% , and obtained similar results for enrichment of categories associated development of skeletal system ( supplementary tables s2 , supplementary material online ) . however , due to ascertainment bias and the small number of genotyped snps , it is difficult to identify the number of genes that have evolved under positive selection in the debao . 2.positive selection analysis of the db . genome - wide distribution of ( a ) di , ( b ) the p values of the merged xp - ehh values , ( c ) and manhattan plot presenting the association p value across the genome in the db . table 1overrepresented go categories among genes showing high di value in dbterm iddescriptionp valuegene numbergo:0009653anatomical structure morphogenesis2.36e-0557go:0009888tissue development4.33e-0542go:0007275multicellular organismal development5.62e-0582go:0030902hindbrain development5.80e-0511go:0009790embryo development6.85e-0535go:0048856anatomical structure development7.07e-0587go:0044767single - organism developmental process1.29e-0490go:0032502developmental process1.53e-0490go:0043009chordate embryonic development1.89e-0426go:0009792embryo development ending in birth or egg hatching2.23e-0426go:0002062chondrocyte differentiation2.51e-049go:0048731system development3.29e-0474go:0048513organ development6.02e-0460go:0072089stem cell proliferation1.12e-0310go:0035107appendage morphogenesis1.35e-0311go:0035108limb morphogenesis1.35e-0311go:0061448connective tissue development2.07e-0312go:0048736appendage development2.64e-0311go:0060173limb development2.64e-0311go:0001944vasculature development2.72e-0320go:0001501skeletal system development2.91e-0317go:0072359circulatory system development4.14e-0326go:0072358cardiovascular system development4.14e-0326go:0001701 in utero embryonic development4.93e-0317go:0007417central nervous system development5.05e-0322go:0061061muscle structure development5.08e-0318go:0006029proteoglycan metabolic process6.30e-036go:0048468cell development7.98e-0338go:0051216cartilage development9.05e-0310go:0007420brain development9.85e-0318go:0048568embryonic organ development1.03e-0217go:0072091regulation of stem cell proliferation1.04e-027go:0009887organ morphogenesis1.13e-0226go:2000648positive regulation of stem cell proliferation1.30e-026go:0048010vascular endothelial growth factor receptor signaling pathway1.44e-025go:0001568blood vessel development1.47e-0218go:0060021palate development1.49e-027go:0007605sensory perception of sound1.84e-028go:0010629negative regulation of gene expression1.85e-0227go:0051301cell division1.89e-0217go:1902679negative regulation of rna biosynthetic process2.08e-0225go:0035295tube development2.16e-0218go:0021549cerebellum development2.43e-026go:0048729tissue morphogenesis2.47e-0219go:0014706striated muscle tissue development2.52e-0213go:0048598embryonic morphogenesis3.21e-0219go:0051253negative regulation of rna metabolic process3.25e-0225go:0048869cellular developmental process3.26e-0258go:0007399nervous system development3.32e-0235go:0050954sensory perception of mechanical stimulus3.33e-028go:0030154cell differentiation3.38e-0254go:0007049cell cycle3.50e-0228go:0007588excretion3.60e-024go:2000113negative regulation of cellular macromolecule biosynthetic process3.94e-0226go:0002009morphogenesis of an epithelium4.04e-0216go:0001570vasculogenesis4.22e-026go:0060537muscle tissue development4.37e-0213go:0016486peptide hormone processing4.54e-023 positive selection analysis of the db . genome - wide distribution of ( a ) di , ( b ) the p values of the merged xp - ehh values , ( c ) and manhattan plot presenting the association p value across the genome in the db . overrepresented go categories among genes showing high di value in db beside high population differentiation , regions under directional selection also show other signatures of variation , such as long range haplotype homozygosity ( sabeti et al . 2006 ) . to identify alleles potentially under selection in the db , we employed xp - ehh , which is based on long - range haplotype homozygosity and is suitable for the detection of recent positive selection and a total of 505 snps have significantly high merged xp - ehh values in the db ( p < 0.01 ) ( fig . a total of 635 genes were found within 150 kp of these snps , and these genes were found to be significantly enriched in go terms for bone and muscle variation ( supplementary table s3 , supplementary material online ) , such as upper limb undergrowth , abnormal cortical bone morphology , aplasia / hypoplasia affecting bones of the axial skeleton , aplasia / hypoplasia involving bones of the feet , abnormality of the musculature of the limbs , limb - girdle muscle weakness , and foot dorsiflexor weakness . we also retrieved 122 genes with more significance of merged xp - ehh ( p < 0.005 ) , which showed similar enrichment in the development , bone and muscle variation , such as regulation of developmental process , skeletal muscle tissue development , spinal cord lesions , and abnormality of the mandible although many candidate selected genes are involved in the development of skeletal system , the selected alleles in db pony identified by the outlier methods can not be directly associated with small stature in db pony , as they might be confounded by other unique phenotypes found in the db . to verify whether the identified loci play a role in the small stature size of the db , we conducted association scans within the db and yl for variation in stature ( fig . 2c ) . after adjustment for fdr , 22 snps showed a significant association with stature ( bonf p value < 0.01 ) , all of which harbored significantly high di values ( top 1% ) and/or significant merged xp - ehh values ( p < 0.01 ) . to control for potential confounding effects of stratification and the nonindependence of related individuals that may bias the association study ( although the above population structure analysis showed no legacy of stratification for each of the chinese breeds and we tried to collect samples from different families or villages ) , we performed an adaptive permutation and confirmed the most significant 22 snps for stature variation ( supplementary table s5 , supplementary material online ) . furthermore , we also excluded the possibility that there could be differences for these 22 snps in their association with stature variation between the db and yl genomic backgrounds , as no significance was found in an interaction analysis ( ge ) ( supplementary table s5 , supplementary material online ) . these 22 snps together explain most of stature variance among the 63 horses from the db and yl breeds ( r = 0.943 ) . for the 22 candidate snps , only two regions ( of less than 1 mb ) contained more than one adjacent snp the first genomic region is located on eca8 and contained two snps : eca8.18101000 and eca8.18120526 . eca8.18101000 shows both the most significant association and the highest di value ( di = 21.90 ) ( fig . 2a and c ) and the major genotype for this snp in the db was a , with a frequency of 80.65% , whereas this genotype had a frequency of only 18.85% , with no homozygous aa genotype individuals observed , in the two other breeds . the proportion of the variance for height explained by a single snp was the highest for this snp , with a r = 0.506 . eca8.18120526 has the fifth highest association significance and the second highest di value , with 70.97% of the genotypes being g in the db , but only 19.30% in the other two breeds , and only one gg homozygote was observed in the mg . both of these snps are located less than 100 kb upstream of the gene tbx3 , a t - box gene that plays a role in the development of the anterior / posterior axis of the tetrapod forelimb ( bamshad et al . tbx3 was not detected in previous studies for short stature ( makvandi - nejad et al . 2012 ; signer - hasler et al . 2012 ; petersen , mickelson , cothran , et al . 2013 ; tetens et al . 2013 ) , suggesting the possibility of independent selection for small stature in the db . the second region with clustered snps is on eca6 and contained three snps with high di values , high merged xp - ehh values , and significant association p values ( fig . although this region contains three genes , msrb3 , lemd3 and hmga2 , hmga2 is the likely candidate gene as it has been previously associated with height variation in human populations ( weedon et al . 2007 ; yang et al . 2010 ) and was studied previously , and found to be associated with stature in horse populations ( makvandi - nejad et al . 2013).hmga2 , like igf1 , has previously been suggested to have experienced convergent evolution in horses and humans ( petersen , mickelson , rendahl , et al . in addition , a previous association study in dogs , where these three genes are also clustered together , found the strongest association signal with body weight to be located near hmga2 , whereas the strongest association signal with ear floppiness was near msrb3 ( boyko et al . the genotypes of all five snps ( two near tbx3 and three near hmga2 ) significantly associate with variation in stature ( pairwise t - test ) ( supplementary fig . more interestingly , although tbx3 does not interact directly with hmga2 , they both directly interact with bmp3 and cdh4 , which regulate the bone synthesis . furthermore , tbx3 and hmga2 together explain 71.8% of the variance in height , with the explained proportion rising to 83.3% when an interaction between these two genes is added . these results therefore strongly suggest that a complex network underlies the evolution of small body size in the db . nearly 1% of all human genes have been implicated in contributing to size variation ( lango allen et al . in contrast , only a small number of genes appear to dominate size variation in domestic animals , such as dogs ( boyko et al . qualitative evidence points to the genetic control of size by many loci with small effect in naturally evolving species , but by few loci with large effect in domesticated species . still , as most genes perform conserved roles in regulating size across species , this should provide opportunities to explore the genetics of size variation in humans and other mammals . although previous studies have identified several genes that may be influential in the determination of horse size , imaging a common genetic mechanism that accounts for all horse populations is difficult as many populations are restricted to localized environment where they were bred for different goals . here we report that the gene tbx3 likely dominates the small stature of the db horse , an ancient small pony specific to china that was selected for transport in mountainous regions . tbx3 has not been associated with body size in other horse populations ( makvandi - nejad et al . moreover , only one gene , hmga2 , from all reported horse - size candidate genes ( makvandi - nejad et al . 2013 ) showed a selective signature in the db , although we can not address whether the selective signature found in this gene was shared by other pony breeds , as the three snps around hmga2 were not genotyped in any of the other pony breeds . therefore , it is likely that the db independently evolved small stature . to test this hypothesis , we also evaluated the di value of db and other ponies from a total of 35 breeds of horses . although the two most differentiated snps in db were removed from this analysis , due to a greater than 10% missing rate among all 35 breeds , the most differentiated snp in the db ( chr8:17883095 , di = 183.346 ) was still located approximately 300 kb upstream of tbx3 . in contrast to the db , none of the other pony breeds showed relatively high di values for this snp . in addition , we also conducted the f3 test ( reich et al . 2009 ) implemented in treemix ( pickrell and pritchard 2012 ) to investigate whether there were any potential migration events among the pony breeds . no evidence of migration between the db and the other pony breeds was found ( supplementary table s6 , supplementary material online ) . taking together , these results suggest the independent evolution of small stature size in db . ihh , which has been reported as a candidate contributing to stature size variation in human ( weedon et al . however , neither a signature of high diversification nor stature - associated significance was found , which may indicate that this gene has a modest effect in a subset of the db population . a caveat of our study is the possible shortcomings in the genotyped snp data , for example , snp ascertainment bias . although , a group of genes were identified , the relationship of the genotype and phenotype still needs additional functional evidence . whole - genome sequences from multiple individuals would be necessary for validating the potential selective targets , which should also lead to the identification of additional key mutations responsible for the body size seen in the db . the geneseek equine snp ( single nucleotide polymorphism ) 65 bead chip panel was used to genotype 32 individuals each from the db , yl , and mg breeds of horse . to assess the phylogenetic relationships of the chinese horses to other horse breeds , we obtained snp genotype data from 32 different breeds from a previous study ( petersen , mickelson , rendahl , et al . 2013 ) , which yielded a total of 21,740 snps that passed our quality filtration ( see materials and methods ) . after merging with the snp data from a przewalski s horse ( orlando et al . 2013 ) , a total of 14,959 snps were used to infer a maximum - likelihood tree of the horse populations using the genome - wide allele frequency from the przewalski s horse as the outgroup ( fig . as expected , all three chinese breeds genotyped here clustered with the mongolian horses ( fig . 1b ) , consistent with their close geographic distribution . the relationships of the ponies , scandinavian breeds , heavy draft horses , recent breeds derived from the thoroughbred , modern us breeds , and the trotting breeds ( fig . 1b ) were in agreement with that described in a previous study ( petersen , mickelson , cothran , et al . landrace breeds , which originated geographically , ranged freely and experience lesser degrees of management , located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . considering the fact that most modern breeds were recently created by infusing arabian , and/or thoroughbred , which is also inherited majorly from arabian , we simply grouped these breeds into an arab group and the remaining into a non - arab group ( materials and methods ) . ancient breeds located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . 1b ) . breeds with short stature were located at disperse locations in the phylogenetic trees , suggesting independent domestication of these short stature breeds . however , when the treemix program ( pickrell and pritchard 2012 ) was used to infer migration events , the tree topology frequently changed with changes in the defined number of migration events ( supplementary fig . s1 , supplementary material online ) , suggesting a complex history for horse domestication with genetic admixtures occurring among breeds . the data also suggest that the exact phylogenetic relationship might be difficult to reveal based only on allele frequency data , particularly here , since genetic admixture occurred and only one wild przewalski s horse was available . ( 2013 ) found that the mangalarga paulista was located basally in a phylogenetic tree constructed by the parsimony method , whereas in contrast , mccue et al . ( 2012 ) identified the norwegian fjord and icelandic as being basal in their phylogenetic tree , and here , we have the norwegian fjord , n swedish , and shetland horses appearing close to the przewalski s horse ( fig . 1b ) ; however , we also detected migrations from the przewalski s horse to these three breeds ( supplementary fig . s1 , supplementary material online).to better analyze the phylogenetic relationships , we next implemented a bayesian model - based approach to assess the individual assignment using the program structure ( pritchard et al . a gradient from non - arab group to arab group was found whether we assumed that there were two ancestral populations ( k = 2 ) . some european draft ( clydesdale , shire ) and pony breeds ( icelandic , miniature , shetland ) separated when four ancestral populations ( k = 4 ) were assumed . horses from mongolia , china and siberia grouped into a distinct cluster when k = 12 , suggesting a much closer relationship among these horses from the middle part of the eurasian continent . when k = 12 , przewalski s horse showed closer relationship and clustered with the chinese breeds . consistently , the neighbor - joining tree based on all individuals showed that the chinese breeds had the closest relationship to the przewalski s horse ( supplementary fig . s2 , supplementary material online ) , which contrasts with the phylogenetic tree inferred by whole population allele frequency data ( fig . these results also suggest that the phylogenetic relationships are difficult to reveal based only on allele frequency data . genetic migrations between the przewalski s horse and the chinese horse breeds were not detected by our treemix analysis , which potentially suggests that there is a true closer relationship between the przewalski s horse and the chinese breeds ( supplementary fig . s1 , supplementary material online ) . to exclude the possibility that the estimated population structures were influenced by the specific algorithmic strategy , we also used principal component analysis ( pca ) to examine the population relationships ( supplementary fig . similar to the above analysis , the first dimension moderately distinguished the arab group and non - arab group ( fig . s4 , supplementary material online ) , whereas the second dimension moderately separated horses with differing statures ( fig . horses from the middle east and asia region clustered together in the central part of the multidimensional scaling plot ( fig . consistent with the above , the przewalski s horse is close to the ancient breeds and non - arab breeds ( fig . s4b , supplementary material online ) , but in two dimensions we could not distinguish clearly the przewalski s horse from the domestic breeds . the individual closest to the przewalski s horse in the pca was the exmoor , but the treemix analysis found strong evidence of admixture between the exmoor and the przewalski s horse ( supplementary fig . consistent with the phylogenetic tree and the structural analysis , the chinese breeds were located within the ancient breeds in the pca analysis ( supplementary fig . s4b , supplementary material online ) , and closer than most breeds to the przewalski s horse ( supplementary fig . s7 , supplementary material online ) , which were comparable to the expected level of heterozygosity ( petersen , mickelson , cothran , et al . in addition , ld , which can be informative for population demography , found a similar decay in each of the three chinese breeds , with their low levels of ld similar to those seen within other landrace breeds ( fig . there were two exceptions : clydesdale and exmoor , which have small population sizes compared with other landrace breeds ( petersen , mickelson , cothran , et al . thus , the higher levels of genetic diversity , the closer relationship to the ancient breeds , and the lower levels of ld in the db than in other recent breeds suggest an ancient origin for the db , consistent with its antiquity record dating to at least 2,000 years ago ( hendricks 2007 ) . the three chinese breeds showed similar roh means , with the peaks of the roh distributions near the same lengths ( supplementary fig . s8a and b , supplementary material online ) , indicative of similar contemporary population sizes and mating systems . taken together , these results indicate that the three chinese breeds have an ancient origin , which is consistent with their anecdotal records of breed history ( wentong 1990 ; china national commission of animal genetic resources 2011 ) . perhaps surprisingly , ld across whole breeds did not rapidly decay , a pattern that is very different from that observed in dogs ( boyko et al . 1c ) , which may indicate that numerous ibd ( identity by descent ) segments are shared across multiple breeds and/or the shared segments are quite large . this pattern is consistent with horse breeding history as many modern breeds originated , at least in part , from the arabian horses , in a relatively short period of time . as the db individuals could not be assigned to a single cluster using the snps detected by all horses , we next assessed the populations of the three chinese breeds as a group using the 52,244 snps passed after quality filtration . the results from both structure and pca clearly distinguished the db from the other two populations ( supplementary fig . s8c and d , supplementary material online ) , which may indicate a differentiation between south china and north china horse populations . given that the db possesses a significantly low stature and their population characteristics imply an ancient history , we sought to identify candidate loci that regulate this phenotype and estimate the origin of this phenotype . population structure analysis implied a much closer relationship among the db , yl , and mg breeds ; therefore , we first evaluated the population differentiation of db from the other two chinese breeds to search for loci that show evidence of positive selection specific to the db , using the statistic di , as described in akey et al . we first extracted the top snps using a cutoff of 0.5% , and found 262 snps , which mapped to 153 genes ( within 150 kb of the snps ) . gene enrichment analysis found some categories associated with development of the skeletal system , such as abnormal morphology of forearm bone , abnormality of the ischium , dumbbell - shaped long bone , and aplasia / hypoplasia involving bones of the feet , but each had very few genes ( supplementary table s1 , supplementary material online ) . when a cutoff of 1% was used , a total of 523 snps ( di > 6.34 ) were identified ( fig . . gene enrichment analysis of the 338 genes closest to these snp found a significant overrepresentation in developmental process , for example , 87 genes involved in anatomical structure development ( go:0048856 ) , 17 genes involved in skeletal system development ( go:0001501 ) , 10 genes in cartilage development ( go:0051216 ) , 11 genes ( hand2 , lef1 , alx3 , col2a1 , zbtb16 , tbx3 , sulf1 , asph , en1 , pcsk5 , chst11 ) involved in limb development ( go:0060173 ) , and limb morphogenesis ( go:0035108 ) ( table 1 ) . we also extracted the top snps using cutoffs of 0.1% , and obtained similar results for enrichment of categories associated development of skeletal system ( supplementary tables s2 , supplementary material online ) . however , due to ascertainment bias and the small number of genotyped snps , it is difficult to identify the number of genes that have evolved under positive selection in the debao . genome - wide distribution of ( a ) di , ( b ) the p values of the merged xp - ehh values , ( c ) and manhattan plot presenting the association p value across the genome in the db . table 1overrepresented go categories among genes showing high di value in dbterm iddescriptionp valuegene numbergo:0009653anatomical structure morphogenesis2.36e-0557go:0009888tissue development4.33e-0542go:0007275multicellular organismal development5.62e-0582go:0030902hindbrain development5.80e-0511go:0009790embryo development6.85e-0535go:0048856anatomical structure development7.07e-0587go:0044767single - organism developmental process1.29e-0490go:0032502developmental process1.53e-0490go:0043009chordate embryonic development1.89e-0426go:0009792embryo development ending in birth or egg hatching2.23e-0426go:0002062chondrocyte differentiation2.51e-049go:0048731system development3.29e-0474go:0048513organ development6.02e-0460go:0072089stem cell proliferation1.12e-0310go:0035107appendage morphogenesis1.35e-0311go:0035108limb morphogenesis1.35e-0311go:0061448connective tissue development2.07e-0312go:0048736appendage development2.64e-0311go:0060173limb development2.64e-0311go:0001944vasculature development2.72e-0320go:0001501skeletal system development2.91e-0317go:0072359circulatory system development4.14e-0326go:0072358cardiovascular system development4.14e-0326go:0001701 in utero embryonic development4.93e-0317go:0007417central nervous system development5.05e-0322go:0061061muscle structure development5.08e-0318go:0006029proteoglycan metabolic process6.30e-036go:0048468cell development7.98e-0338go:0051216cartilage development9.05e-0310go:0007420brain development9.85e-0318go:0048568embryonic organ development1.03e-0217go:0072091regulation of stem cell proliferation1.04e-027go:0009887organ morphogenesis1.13e-0226go:2000648positive regulation of stem cell proliferation1.30e-026go:0048010vascular endothelial growth factor receptor signaling pathway1.44e-025go:0001568blood vessel development1.47e-0218go:0060021palate development1.49e-027go:0007605sensory perception of sound1.84e-028go:0010629negative regulation of gene expression1.85e-0227go:0051301cell division1.89e-0217go:1902679negative regulation of rna biosynthetic process2.08e-0225go:0035295tube development2.16e-0218go:0021549cerebellum development2.43e-026go:0048729tissue morphogenesis2.47e-0219go:0014706striated muscle tissue development2.52e-0213go:0048598embryonic morphogenesis3.21e-0219go:0051253negative regulation of rna metabolic process3.25e-0225go:0048869cellular developmental process3.26e-0258go:0007399nervous system development3.32e-0235go:0050954sensory perception of mechanical stimulus3.33e-028go:0030154cell differentiation3.38e-0254go:0007049cell cycle3.50e-0228go:0007588excretion3.60e-024go:2000113negative regulation of cellular macromolecule biosynthetic process3.94e-0226go:0002009morphogenesis of an epithelium4.04e-0216go:0001570vasculogenesis4.22e-026go:0060537muscle tissue development4.37e-0213go:0016486peptide hormone processing4.54e-023 positive selection analysis of the db . genome - wide distribution of ( a ) di , ( b ) the p values of the merged xp - ehh values , ( c ) and manhattan plot presenting the association p value across the genome in the db . overrepresented go categories among genes showing high di value in db beside high population differentiation , regions under directional selection also show other signatures of variation , such as long range haplotype homozygosity ( sabeti et al . 2006 ) . to identify alleles potentially under selection in the db , we employed xp - ehh , which is based on long - range haplotype homozygosity and is suitable for the detection of recent positive selection and is robust to the ascertainment bias of snps ( sabeti et al . a total of 505 snps have significantly high merged xp - ehh values in the db ( p < 0.01 ) ( fig . a total of 635 genes were found within 150 kp of these snps , and these genes were found to be significantly enriched in go terms for bone and muscle variation ( supplementary table s3 , supplementary material online ) , such as upper limb undergrowth , abnormal cortical bone morphology , aplasia / hypoplasia affecting bones of the axial skeleton , aplasia / hypoplasia involving bones of the feet , abnormality of the musculature of the limbs , limb - girdle muscle weakness , and foot dorsiflexor weakness . we also retrieved 122 genes with more significance of merged xp - ehh ( p < 0.005 ) , which showed similar enrichment in the development , bone and muscle variation , such as regulation of developmental process , skeletal muscle tissue development , spinal cord lesions , and abnormality of the mandible although many candidate selected genes are involved in the development of skeletal system , the selected alleles in db pony identified by the outlier methods can not be directly associated with small stature in db pony , as they might be confounded by other unique phenotypes found in the db . to verify whether the identified loci play a role in the small stature size of the db , we conducted association scans within the db and yl for variation in stature ( fig . 2c ) . after adjustment for fdr , 22 snps showed a significant association with stature ( bonf p value < 0.01 ) , all of which harbored significantly high di values ( top 1% ) and/or significant merged xp - ehh values ( p < 0.01 ) . to control for potential confounding effects of stratification and the nonindependence of related individuals that may bias the association study ( although the above population structure analysis showed no legacy of stratification for each of the chinese breeds and we tried to collect samples from different families or villages ) , we performed an adaptive permutation and confirmed the most significant 22 snps for stature variation ( supplementary table s5 , supplementary material online ) . furthermore , we also excluded the possibility that there could be differences for these 22 snps in their association with stature variation between the db and yl genomic backgrounds , as no significance was found in an interaction analysis ( ge ) ( supplementary table s5 , supplementary material online ) . these 22 snps together explain most of stature variance among the 63 horses from the db and yl breeds ( r = 0.943 ) . for the 22 candidate snps , only two regions ( of less than 1 mb ) contained more than one adjacent snp . the first genomic region is located on eca8 and contained two snps : eca8.18101000 and eca8.18120526 . eca8.18101000 shows both the most significant association and the highest di value ( di = 21.90 ) ( fig . 2a and c ) and the major genotype for this snp in the db was a , with a frequency of 80.65% , whereas this genotype had a frequency of only 18.85% , with no homozygous aa genotype individuals observed , in the two other breeds . the proportion of the variance for height explained by a single snp was the highest for this snp , with a r = 0.506 . eca8.18120526 has the fifth highest association significance and the second highest di value , with 70.97% of the genotypes being g in the db , but only 19.30% in the other two breeds , and only one gg homozygote was observed in the mg . both of these snps are located less than 100 kb upstream of the gene tbx3 , a t - box gene that plays a role in the development of the anterior / posterior axis of the tetrapod forelimb ( bamshad et al . tbx3 was not detected in previous studies for short stature ( makvandi - nejad et al . 2012 ; signer - hasler et al . 2012 ; petersen , mickelson , cothran , et al . 2013 ; tetens et al . 2013 ) , suggesting the possibility of independent selection for small stature in the db . the second region with clustered snps is on eca6 and contained three snps with high di values , high merged xp - ehh values , and significant association p values ( fig . although this region contains three genes , msrb3 , lemd3 and hmga2 , hmga2 is the likely candidate gene as it has been previously associated with height variation in human populations ( weedon et al . 2007 ; yang et al . 2010 ) and was studied previously , and found to be associated with stature in horse populations ( makvandi - nejad et al . 2013).hmga2 , like igf1 , has previously been suggested to have experienced convergent evolution in horses and humans ( petersen , mickelson , rendahl , et al . in addition , a previous association study in dogs , where these three genes are also clustered together , found the strongest association signal with body weight to be located near hmga2 , whereas the strongest association signal with ear floppiness was near msrb3 ( boyko et al . the genotypes of all five snps ( two near tbx3 and three near hmga2 ) significantly associate with variation in stature ( pairwise t - test ) ( supplementary fig . more interestingly , although tbx3 does not interact directly with hmga2 , they both directly interact with bmp3 and cdh4 , which regulate the bone synthesis . furthermore , tbx3 and hmga2 together explain 71.8% of the variance in height , with the explained proportion rising to 83.3% when an interaction between these two genes is added . these results therefore strongly suggest that a complex network underlies the evolution of small body size in the db . nearly 1% of all human genes have been implicated in contributing to size variation ( lango allen et al . in contrast , only a small number of genes appear to dominate size variation in domestic animals , such as dogs ( boyko et al . 2010 ; rimbault et al . qualitative evidence points to the genetic control of size by many loci with small effect in naturally evolving species , but by few loci with large effect in domesticated species . still , as most genes perform conserved roles in regulating size across species , this should provide opportunities to explore the genetics of size variation in humans and other mammals . although previous studies have identified several genes that may be influential in the determination of horse size , imaging a common genetic mechanism that accounts for all horse populations is difficult as many populations are restricted to localized environment where they were bred for different goals . here we report that the gene tbx3 likely dominates the small stature of the db horse , an ancient small pony specific to china that was selected for transport in mountainous regions . tbx3 has not been associated with body size in other horse populations ( makvandi - nejad et al . moreover , only one gene , hmga2 , from all reported horse - size candidate genes ( makvandi - nejad et al . 2012 ; petersen , mickelson , rendahl , et al . 2013 ) showed a selective signature in the db , although we can not address whether the selective signature found in this gene was shared by other pony breeds , as the three snps around hmga2 were not genotyped in any of the other pony breeds . therefore , it is likely that the db independently evolved small stature . to test this hypothesis , we also evaluated the di value of db and other ponies from a total of 35 breeds of horses . although the two most differentiated snps in db were removed from this analysis , due to a greater than 10% missing rate among all 35 breeds , the most differentiated snp in the db ( chr8:17883095 , di = 183.346 ) was still located approximately 300 kb upstream of tbx3 . in contrast to the db , none of the other pony breeds showed relatively high di values for this snp . 2009 ) implemented in treemix ( pickrell and pritchard 2012 ) to investigate whether there were any potential migration events among the pony breeds . no evidence of migration between the db and the other pony breeds was found ( supplementary table s6 , supplementary material online ) . taking together , these results suggest the independent evolution of small stature size in db . ihh , which has been reported as a candidate contributing to stature size variation in human ( weedon et al . however , neither a signature of high diversification nor stature - associated significance was found , which may indicate that this gene has a modest effect in a subset of the db population . a caveat of our study is the possible shortcomings in the genotyped snp data , for example , snp ascertainment bias . although , a group of genes were identified , the relationship of the genotype and phenotype still needs additional functional evidence . whole - genome sequences from multiple individuals would be necessary for validating the potential selective targets , which should also lead to the identification of additional key mutations responsible for the body size seen in the db . supplementary figures s1s9 and tables s1s6 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .

body size , one of the most important quantitative traits under evolutionary scrutiny , varies considerably among species and among populations within species . revealing the genetic basis underlying this variation is very important , particularly in humans where there is a close relationship with diseases and in domestic animals as the selective patterns are associated with improvements in production traits . the debao pony is a horse breed with small body size that is unique to china ; however , it is unknown whether the size - related candidate genes identified in western breeds also account for the small body size of the debao pony . here , we compared individual horses from the debao population with other two chinese horse populations using single nucleotide polymorphisms ( snps ) identified with the equine snp 65 bead chip . the previously reported size - related candidate gene hmga2 showed a significant signature for selection , consistent with its role observed in human populations . more interestingly , we found a candidate gene tbx3 , which had not been observed in previous studies on horse body size that displayed the highest differentiation and most significant association , and thus likely is the dominating factor for the small stature of the debao pony . further comparison between the debao pony and other breeds of horses from around the world demonstrated that tbx3 was selected independently in the debao pony , suggesting that there were multiple origins of small stature in the horse .

Introduction Materials and Methods Results and Discussion Phylogenetic Analysis of the DB Signatures of Positive Selection in the DB Supplementary Material

body size , one of the most important quantitative traits under evolutionary scrutiny , varies considerably at both the inter- and intraspecific levels ( blanckenhorn et al . understanding the genetic foundation of body size variation is particularly important , especially in humans for studies on the genetic mechanisms underlying associated diseases and in domesticated animals for understanding the patterns of artificial selection and for improving production traits . compared with populations that only experience natural selection , tremendous variation in body size is observed in domesticated species , such as dogs and chickens , which have experienced strong artificial selective pressures aimed at this trait for improvements or for exaggeration . for example , igf1 together with almost 700 other genes explains only approximately 16% of the height variation observed in humans ( lango allen et al . 2010 ) , but a single mutation within igf1 is the dominant explanation for small body size in several different breeds of dogs ( sutter et al . the official definition of a pony is a horse that measures less than 14.2 hands ( 58 inches , 147 cm ) at the withers ( mattern 2010 ) . given the restricted geographic distributions of these different breeds , it is possible that small stature was independently selected across these distinct horse breed lineages . , horse chromosomes 3 , 6 , 9 , and 11 ) that contribute to size variation in breeds developed in europe , america and the middle east , where frequent migration occurred during the breeding process , it remains unknown if these loci account for the size variation observed in all horse breeds ( makvandi - nejad et al . , we conducted a genomic scan to search for signatures of positive selection for diminutive body size in the debao pony ( db ) and selection for the relatively higher stature of the yili ( yl ) and inner mongolian ( mg ) horse breeds from northern china ( fig . our objectives were to 1 ) investigate the phylogenic relationship between the three chinese horses and previously studied horses to estimate the origin of the db , 2 ) compare the population genomic structure of the db with those of other horses to identify genetic mechanisms underlying the small size of the db , and 3 ) identify if introgression of size - related genes occurred among different horse breeds to determine whether small stature had a single or multiple origins . a total of 96 horse individuals from three horse breeds ( db [ n = 32 ] , yl [ n = 32 ] , and mg [ n = 32 ] ) were genotyped using the geneseek equine snp 65 bead chip panel . genotyped snps data from 32 other breeds of horses ( n = 729 ) from previous studies ( petersen , mickelson , cothran , et al . with a pruned data set , which discarded snps in linkage disequilibrium ( ld ) across breeds ( plink indep 50 5 2 ) , we implemented a bayesian model - based approach to assess the population relationship of the world - wide horses using the program structure ( pritchard et al . the geneseek equine snp ( single nucleotide polymorphism ) 65 bead chip panel was used to genotype 32 individuals each from the db , yl , and mg breeds of horse . 2013 ) , a total of 14,959 snps were used to infer a maximum - likelihood tree of the horse populations using the genome - wide allele frequency from the przewalski s horse as the outgroup ( fig . landrace breeds , which originated geographically , ranged freely and experience lesser degrees of management , located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . ancient breeds located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . ( 2012 ) identified the norwegian fjord and icelandic as being basal in their phylogenetic tree , and here , we have the norwegian fjord , n swedish , and shetland horses appearing close to the przewalski s horse ( fig . 1b ) ; however , we also detected migrations from the przewalski s horse to these three breeds ( supplementary fig . horses from mongolia , china and siberia grouped into a distinct cluster when k = 12 , suggesting a much closer relationship among these horses from the middle part of the eurasian continent . genetic migrations between the przewalski s horse and the chinese horse breeds were not detected by our treemix analysis , which potentially suggests that there is a true closer relationship between the przewalski s horse and the chinese breeds ( supplementary fig . horses from the middle east and asia region clustered together in the central part of the multidimensional scaling plot ( fig . thus , the higher levels of genetic diversity , the closer relationship to the ancient breeds , and the lower levels of ld in the db than in other recent breeds suggest an ancient origin for the db , consistent with its antiquity record dating to at least 2,000 years ago ( hendricks 2007 ) . perhaps surprisingly , ld across whole breeds did not rapidly decay , a pattern that is very different from that observed in dogs ( boyko et al . population structure analysis implied a much closer relationship among the db , yl , and mg breeds ; therefore , we first evaluated the population differentiation of db from the other two chinese breeds to search for loci that show evidence of positive selection specific to the db , using the statistic di , as described in akey et al . we first extracted the top snps using a cutoff of 0.5% , and found 262 snps , which mapped to 153 genes ( within 150 kb of the snps ) . gene enrichment analysis of the 338 genes closest to these snp found a significant overrepresentation in developmental process , for example , 87 genes involved in anatomical structure development ( go:0048856 ) , 17 genes involved in skeletal system development ( go:0001501 ) , 10 genes in cartilage development ( go:0051216 ) , 11 genes ( hand2 , lef1 , alx3 , col2a1 , zbtb16 , tbx3 , sulf1 , asph , en1 , pcsk5 , chst11 ) involved in limb development ( go:0060173 ) , and limb morphogenesis ( go:0035108 ) ( table 1 ) . however , due to ascertainment bias and the small number of genotyped snps , it is difficult to identify the number of genes that have evolved under positive selection in the debao . to identify alleles potentially under selection in the db , we employed xp - ehh , which is based on long - range haplotype homozygosity and is suitable for the detection of recent positive selection and a total of 505 snps have significantly high merged xp - ehh values in the db ( p < 0.01 ) ( fig . we also retrieved 122 genes with more significance of merged xp - ehh ( p < 0.005 ) , which showed similar enrichment in the development , bone and muscle variation , such as regulation of developmental process , skeletal muscle tissue development , spinal cord lesions , and abnormality of the mandible although many candidate selected genes are involved in the development of skeletal system , the selected alleles in db pony identified by the outlier methods can not be directly associated with small stature in db pony , as they might be confounded by other unique phenotypes found in the db . to verify whether the identified loci play a role in the small stature size of the db , we conducted association scans within the db and yl for variation in stature ( fig . after adjustment for fdr , 22 snps showed a significant association with stature ( bonf p value < 0.01 ) , all of which harbored significantly high di values ( top 1% ) and/or significant merged xp - ehh values ( p < 0.01 ) . to control for potential confounding effects of stratification and the nonindependence of related individuals that may bias the association study ( although the above population structure analysis showed no legacy of stratification for each of the chinese breeds and we tried to collect samples from different families or villages ) , we performed an adaptive permutation and confirmed the most significant 22 snps for stature variation ( supplementary table s5 , supplementary material online ) . furthermore , we also excluded the possibility that there could be differences for these 22 snps in their association with stature variation between the db and yl genomic backgrounds , as no significance was found in an interaction analysis ( ge ) ( supplementary table s5 , supplementary material online ) . eca8.18101000 shows both the most significant association and the highest di value ( di = 21.90 ) ( fig . eca8.18120526 has the fifth highest association significance and the second highest di value , with 70.97% of the genotypes being g in the db , but only 19.30% in the other two breeds , and only one gg homozygote was observed in the mg . both of these snps are located less than 100 kb upstream of the gene tbx3 , a t - box gene that plays a role in the development of the anterior / posterior axis of the tetrapod forelimb ( bamshad et al . tbx3 was not detected in previous studies for short stature ( makvandi - nejad et al . 2013 ) , suggesting the possibility of independent selection for small stature in the db . although this region contains three genes , msrb3 , lemd3 and hmga2 , hmga2 is the likely candidate gene as it has been previously associated with height variation in human populations ( weedon et al . 2010 ) and was studied previously , and found to be associated with stature in horse populations ( makvandi - nejad et al . these results therefore strongly suggest that a complex network underlies the evolution of small body size in the db . although previous studies have identified several genes that may be influential in the determination of horse size , imaging a common genetic mechanism that accounts for all horse populations is difficult as many populations are restricted to localized environment where they were bred for different goals . here we report that the gene tbx3 likely dominates the small stature of the db horse , an ancient small pony specific to china that was selected for transport in mountainous regions . tbx3 has not been associated with body size in other horse populations ( makvandi - nejad et al . 2013 ) showed a selective signature in the db , although we can not address whether the selective signature found in this gene was shared by other pony breeds , as the three snps around hmga2 were not genotyped in any of the other pony breeds . to test this hypothesis , we also evaluated the di value of db and other ponies from a total of 35 breeds of horses . whole - genome sequences from multiple individuals would be necessary for validating the potential selective targets , which should also lead to the identification of additional key mutations responsible for the body size seen in the db . the geneseek equine snp ( single nucleotide polymorphism ) 65 bead chip panel was used to genotype 32 individuals each from the db , yl , and mg breeds of horse . 2013 ) , a total of 14,959 snps were used to infer a maximum - likelihood tree of the horse populations using the genome - wide allele frequency from the przewalski s horse as the outgroup ( fig . landrace breeds , which originated geographically , ranged freely and experience lesser degrees of management , located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . ancient breeds located basally in the tree with close relationship with the wild horse , and many of the modern breeds clustered together ( fig . ( 2012 ) identified the norwegian fjord and icelandic as being basal in their phylogenetic tree , and here , we have the norwegian fjord , n swedish , and shetland horses appearing close to the przewalski s horse ( fig . 1b ) ; however , we also detected migrations from the przewalski s horse to these three breeds ( supplementary fig . horses from mongolia , china and siberia grouped into a distinct cluster when k = 12 , suggesting a much closer relationship among these horses from the middle part of the eurasian continent . genetic migrations between the przewalski s horse and the chinese horse breeds were not detected by our treemix analysis , which potentially suggests that there is a true closer relationship between the przewalski s horse and the chinese breeds ( supplementary fig . horses from the middle east and asia region clustered together in the central part of the multidimensional scaling plot ( fig . consistent with the phylogenetic tree and the structural analysis , the chinese breeds were located within the ancient breeds in the pca analysis ( supplementary fig . thus , the higher levels of genetic diversity , the closer relationship to the ancient breeds , and the lower levels of ld in the db than in other recent breeds suggest an ancient origin for the db , consistent with its antiquity record dating to at least 2,000 years ago ( hendricks 2007 ) . as the db individuals could not be assigned to a single cluster using the snps detected by all horses , we next assessed the populations of the three chinese breeds as a group using the 52,244 snps passed after quality filtration . population structure analysis implied a much closer relationship among the db , yl , and mg breeds ; therefore , we first evaluated the population differentiation of db from the other two chinese breeds to search for loci that show evidence of positive selection specific to the db , using the statistic di , as described in akey et al . we first extracted the top snps using a cutoff of 0.5% , and found 262 snps , which mapped to 153 genes ( within 150 kb of the snps ) . gene enrichment analysis found some categories associated with development of the skeletal system , such as abnormal morphology of forearm bone , abnormality of the ischium , dumbbell - shaped long bone , and aplasia / hypoplasia involving bones of the feet , but each had very few genes ( supplementary table s1 , supplementary material online ) . gene enrichment analysis of the 338 genes closest to these snp found a significant overrepresentation in developmental process , for example , 87 genes involved in anatomical structure development ( go:0048856 ) , 17 genes involved in skeletal system development ( go:0001501 ) , 10 genes in cartilage development ( go:0051216 ) , 11 genes ( hand2 , lef1 , alx3 , col2a1 , zbtb16 , tbx3 , sulf1 , asph , en1 , pcsk5 , chst11 ) involved in limb development ( go:0060173 ) , and limb morphogenesis ( go:0035108 ) ( table 1 ) . however , due to ascertainment bias and the small number of genotyped snps , it is difficult to identify the number of genes that have evolved under positive selection in the debao . to identify alleles potentially under selection in the db , we employed xp - ehh , which is based on long - range haplotype homozygosity and is suitable for the detection of recent positive selection and is robust to the ascertainment bias of snps ( sabeti et al . we also retrieved 122 genes with more significance of merged xp - ehh ( p < 0.005 ) , which showed similar enrichment in the development , bone and muscle variation , such as regulation of developmental process , skeletal muscle tissue development , spinal cord lesions , and abnormality of the mandible although many candidate selected genes are involved in the development of skeletal system , the selected alleles in db pony identified by the outlier methods can not be directly associated with small stature in db pony , as they might be confounded by other unique phenotypes found in the db . to verify whether the identified loci play a role in the small stature size of the db , we conducted association scans within the db and yl for variation in stature ( fig . to control for potential confounding effects of stratification and the nonindependence of related individuals that may bias the association study ( although the above population structure analysis showed no legacy of stratification for each of the chinese breeds and we tried to collect samples from different families or villages ) , we performed an adaptive permutation and confirmed the most significant 22 snps for stature variation ( supplementary table s5 , supplementary material online ) . furthermore , we also excluded the possibility that there could be differences for these 22 snps in their association with stature variation between the db and yl genomic backgrounds , as no significance was found in an interaction analysis ( ge ) ( supplementary table s5 , supplementary material online ) . eca8.18101000 shows both the most significant association and the highest di value ( di = 21.90 ) ( fig . eca8.18120526 has the fifth highest association significance and the second highest di value , with 70.97% of the genotypes being g in the db , but only 19.30% in the other two breeds , and only one gg homozygote was observed in the mg . both of these snps are located less than 100 kb upstream of the gene tbx3 , a t - box gene that plays a role in the development of the anterior / posterior axis of the tetrapod forelimb ( bamshad et al . tbx3 was not detected in previous studies for short stature ( makvandi - nejad et al . 2013 ) , suggesting the possibility of independent selection for small stature in the db . although this region contains three genes , msrb3 , lemd3 and hmga2 , hmga2 is the likely candidate gene as it has been previously associated with height variation in human populations ( weedon et al . 2010 ) and was studied previously , and found to be associated with stature in horse populations ( makvandi - nejad et al . these results therefore strongly suggest that a complex network underlies the evolution of small body size in the db . although previous studies have identified several genes that may be influential in the determination of horse size , imaging a common genetic mechanism that accounts for all horse populations is difficult as many populations are restricted to localized environment where they were bred for different goals . here we report that the gene tbx3 likely dominates the small stature of the db horse , an ancient small pony specific to china that was selected for transport in mountainous regions . tbx3 has not been associated with body size in other horse populations ( makvandi - nejad et al . 2013 ) showed a selective signature in the db , although we can not address whether the selective signature found in this gene was shared by other pony breeds , as the three snps around hmga2 were not genotyped in any of the other pony breeds . to test this hypothesis , we also evaluated the di value of db and other ponies from a total of 35 breeds of horses . ihh , which has been reported as a candidate contributing to stature size variation in human ( weedon et al . however , neither a signature of high diversification nor stature - associated significance was found , which may indicate that this gene has a modest effect in a subset of the db population . whole - genome sequences from multiple individuals would be necessary for validating the potential selective targets , which should also lead to the identification of additional key mutations responsible for the body size seen in the db .

the contribution of a. baumannii to hospital acquired infections has increased over the past three decades ( 1 , 2 ) . this organism causes of serious infectious diseases such as pneumonia , urinary tract infections , endocarditis , wound infections , meningitis , and septicemia ( 3 ) . it is becoming evident that biofilm forming ability can be considered one of the main virulence factor common to a large number of a. baumannii clinical isolates ( 4 ) . recent evidence has shown that biofilm formation at the solid - liquid interface is at least three times higher in a. baumannii than in the other acinetobacter species ( 8091% versus 524% ) , giving rise to a thick pellicle clearly visible on the top of broth cultures ( 5 ) . acylhomoserine lactones ( ahls ) are major class of auto - inducer signals used by gram negative bacteria to regulate diverse physiological functions such as virulence factors and biofilm ( 6 ) . quorum sensing ( qs ) was found to regulate multidrug resistance ( mdr ) in two ways , one involve up regulation of biofilm associated surface exopolysaccharides ( eps ) matrix and by up regulation of efflux pump genes ( 7 ) . the production of an eps matrix is one of the distinguishing characteristic of biofilm and it has been suggested that eps prevents the access of antibiotics into the bacterial community . there is a strong association between mdr and biofilms in this bacterium where in majority of clinical isolates , which were strong biofilm exhibiting mdr pattern . in a. baumannii , major gene , ahl found to be a major component for biofilm formation in a. baumannii that has been reported by various authors ( 9 ) . n-(3-hydroxydodecanoyl)-l - homoserine lactone ( 3-hydroxy - c12-hsl ) is the only qs molecule identified in this bacterium , though mass spectrometry suggested that other ahl molecules may also present at significantly lower amounts . however , contribution of quorum sensors to the overall pathogenic potential in the genus is currently unknown ( 10 , 11 ) . in one most recent study , different species of a. baumannii were analyzed for the production of ahl and it was shown that qs sensors were not homogenously distributed among species , though one particular ahl was specifically present in the most strains of a. calcoaceticus - a. baumannii complex ( 12 ) . in the present study we aimed to clone the luxi gene encoding n - acyl - homoserine synthase detected in clinical isolates of a. baumannii and study its expression in e. coli transformants . sixty five isolates of a. baumannii were cultured from patients hospitalized in intensive care units of three hospitals in kerman city , iran during february august 2013 . the individual colonies grown on blood and luria - bertani ( lb ) agar were allocated to the genus acinetobacter according to biochemical characteristics . strains were identified as a. baumannii according to the analytical profile index ( api ) 20ne protocol ( biomrieux , france ) and the presence of blaoxa-51 gene ( 13 ) . the susceptibility of isolates to three different classes of antibiotics ( -lactams , quinolones and aminoglycosides ) were determined according to the clinical and laboratory standards institute guidelines ( clsi ) procedures ( 2 ) . micro - broth dilution method was also used to determine the minimum inhibitory concentrations ( mics ) of antibiotics ( 5 ) . four a. baumannii hospital strains which demonstrated strong biofilm ( strains 16 , 23 , 40 and 55 ) were selected in this investigation . the bacterial cells were grown overnight in 5 ml luria - bertani ( lb ) broth ( under shaking condition ( 200 rpm ) . the presence of luxi gene in these strains was detected by conventional pcr using following primers luxi - f : 5-cttcatatgaatattattgctggatttc-3 luxi - r : 5-gctcgagcctatctaaatacacatcaatca - 3 based on the sequences of target gene . pcr was carried out in the biometra t3000 thermocycler ( biometra , gottingen , germany ) and amplification started with an initial denaturation step at 95 c for 2 min followed by 30 cycles of denaturation at 95 c , annealing at 58 c for 45 s and extension at 72 c for 1 min . subsequently , the ahl production was measured in the strains showed high biofilm formation by colorimetric ( liquid - liquid extraction ) method and compared with control strain a. baumannii atcc 19606 . the genomic dna was extracted using bacterial rtp extraction kit ( invitek - germany ) . pcr amplification was carried out using pfu - polymerase ( fermentas , vilnius , lithuania ) , and presence of luxi gene was detected using 1% agarose gel ( merck - germany ) electrophoresis . the recombinant was constructed by cloning a full - length 370bp pcr product luxi gene into ptg19 plasmid vector which has single 3-t overhangs on both ends and allows direct , high percentage of recombinant clones in low background . transformations of the recombinant construct to e. coli k12 dh5 mutants [ dlaczdelta m15 delta ( laczya - argf ) u169 reca1 enda1 hsdr17 ( rk - mk+ ) supe44 thi-1 gyra96rela1 ] was carried out with 50 mm cacl2 in ice powder as described previously ( 15 ) . after overnight growth , presence of luxi gene in recombinant dna was confirmed by pcr amplification technique . plasmid dna was further extracted from the e. coli transformants by feldan plasmid dna extraction miniprep kit ( bio basic , amherst ny usa ) . the luxi dna fragment was then extracted directly from gel using gel extraction kit and ligated into pet28 by t4 dna ligase according to the manufacturer s instructions ( fermentas , lithuania ) . the subcloned recombinants was then transformed into e. coli bl21 ( de3 ) strain [ fhua2 [ lon ] ompt gal ( de3 ) [ dcm ] hsds de3 = sbamhio ecori - b int : : ( laci : : placuv5::t7 gene1 ) i21 nin5 ] using 50 mm cacl2 in ice powder then subjected to a heat shock at 42 c for 50 s to enable dna uptake . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . to study the expression of the luxi gene , we performed qrt - pcr for total rna extraction from well isolated colonies . briefly , a loopful of well isolated colony of a. baumannii , e. coli bl21 transformants and e. coli bl21 as a control strain was inoculated into a 100ml erlenmeyer flask containing 20 ml sterile lb broth . the microbial cells ( 1 ml ) were centrifuged at 4 c for 10 min and the cell pellets were used for rna extractions using accuzol kit as described by the manufacturer ( bioneer , seoul , korea ) . synthesis of cdna was performed using accupowerr cycle script rt premix ( dn6 ) kit ( bioneer ) . about 1 g of rna template was added to a 1.5 ml microfuge tube ( eppendorf , germany ) then 20 l of depc water was added to each tube . to this preparation , 30 l of the lyophilized transparent pellet was dissolved by vortexing and briefly spinned down . the prepared solution was incubated at 45 c for 1 h and the tubes were placed at 95 c for 5 min for inactivation of reverse transcriptase activity . for amplification purpose , 25 l of accupowerr 29 green star master mix solution ( bioneer , seoul , korea ) , 1 l of each primers , 1 l of rox dye , and 5 l of cdna template were added . quantification of luxi gene was performed using the abi step one real - time pcr system ( applied biosystems , foster city , ca , usa ) with the following cycle profile : 1 cycle at 95 c for 3 min , followed by 40 cycles at 95 c for 20 s , annealing temperature 58 c for 20 s , and extension at 72 c for 20 s. in addition , we performed one cycle of melting curve . the gene expression was calculated as a fold change ( rq ) between target genes and matched reference 16s rrna levels as follows : rq = 2 where ct equal the difference between the ct values for the analyzed gene and ct for the 16s rrna reference gene amplification ( 16 ) . the amount of ahl in the spent culture supernatant was studied by colorimetric ( liquid - liquid extraction ) method as described previously ( 13 ) . to assess the ahl functional groups , we performed fourier transform infra - red ( ft - ir ) , as suggested before ( 17 , 18 ) . briefly , a drop of the ahl was placed on one of the kbr grid / plates . then , the second plate was placed on top and subjected to ir using brauker tensor 70 ft - ir spector - photometer . presence of various functional groups was detected in the range of 5004000 cm wave numbers . pearson or fisher s exact tests were performed to compare the number of isolates producing ahl . statistical analyses were performed using spss 17.0 ( spss , chicago , il , usa ) ; p 0.05 was considered as level of significance for the two - tailed tests . sixty five isolates of a. baumannii were cultured from patients hospitalized in intensive care units of three hospitals in kerman city , iran during february august 2013 . the individual colonies grown on blood and luria - bertani ( lb ) agar were allocated to the genus acinetobacter according to biochemical characteristics . strains were identified as a. baumannii according to the analytical profile index ( api ) 20ne protocol ( biomrieux , france ) and the presence of blaoxa-51 gene ( 13 ) . the susceptibility of isolates to three different classes of antibiotics ( -lactams , quinolones and aminoglycosides ) were determined according to the clinical and laboratory standards institute guidelines ( clsi ) procedures ( 2 ) . micro - broth dilution method was also used to determine the minimum inhibitory concentrations ( mics ) of antibiotics ( 5 ) . four a. baumannii hospital strains which demonstrated strong biofilm ( strains 16 , 23 , 40 and 55 ) were selected in this investigation . the bacterial cells were grown overnight in 5 ml luria - bertani ( lb ) broth ( under shaking condition ( 200 rpm ) . the presence of luxi gene in these strains was detected by conventional pcr using following primers luxi - f : 5-cttcatatgaatattattgctggatttc-3 luxi - r : 5-gctcgagcctatctaaatacacatcaatca - 3 based on the sequences of target gene . pcr was carried out in the biometra t3000 thermocycler ( biometra , gottingen , germany ) and amplification started with an initial denaturation step at 95 c for 2 min followed by 30 cycles of denaturation at 95 c , annealing at 58 c for 45 s and extension at 72 c for 1 min . subsequently , the ahl production was measured in the strains showed high biofilm formation by colorimetric ( liquid - liquid extraction ) method and compared with control strain a. baumannii atcc 19606 . the genomic dna was extracted using bacterial rtp extraction kit ( invitek - germany ) . pcr amplification was carried out using pfu - polymerase ( fermentas , vilnius , lithuania ) , and presence of luxi gene was detected using 1% agarose gel ( merck - germany ) electrophoresis . the recombinant was constructed by cloning a full - length 370bp pcr product luxi gene into ptg19 plasmid vector which has single 3-t overhangs on both ends and allows direct , high percentage of recombinant clones in low background . transformations of the recombinant construct to e. coli k12 dh5 mutants [ dlaczdelta m15 delta ( laczya - argf ) u169 reca1 enda1 hsdr17 ( rk - mk+ ) supe44 thi-1 gyra96rela1 ] was carried out with 50 mm cacl2 in ice powder as described previously ( 15 ) . after overnight growth , presence of luxi gene in recombinant dna was confirmed by pcr amplification technique . plasmid dna was further extracted from the e. coli transformants by feldan plasmid dna extraction miniprep kit ( bio basic , amherst ny usa ) . the luxi dna fragment was then extracted directly from gel using gel extraction kit and ligated into pet28 by t4 dna ligase according to the manufacturer s instructions ( fermentas , lithuania ) . the subcloned recombinants was then transformed into e. coli bl21 ( de3 ) strain [ fhua2 [ lon ] ompt gal ( de3 ) [ dcm ] hsds de3 = sbamhio ecori - b int : : ( laci : : placuv5::t7 gene1 ) i21 nin5 ] using 50 mm cacl2 in ice powder then subjected to a heat shock at 42 c for 50 s to enable dna uptake . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . to study the expression of the luxi gene , we performed qrt - pcr for total rna extraction from well isolated colonies . briefly , a loopful of well isolated colony of a. baumannii , e. coli bl21 transformants and e. coli bl21 as a control strain was inoculated into a 100ml erlenmeyer flask containing 20 ml sterile lb broth . the microbial cells ( 1 ml ) were centrifuged at 4 c for 10 min and the cell pellets were used for rna extractions using accuzol kit as described by the manufacturer ( bioneer , seoul , korea ) . synthesis of cdna was performed using accupowerr cycle script rt premix ( dn6 ) kit ( bioneer ) . about 1 g of rna template was added to a 1.5 ml microfuge tube ( eppendorf , germany ) then 20 l of depc water was added to each tube . to this preparation , 30 l of the lyophilized transparent pellet was dissolved by vortexing and briefly spinned down . the prepared solution was incubated at 45 c for 1 h and the tubes were placed at 95 c for 5 min for inactivation of reverse transcriptase activity . for amplification purpose , 25 l of accupowerr 29 green star master mix solution ( bioneer , seoul , korea ) , 1 l of each primers , 1 l of rox dye , and 5 l of cdna template were added . quantification of luxi gene was performed using the abi step one real - time pcr system ( applied biosystems , foster city , ca , usa ) with the following cycle profile : 1 cycle at 95 c for 3 min , followed by 40 cycles at 95 c for 20 s , annealing temperature 58 c for 20 s , and extension at 72 c for 20 s. in addition , we performed one cycle of melting curve . the gene expression was calculated as a fold change ( rq ) between target genes and matched reference 16s rrna levels as follows : rq = 2 where ct equal the difference between the ct values for the analyzed gene and ct for the 16s rrna reference gene amplification ( 16 ) . the amount of ahl in the spent culture supernatant was studied by colorimetric ( liquid - liquid extraction ) method as described previously ( 13 ) . to assess the ahl functional groups , we performed fourier transform infra - red ( ft - ir ) , as suggested before ( 17 , 18 ) . briefly , a drop of the ahl was placed on one of the kbr grid / plates . then , the second plate was placed on top and subjected to ir using brauker tensor 70 ft - ir spector - photometer . presence of various functional groups was detected in the range of 5004000 cm wave numbers . pearson or fisher s exact tests were performed to compare the number of isolates producing ahl . statistical analyses were performed using spss 17.0 ( spss , chicago , il , usa ) ; p 0.05 was considered as level of significance for the two - tailed tests . quantification of biofilm formation by microtiter plate method introduced 35.3% ( n = 23 ) of the isolates as strong biofilm producers . therefore , we amplified the luxi gene from four strong biofilm producing a. baumannii by pcr and the products were detected by agarose gel electrophoresis ( fig . 1 ) . at the same time presence of ahl was verified in these strains . for cloning purposes , the reference sequence ( genbank accession number nc_014259.1 ) of luxi gene used in this study obtained from ncbi database ( http://www.ncbi.nlm.nih.gov/blast ) . the amplified gene was cloned into ptg19 plasmid vector and recombinant dna was then transformed to e. coli dh5 luxi - strain . by this strategy , the linearized ptg19 vector with 3-dt overhangs prevent vector re - circularisation , therefore resulting in high percentage of recombinant clones in low background . following transformation of e. coli competent cells with the recombinant dna , the presence of luxi gene was detected by pcr . purified luxi dna fragment was directly isolated from agarose gel and subcloned into expression pet28a vector double digested with restriction enzymes ndei and xhoi . genetic map of plasmid pet28a used for cloning luxi gene and the sites of restriction enzymes ndei ( 238 ) and xhoi ( 158 ) are shown in the fig . the subcloned containing luxi gene ( pet28a + luxi recombinant ) was then transformed to e. coli bl21 ( luxi- ) . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . presence of luxi gene in bl21 transformants was confirmed by colony pcr and corresponding 554bp dna band was observed in the agarose gel electrophoresis . the results were compared with e. coli bl21 containing plasmid pet28a alone and a. baumannii wild type . agarose gel electrophoresis of luxi gene detected by pcr in a. baumannii producing high quantity of biofilm . cloning strategy for luxi gene detected in a. baumannii producing high biofilm used in this study . the expression of the luxi gene in e. coli bl21 cells and the transformants was compared by qpcr assay . we observed more than fourfold overexpression in luxi gene in the transformants cells containing pet28a + luxi as compare to control ( p 0.05 ) ( fig . qrt - pcr amplification plot of a ) luxi in a. baumannii strain 23 b ) and e. coli bl21 transformants containing luxi . the results were conducted in duplicate and similar observation was made . nc = negative control . to verify the presence of entire luxi gene in transforming cells , we measured the amount of ahl in the spent culture supernatant by colorimetric method . the a. baumannii isolate 23 showed highest amount of ahl activity while , isolate 22 exhibited ahl activity below threshold level ( od0.985 ) . similarly , our bl21 transformants showed similar od reading for ahl production as compared to a. baumannii isolate 23 ( fig . n - acyl homoserine lactone ( ahl ) activities of a. baumannii isolates collected in this study . furthermore , the association between ahl production and luxi gene was supported by ft - ir . analysis of ahl structure by ft - ir revealed the existence of a peak at 1764 cm due to carbonyl ( c = o ) of the lactone ring bonded to functional amide group ( n - h ) group at approximately 1659 cm in form of the stretching bond ( fig . the ir spectrum of the ahl appeared at the same wave number of the reported literature and confirmed the existence of ahl obtained in this study . the lactone ring and amide group were shown at 1764.69 cm and 1659.23 cm wave number . quantification of biofilm formation by microtiter plate method introduced 35.3% ( n = 23 ) of the isolates as strong biofilm producers . therefore , we amplified the luxi gene from four strong biofilm producing a. baumannii by pcr and the products were detected by agarose gel electrophoresis ( fig . 1 ) . at the same time presence of ahl was verified in these strains . for cloning purposes , the reference sequence ( genbank accession number nc_014259.1 ) of luxi gene used in this study obtained from ncbi database ( http://www.ncbi.nlm.nih.gov/blast ) . the amplified gene was cloned into ptg19 plasmid vector and recombinant dna was then transformed to e. coli dh5 luxi - strain . by this strategy , the linearized ptg19 vector with 3-dt overhangs prevent vector re - circularisation , therefore resulting in high percentage of recombinant clones in low background . following transformation of e. coli competent cells with the recombinant dna , the presence of luxi gene was detected by pcr . purified luxi dna fragment was directly isolated from agarose gel and subcloned into expression pet28a vector double digested with restriction enzymes ndei and xhoi . genetic map of plasmid pet28a used for cloning luxi gene and the sites of restriction enzymes ndei ( 238 ) and xhoi ( 158 ) are shown in the fig . the subcloned containing luxi gene ( pet28a + luxi recombinant ) was then transformed to e. coli bl21 ( luxi- ) . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . presence of luxi gene in bl21 transformants was confirmed by colony pcr and corresponding 554bp dna band was observed in the agarose gel electrophoresis . the results were compared with e. coli bl21 containing plasmid pet28a alone and a. baumannii wild type . agarose gel electrophoresis of luxi gene detected by pcr in a. baumannii producing high quantity of biofilm . cloning strategy for luxi gene detected in a. baumannii producing high biofilm used in this study . the expression of the luxi gene in e. coli bl21 cells and the transformants was compared by qpcr assay . we observed more than fourfold overexpression in luxi gene in the transformants cells containing pet28a + luxi as compare to control ( p 0.05 ) ( fig . qrt - pcr amplification plot of a ) luxi in a. baumannii strain 23 b ) and e. coli bl21 transformants containing luxi . the results were conducted in duplicate and similar observation was made . nc = negative control . to verify the presence of entire luxi gene in transforming cells , we measured the amount of ahl in the spent culture supernatant by colorimetric method . the a. baumannii isolate 23 showed highest amount of ahl activity while , isolate 22 exhibited ahl activity below threshold level ( od0.985 ) . similarly , our bl21 transformants showed similar od reading for ahl production as compared to a. baumannii isolate 23 ( fig . n - acyl homoserine lactone ( ahl ) activities of a. baumannii isolates collected in this study . furthermore , the association between ahl production and luxi gene was supported by ft - ir . analysis of ahl structure by ft - ir revealed the existence of a peak at 1764 cm due to carbonyl ( c = o ) of the lactone ring bonded to functional amide group ( n - h ) group at approximately 1659 cm in form of the stretching bond ( fig . the ir spectrum of the ahl appeared at the same wave number of the reported literature and confirmed the existence of ahl obtained in this study . the lactone ring and amide group were shown at 1764.69 cm and 1659.23 cm wave number . infections caused by a. baumannii are reported from many hospitals and unfortunately all of them associated with mdr strains ( 19 ) . recently , some hospitals reported infections with extensive drug resistance ( xdr ) and pan - drug strains of this bacterium with high rate of mortality ( 19 ) . the important feature that enables a. baumannii to cause this much damage is due to ability to form biofilm initiated by qs system ( 20 ) . a more recent study in a university hospital found that hospitalization in an icu and previous administration of antibiotics were associated with acinetobacter colonization at various body sites in 3.2 to 10.8 per 1.000 patients , with acinetobacter infection accounting for 0.3% of endemic nosocomial infections in critically ill patients and for 20% of nosocomial bacteremia hospital wide ( 21 ) . unlike p. aeruginosa that possesses two paired qs systems ( lasr / lasi and rhlr / rhli ) and one orphan qs regulator qscr , a. baumannii was reported to have one paired qs system : abar / abai ( 22 ) . the most recently , suggest iron play an important role in ahl production ( 13 ) . in this work , the gene for putative ahl synthase obtained from strong biofilm forming wild type strain of a. baumannii recovered from icu patients has been successfully cloned into ptg19 plasmid vector and the presence of the gene in e. coli dh5 luxi - transformants was detected by pcr method . the 5.4 kb pet28a vector was double digested with restriction enzymes in order to insert the entire gene to insertion site of the vector . this cloning strategy resulted high transcription yield and was confirmed by qrt - pcr analysis in the recombinant cells with total rna extracted . unlike the non - recombinant e. coli bl21 cells , the expression of luxi gene in pet28a + luxi cells was more than fourfold higher in transformants suggesting correct design of cloning strategy . the presence of ahl activity was further confirmed in the transformants by colorimetric method and od reading was similar to wild type strains of a. baumannii 23 . this indicate that luxi gene is up regulated by plasmid vector promoter and in fact produce entire ahl compound and no other dna element play role in synthesis process . to obtain convincing results of cloning intact luxi gene , we analyzed the structure of ahl in wild type as well as transformants by ft - ir spectroscopy . investigation by different researchers showed an abai mutant has a reduced biofilm - forming and motility phenotype ( 22 ) . similarly , ahl - dependent cross - talk between a. baumannii and p. aeruginosa can occur as the ahl of either species can induce the heterologous promoter in a mixed infection ( 23 ) . our research also underlines that the ft - ir provided direct evidence of cloning whole luxi gene . a key consideration in structure of ahl was a starching bond between amide functional group and lactone ring . the ft - ir confirmed the structure of ahl and suggested that luxi is only gene involved in ahl production not regulation . ahls produced by different bacteria differ in the length of the r - group side - chain ( 24 ) . niuet al . ( 25 ) reported cloning of abai gene in a. baumannii , and directed ahl production in recombinant e. coli . the abai protein was similar to members of the luxi family of autoinducer synthases and was predicted to be the only autoinducer synthase encoded by a. baumannii . it has been suggested that , the expression of abai at the transcriptional level was activated by ethyl acetate extracts of culture supernatants or by synthetic 3-hydroxy - c12-hsl while , an abai::km mutant failed to produce any detectable ahl signals and was impaired in biofilm development ( 26 ) . in other cloning strategy , the 552 bp aciigene from a. baumannii was amplified by pcr and cloned into pet28a overexpression vector , producing pet28a - acii , with a 6 his - tag driven by a t7 promoter . e. coli bl21 was transformed with this recombinant plasmid and the recombinant acii gene was overexpressed upon iptg induction ( 27 ) . on the other hand , a transcriptional analysis revealed that biofilm inducing molecules , an ahl and a -lactam antibiotic , strongly induced not only biofilm formation but also adenosine deaminase gene expression , suggesting that an elaborate gene regulation network for biofilm formation is present in the -lactam antibiotic resistant bacterium ( 28 ) . ( 28 ) reported the cloning and characterization of the luxi homologue from acinetobacter sp . strain gg2 , and confirmation of its ahls production . it is interesting to know that a. baumannii strains could recognize and respond to p. aeruginosa qs molecules , indicating a possible inter - species communication . most recent study on cloning of putative ahl synthase from enterobacter cancerogenus m004 ( designated asecni ) has been successfully cloned and characterized . when e. coli harboring the pet28a - ecni and induced by iptg for 8 h , its spent supernatant was profiled using lc - ms / ms confirmed the profiles of both 3-oxo - c6 hsl and 3-oxo - c8 hsl suggesting that ecni is indeed the ahlsynthase of e. cancerogenus m004 ( 29 ) . our data showed that cloning of luxi in pet28a gives very efficient ahl production in e. coli transformants similar to wild type a. baumannii strains and perhaps in other bacteria . we propose that , abai has a primary role in ahl signal in a. baumannii and composed of 3-hydroxy - c12-hsl . it was the first time to use two cloning vector for expression of luxi gene . the limitation of this work were high expense and tedious experiments to actually confirm the proper expression of luxi gene .

background and objectives : in present study we aimed to clone the luxi gene encoding n - acyl - homoserine synthase detected in clinical isolates of acinetobacter baumannii and study its expression in escherichia coli transformants.materials and methods : four a. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation . the presence of luxi gene was detected using pcr technique . purified pcr product dna was initially cloned into ptg19 and transformed to e. coli dh5. the gene was then recovered from agarose gel and ligated by t4 dna ligase into pet28a expression vector using ndei and xhoi enzymes . pet28a + luxi was transformed into e. coli bl21 ( de3 ) . the luxi putative gene was further detected in the transformants by colony pcr . expression of the luxi gene in the recombinant e. coli bl21 cells was studied by quantitative real time pcr ( qrt - pcr ) and the presence of n - acylhomoserine lactone ( ahl ) was checked by colorimetric assay and fourier transform infra - red ( ft - ir ) spectroscopy.results:we successfully cloned ahl gene from a. baumannii strain 23 to pet28a expression vector . there was four fold increases in expression of luxi in the transformants ( p 0.05 ) . it was found that , strain 23 and the transformants showed highest amount of ahl activity ( od = 1.524 ) . the ft - ir analysis indicated stretching c = o bond of the lactone ring and primary amides ( n = h ) at 1764.69 cm1 and 1659.23 cm1 respectively.conclusion:from above results we concluded that , luxi in a. baumannii is indeed responsible for ahl production and not regulation and pet28a vector allows efficient ahl expression in e. coli bl21 transformants .

INTRODUCTION MATERIALS AND METHODS Bacterial isolation and identification. Detection of Cloning of putative Measurement of AHL expression by quantitative real time PCR (qRT-PCR). N-acyl-homoserine lactone assay. Determination of AHL functional groups. Statistical Analyses. RESULTS Cloning strategy. Quantitative real time PCR analysis. None Analysis of AHL structure by FT-IR. DISCUSSION CONCLUSIONS

recent evidence has shown that biofilm formation at the solid - liquid interface is at least three times higher in a. baumannii than in the other acinetobacter species ( 8091% versus 524% ) , giving rise to a thick pellicle clearly visible on the top of broth cultures ( 5 ) . there is a strong association between mdr and biofilms in this bacterium where in majority of clinical isolates , which were strong biofilm exhibiting mdr pattern . in one most recent study , different species of a. baumannii were analyzed for the production of ahl and it was shown that qs sensors were not homogenously distributed among species , though one particular ahl was specifically present in the most strains of a. calcoaceticus - a. baumannii complex ( 12 ) . in the present study we aimed to clone the luxi gene encoding n - acyl - homoserine synthase detected in clinical isolates of a. baumannii and study its expression in e. coli transformants . strains were identified as a. baumannii according to the analytical profile index ( api ) 20ne protocol ( biomrieux , france ) and the presence of blaoxa-51 gene ( 13 ) . four a. baumannii hospital strains which demonstrated strong biofilm ( strains 16 , 23 , 40 and 55 ) were selected in this investigation . the presence of luxi gene in these strains was detected by conventional pcr using following primers luxi - f : 5-cttcatatgaatattattgctggatttc-3 luxi - r : 5-gctcgagcctatctaaatacacatcaatca - 3 based on the sequences of target gene . subsequently , the ahl production was measured in the strains showed high biofilm formation by colorimetric ( liquid - liquid extraction ) method and compared with control strain a. baumannii atcc 19606 . pcr amplification was carried out using pfu - polymerase ( fermentas , vilnius , lithuania ) , and presence of luxi gene was detected using 1% agarose gel ( merck - germany ) electrophoresis . the recombinant was constructed by cloning a full - length 370bp pcr product luxi gene into ptg19 plasmid vector which has single 3-t overhangs on both ends and allows direct , high percentage of recombinant clones in low background . transformations of the recombinant construct to e. coli k12 dh5 mutants [ dlaczdelta m15 delta ( laczya - argf ) u169 reca1 enda1 hsdr17 ( rk - mk+ ) supe44 thi-1 gyra96rela1 ] was carried out with 50 mm cacl2 in ice powder as described previously ( 15 ) . after overnight growth , presence of luxi gene in recombinant dna was confirmed by pcr amplification technique . plasmid dna was further extracted from the e. coli transformants by feldan plasmid dna extraction miniprep kit ( bio basic , amherst ny usa ) . the luxi dna fragment was then extracted directly from gel using gel extraction kit and ligated into pet28 by t4 dna ligase according to the manufacturer s instructions ( fermentas , lithuania ) . the subcloned recombinants was then transformed into e. coli bl21 ( de3 ) strain [ fhua2 [ lon ] ompt gal ( de3 ) [ dcm ] hsds de3 = sbamhio ecori - b int : : ( laci : : placuv5::t7 gene1 ) i21 nin5 ] using 50 mm cacl2 in ice powder then subjected to a heat shock at 42 c for 50 s to enable dna uptake . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . to study the expression of the luxi gene , we performed qrt - pcr for total rna extraction from well isolated colonies . briefly , a loopful of well isolated colony of a. baumannii , e. coli bl21 transformants and e. coli bl21 as a control strain was inoculated into a 100ml erlenmeyer flask containing 20 ml sterile lb broth . quantification of luxi gene was performed using the abi step one real - time pcr system ( applied biosystems , foster city , ca , usa ) with the following cycle profile : 1 cycle at 95 c for 3 min , followed by 40 cycles at 95 c for 20 s , annealing temperature 58 c for 20 s , and extension at 72 c for 20 s. in addition , we performed one cycle of melting curve . the amount of ahl in the spent culture supernatant was studied by colorimetric ( liquid - liquid extraction ) method as described previously ( 13 ) . to assess the ahl functional groups , we performed fourier transform infra - red ( ft - ir ) , as suggested before ( 17 , 18 ) . presence of various functional groups was detected in the range of 5004000 cm wave numbers . strains were identified as a. baumannii according to the analytical profile index ( api ) 20ne protocol ( biomrieux , france ) and the presence of blaoxa-51 gene ( 13 ) . four a. baumannii hospital strains which demonstrated strong biofilm ( strains 16 , 23 , 40 and 55 ) were selected in this investigation . the presence of luxi gene in these strains was detected by conventional pcr using following primers luxi - f : 5-cttcatatgaatattattgctggatttc-3 luxi - r : 5-gctcgagcctatctaaatacacatcaatca - 3 based on the sequences of target gene . subsequently , the ahl production was measured in the strains showed high biofilm formation by colorimetric ( liquid - liquid extraction ) method and compared with control strain a. baumannii atcc 19606 . pcr amplification was carried out using pfu - polymerase ( fermentas , vilnius , lithuania ) , and presence of luxi gene was detected using 1% agarose gel ( merck - germany ) electrophoresis . the recombinant was constructed by cloning a full - length 370bp pcr product luxi gene into ptg19 plasmid vector which has single 3-t overhangs on both ends and allows direct , high percentage of recombinant clones in low background . transformations of the recombinant construct to e. coli k12 dh5 mutants [ dlaczdelta m15 delta ( laczya - argf ) u169 reca1 enda1 hsdr17 ( rk - mk+ ) supe44 thi-1 gyra96rela1 ] was carried out with 50 mm cacl2 in ice powder as described previously ( 15 ) . after overnight growth , presence of luxi gene in recombinant dna was confirmed by pcr amplification technique . plasmid dna was further extracted from the e. coli transformants by feldan plasmid dna extraction miniprep kit ( bio basic , amherst ny usa ) . the luxi dna fragment was then extracted directly from gel using gel extraction kit and ligated into pet28 by t4 dna ligase according to the manufacturer s instructions ( fermentas , lithuania ) . the subcloned recombinants was then transformed into e. coli bl21 ( de3 ) strain [ fhua2 [ lon ] ompt gal ( de3 ) [ dcm ] hsds de3 = sbamhio ecori - b int : : ( laci : : placuv5::t7 gene1 ) i21 nin5 ] using 50 mm cacl2 in ice powder then subjected to a heat shock at 42 c for 50 s to enable dna uptake . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . to study the expression of the luxi gene , we performed qrt - pcr for total rna extraction from well isolated colonies . briefly , a loopful of well isolated colony of a. baumannii , e. coli bl21 transformants and e. coli bl21 as a control strain was inoculated into a 100ml erlenmeyer flask containing 20 ml sterile lb broth . quantification of luxi gene was performed using the abi step one real - time pcr system ( applied biosystems , foster city , ca , usa ) with the following cycle profile : 1 cycle at 95 c for 3 min , followed by 40 cycles at 95 c for 20 s , annealing temperature 58 c for 20 s , and extension at 72 c for 20 s. in addition , we performed one cycle of melting curve . the amount of ahl in the spent culture supernatant was studied by colorimetric ( liquid - liquid extraction ) method as described previously ( 13 ) . to assess the ahl functional groups , we performed fourier transform infra - red ( ft - ir ) , as suggested before ( 17 , 18 ) . presence of various functional groups was detected in the range of 5004000 cm wave numbers . quantification of biofilm formation by microtiter plate method introduced 35.3% ( n = 23 ) of the isolates as strong biofilm producers . therefore , we amplified the luxi gene from four strong biofilm producing a. baumannii by pcr and the products were detected by agarose gel electrophoresis ( fig . for cloning purposes , the reference sequence ( genbank accession number nc_014259.1 ) of luxi gene used in this study obtained from ncbi database ( http://www.ncbi.nlm.nih.gov/blast ) . the amplified gene was cloned into ptg19 plasmid vector and recombinant dna was then transformed to e. coli dh5 luxi - strain . following transformation of e. coli competent cells with the recombinant dna , the presence of luxi gene was detected by pcr . purified luxi dna fragment was directly isolated from agarose gel and subcloned into expression pet28a vector double digested with restriction enzymes ndei and xhoi . genetic map of plasmid pet28a used for cloning luxi gene and the sites of restriction enzymes ndei ( 238 ) and xhoi ( 158 ) are shown in the fig . the subcloned containing luxi gene ( pet28a + luxi recombinant ) was then transformed to e. coli bl21 ( luxi- ) . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . presence of luxi gene in bl21 transformants was confirmed by colony pcr and corresponding 554bp dna band was observed in the agarose gel electrophoresis . the results were compared with e. coli bl21 containing plasmid pet28a alone and a. baumannii wild type . agarose gel electrophoresis of luxi gene detected by pcr in a. baumannii producing high quantity of biofilm . cloning strategy for luxi gene detected in a. baumannii producing high biofilm used in this study . the expression of the luxi gene in e. coli bl21 cells and the transformants was compared by qpcr assay . we observed more than fourfold overexpression in luxi gene in the transformants cells containing pet28a + luxi as compare to control ( p 0.05 ) ( fig . qrt - pcr amplification plot of a ) luxi in a. baumannii strain 23 b ) and e. coli bl21 transformants containing luxi . to verify the presence of entire luxi gene in transforming cells , we measured the amount of ahl in the spent culture supernatant by colorimetric method . the a. baumannii isolate 23 showed highest amount of ahl activity while , isolate 22 exhibited ahl activity below threshold level ( od0.985 ) . similarly , our bl21 transformants showed similar od reading for ahl production as compared to a. baumannii isolate 23 ( fig . n - acyl homoserine lactone ( ahl ) activities of a. baumannii isolates collected in this study . furthermore , the association between ahl production and luxi gene was supported by ft - ir . analysis of ahl structure by ft - ir revealed the existence of a peak at 1764 cm due to carbonyl ( c = o ) of the lactone ring bonded to functional amide group ( n - h ) group at approximately 1659 cm in form of the stretching bond ( fig . the lactone ring and amide group were shown at 1764.69 cm and 1659.23 cm wave number . quantification of biofilm formation by microtiter plate method introduced 35.3% ( n = 23 ) of the isolates as strong biofilm producers . therefore , we amplified the luxi gene from four strong biofilm producing a. baumannii by pcr and the products were detected by agarose gel electrophoresis ( fig . the amplified gene was cloned into ptg19 plasmid vector and recombinant dna was then transformed to e. coli dh5 luxi - strain . following transformation of e. coli competent cells with the recombinant dna , the presence of luxi gene was detected by pcr . purified luxi dna fragment was directly isolated from agarose gel and subcloned into expression pet28a vector double digested with restriction enzymes ndei and xhoi . genetic map of plasmid pet28a used for cloning luxi gene and the sites of restriction enzymes ndei ( 238 ) and xhoi ( 158 ) are shown in the fig . the subcloned containing luxi gene ( pet28a + luxi recombinant ) was then transformed to e. coli bl21 ( luxi- ) . this allowed the creation of a translational fusion adding six histidine residues to the carboxyl terminus of the protein and placed expression of the luxi gene under the control of a t7 promoter . presence of luxi gene in bl21 transformants was confirmed by colony pcr and corresponding 554bp dna band was observed in the agarose gel electrophoresis . agarose gel electrophoresis of luxi gene detected by pcr in a. baumannii producing high quantity of biofilm . cloning strategy for luxi gene detected in a. baumannii producing high biofilm used in this study . the expression of the luxi gene in e. coli bl21 cells and the transformants was compared by qpcr assay . we observed more than fourfold overexpression in luxi gene in the transformants cells containing pet28a + luxi as compare to control ( p 0.05 ) ( fig . qrt - pcr amplification plot of a ) luxi in a. baumannii strain 23 b ) and e. coli bl21 transformants containing luxi . to verify the presence of entire luxi gene in transforming cells , we measured the amount of ahl in the spent culture supernatant by colorimetric method . the a. baumannii isolate 23 showed highest amount of ahl activity while , isolate 22 exhibited ahl activity below threshold level ( od0.985 ) . similarly , our bl21 transformants showed similar od reading for ahl production as compared to a. baumannii isolate 23 ( fig . n - acyl homoserine lactone ( ahl ) activities of a. baumannii isolates collected in this study . furthermore , the association between ahl production and luxi gene was supported by ft - ir . analysis of ahl structure by ft - ir revealed the existence of a peak at 1764 cm due to carbonyl ( c = o ) of the lactone ring bonded to functional amide group ( n - h ) group at approximately 1659 cm in form of the stretching bond ( fig . the ir spectrum of the ahl appeared at the same wave number of the reported literature and confirmed the existence of ahl obtained in this study . the lactone ring and amide group were shown at 1764.69 cm and 1659.23 cm wave number . in this work , the gene for putative ahl synthase obtained from strong biofilm forming wild type strain of a. baumannii recovered from icu patients has been successfully cloned into ptg19 plasmid vector and the presence of the gene in e. coli dh5 luxi - transformants was detected by pcr method . this cloning strategy resulted high transcription yield and was confirmed by qrt - pcr analysis in the recombinant cells with total rna extracted . unlike the non - recombinant e. coli bl21 cells , the expression of luxi gene in pet28a + luxi cells was more than fourfold higher in transformants suggesting correct design of cloning strategy . the presence of ahl activity was further confirmed in the transformants by colorimetric method and od reading was similar to wild type strains of a. baumannii 23 . to obtain convincing results of cloning intact luxi gene , we analyzed the structure of ahl in wild type as well as transformants by ft - ir spectroscopy . similarly , ahl - dependent cross - talk between a. baumannii and p. aeruginosa can occur as the ahl of either species can induce the heterologous promoter in a mixed infection ( 23 ) . our research also underlines that the ft - ir provided direct evidence of cloning whole luxi gene . the ft - ir confirmed the structure of ahl and suggested that luxi is only gene involved in ahl production not regulation . ( 25 ) reported cloning of abai gene in a. baumannii , and directed ahl production in recombinant e. coli . the abai protein was similar to members of the luxi family of autoinducer synthases and was predicted to be the only autoinducer synthase encoded by a. baumannii . in other cloning strategy , the 552 bp aciigene from a. baumannii was amplified by pcr and cloned into pet28a overexpression vector , producing pet28a - acii , with a 6 his - tag driven by a t7 promoter . e. coli bl21 was transformed with this recombinant plasmid and the recombinant acii gene was overexpressed upon iptg induction ( 27 ) . our data showed that cloning of luxi in pet28a gives very efficient ahl production in e. coli transformants similar to wild type a. baumannii strains and perhaps in other bacteria . we propose that , abai has a primary role in ahl signal in a. baumannii and composed of 3-hydroxy - c12-hsl . it was the first time to use two cloning vector for expression of luxi gene .

the early symptoms of pancreatic cancer , such as abdominal pain , weight loss , fatigue , jaundice , and nausea , are nonspecific and may occur late in the course of the disease . as a result , pancreatic cancer is usually diagnosed at an advanced stage , frequently after the tumor has already metastasized . pancreatic cancer is insensitive to pharmacological and radiological intervention and often recurs after apparently curative surgery . this observation has led to the following two hypotheses : i. pancreatic cancer causes diabetes and ii . numerous studies have been performed in order to elucidate the relationship between these two diseases . the majority of diabetes associated with pancreatic cancer is diagnosed either concomitantly with the cancer or during the two years before the cancer is found ; 71% of the glucose intolerance found in pancreatic cancer patients is unknown before the cancer is diagnosed . these suggest that recently - developed glucose intolerance or diabetes may be a consequence of pancreatic cancer and that recent onset of glucose intolerance or diabetes may be an early sign of pancreatic cancer . several studies have demonstrated that diabetes in pancreatic cancer patients is characterized by peripheral insulin resistance . insulin resistance is also found in non - diabetic or glucose intolerant pancreatic cancer patients , though to a lesser degree . insulin sensitivity and overall diabetic state in pancreatic cancer patients who undergo tumor resection are markedly improved three months after the surgery . these data suggest that pancreatic tumors are causally related to the insulin resistance and diabetes seen in pancreatic cancer patients . in their study of sera from patients with pancreatic cancer and culture media conditioned by human pancreatic cancer cells , basso et al . found a 2030 mw peptide that they considered to be a putative pancreatic cancer associated diabetogenic factor . a number of investigators have studied insulin resistance at the organ , tissue , and cellular levels in pancreatic cancer [ 7 - 13 ] . studies of the initial steps in the insulin signaling cascade in human skeletal muscles showed no significant differences in insulin receptor binding , tyrosine kinase activity , and insulin receptor substrate-1 content between pancreatic cancer patients and healthy controls . however , phosphatidylinositol 3-kinase ( pi3-k ) activity and glucose transport , which are located downstream to the initial insulin signaling steps , were impaired in pancreatic cancer patients . in addition , glycogen synthase activity was reduced in skeletal muscles of humans and rodents with pancreatic carcinoma and in isolated rat skeletal muscles exposed to human pancreatic tumor extracts in vitro . these data show that the insulin signaling cascade in skeletal muscle is impaired at multiple steps by pancreatic cancer . an italian group has performed a series of studies to investigate the effects of pancreatic cancer cells on hepatic insulin sensitivity . when mice were treated with culture medium conditioned by the human pancreatic cancer cell line mia paca2 , blood glucose was elevated compared to the control value seen in mice treated with unconditioned medium . in addition , isolated rat hepatocytes showed impaired glycolysis when incubated in culture media conditioned by four human pancreatic cancer cell lines . because the islet mass destroyed by the tumor is only a small proportion of the whole islet mass , the islet dysfunction is unlikely to be the result of decreased total islet volume . in fact , endocrine pancreatic function can be maintained even with a larger loss of pancreatic islets . insulin release was also reduced when isolated rat pancreatic islets were incubated in culture media conditioned by the human pancreatic cancer cell lines panc-1 and hpaf or co - cultured with panc-1 and hpaf cells . studies of chemically - induced pancreatic cancer in hamsters found that glucose - stimulated insulin release was impaired in vivo but not in isolated perfused pancreata . ishikawa et al . found an increase in proinsulin relative to insulin in pancreatic cancer patients , suggesting that the maturation of proinsulin may also be affected by the tumor . islet hormone profiles are changed in the circulation of pancreatic cancer patients , suggesting that secretion by different types of islet cells is disrupted by pancreatic cancer . changes in islet hormone concentrations in the circulation can also be seen in hamsters after induction of pancreatic cancer . human pancreatic islets adjacent to pancreatic carcinoma show morphological abnormalities characterized by abnormal co - localization of islet hormones in islet cells . the diabetogenic potential of islet amyloid polypeptide ( iapp or amylin ) has been investigated by several groups . iapp is normally produced in islet beta cells and co - released with insulin at a constant ratio . in 1994 , permert et al . found elevated circulating levels of iapp in patients with pancreatic cancer . in contrast , the expression of iapp mrna in these islets is unchanged , suggesting normal production but increased release of iapp . the molar ratio of iapp / insulin was increased when rat pancreatic islets were co - cultured with panc-1 and hpaf cells or cultured in media conditioned by these cell lines . the ratio was normalized after the co - cultured cancer cells were removed . in a similar co - culture model , ding et al . found that culture media conditioned by human pancreatic cancer cells contained a soluble molecule that selectively enhanced iapp release from brin - bd11 beta cells . increased iapp / insulin ratios were also seen in rats with azaserine - induced acinar pancreatic tumors and in hamsters with ductular pancreatic tumors induced by carcinogen n - nitrosobis(2-oxopropyl)amine ( bop ) . however , exposure of isolated rat pancreatic islets to hamster pancreatic cancer cells did not change the secretion of insulin and iapp . a physiological study of isolated rat pancreatic islets has shown that endogenous iapp reduces arginine - stimulated insulin , glucagon , and somatostatin release . also , the improvement in glucose tolerance seen after tumor removal is associated with normalization of iapp levels in the circulation . therefore , the increased iapp release seen in pancreatic cancer patients may be responsible , at least in part , for the islet dysfunction seen in these individuals . however , when iapp is infused in rats to create circulating concentrations comparable to the circulating iapp levels in pancreatic cancer patients , the rats have normal glucose disposal . thus , the increased iapp secretion found in pancreatic cancer patients is unlikely to be responsible for their peripheral insulin resistance . everhart et al . examined 30 of the epidemiological studies that have looked at the association between diabetes and pancreatic cancer and used 20 of them in a meta - analysis . the pooled relative risk from these studies was 2.1 for diabetes with a duration of at least l year prior to cancer diagnosis or death and 2.0 for diabetes with a duration of at least 5 years . the authors concluded that pancreatic cancer could be added to the list of complications of diabetes . several epidemiological studies have analyzed relative risks associated with the different periods of time after the diagnosis of diabetes and have found a relatively modest but persistent increased risk of death from pancreatic cancer even when the diagnosis of diabetes preceded death by many years [ 32 - 37 ] . a population - based case - control study in the united states with 526 incident cases and 2,153 population controls showed a significant positive trend ( p = 0.016 ) in risk with increasing years prior to diagnosis of cancer . in other studies , the relative risk decreased with increasing follow - up time but remained significant . however , other epidemiological studies have concluded that diabetes is not a risk factor for pancreatic cancer or else that it is not a risk factor if recently - diagnosed cases are excluded [ 6,38 - 40 ] . studies of the relationship between diabetes and pancreatic cancer are complicated by the fact that diabetes has two major forms that are different entities in terms of pathophysiology . a number of studies have suggested that type i diabetes is not associated with an increased risk for pancreatic cancer [ 37 - 39 ] . most epidemiological studies , however , have not distinguished between type i and type ii diabetes . it is likely that the large majority of diabetics in the studies have type ii diabetes because this form of the disease constitutes 8090% of the cases and is typically found in older individuals . in patients with type ii diabetes ( non - insulin - dependent diabetes ) , the pancreas is generally exposed to substantial hyperinsulinemia for years , suggesting that insulin may be involved in the association between long - standing diabetes and pancreatic cancer . a number of experiments have tested the hypothesis that insulin may stimulate the growth of pancreatic cancers . binding studies have shown the presence of insulin receptors on pancreatic cancer cells [ 42 - 45 ] . in vitro studies have shown that insulin promotes growth of the hamster pancreatic cancer cell line h2 t , the rat acinar pancreatic cancer cell line ar42j , and numerous human pancreatic cancer cells lines [ 44,46 - 51 ] . however , the human pancreatic cancer cell line soj-6 was not stimulated by insulin , and one of the studies using panc-1 cells reported no response to exogenous insulin . in addition to hyperinsulinemia , the increased blood glucose and free fatty acids in diabetes may also promote the growth of pancreatic cancer . the genesis of the cancer is also influenced by the endocrine pancreas . in vivo studies concerning the effects of administration of exogenous insulin and/or induction of diabetes on pancreatic cancer have provided inconsistent data that reflect the complex interactions that may be involved in tumor growth [ 53 - 56 ] . exogenous insulin significantly reduced the induction of benign and malignant pancreatic lesions in hamsters when given 2 hours before bop , but the reduction in incidence was not significant when insulin was given simultaneously with bop or 2 hours after bop . cancer incidence in hamsters receiving insulin twice daily starting before bop administration and continuing through the experimental period did not differ significantly from that in controls that received bop only . when hamsters were given streptozotocin ( sz ) injection to diminish insulin cells and given insulin from the following day untill the end of the experiment , the inhibition of carcinogenesis in hamsters receiving sz+bop+insulin treatment was greater than that seen in the sz+bop group , compared to group treated by bop only . because insulin administration was associated with inhibition of beta cell regeneration and persistence of severe diabetes in hamsters treated with sz , the investigators in the sz / bop / insulin study concluded that intact islet cells , rather than the availability of insulin , are prerequisite for triggering the neoplastic effects of bop . the association of intact islets with pancreatic cancer induction is also shown in transplantation studies in which tumors develop in the submandibular gland after bop treatment if normal islets are transplanted to that site but not when pancreatic ductal cells , thyroid , heart muscle , or starch are introduced into the gland [ 58 - 60 ] . submandibular gland tumor incidence was not changed when hamsters were pre - treated with sz before islet transplantation . a study of pancreatic cancer in hamsters fed a high - fat diet that potentiated pancreatic cancer provided data suggesting that islet proliferation associated with insulin resistance enhances carcinogenesis . in that study , high - fat - fed hamsters had elevated insulin levels but normal glucose levels , which was consistent with a state of insulin resistance . the turn - over rate of cells in islets is significantly increased in the high - fat animals , suggesting a compensatory islet cell proliferation . administration of metformin , starting 2 weeks before the administration of bop and continuing throughout the experiment , normalized insulin concentrations and the rate of islet cell turnover . malignant pancreatic lesions were found in 50% of the high - fat / bop animals and none in the high - fat / bop / metformin group ( p < 0.05 ) . the majority of diabetes associated with pancreatic cancer is diagnosed either concomitantly with the cancer or during the two years before the cancer is found ; 71% of the glucose intolerance found in pancreatic cancer patients is unknown before the cancer is diagnosed . these suggest that recently - developed glucose intolerance or diabetes may be a consequence of pancreatic cancer and that recent onset of glucose intolerance or diabetes may be an early sign of pancreatic cancer . several studies have demonstrated that diabetes in pancreatic cancer patients is characterized by peripheral insulin resistance . insulin resistance is also found in non - diabetic or glucose intolerant pancreatic cancer patients , though to a lesser degree . insulin sensitivity and overall diabetic state in pancreatic cancer patients who undergo tumor resection are markedly improved three months after the surgery . these data suggest that pancreatic tumors are causally related to the insulin resistance and diabetes seen in pancreatic cancer patients . in their study of sera from patients with pancreatic cancer and culture media conditioned by human pancreatic cancer cells , basso et al . found a 2030 mw peptide that they considered to be a putative pancreatic cancer associated diabetogenic factor . a number of investigators have studied insulin resistance at the organ , tissue , and cellular levels in pancreatic cancer [ 7 - 13 ] . studies of the initial steps in the insulin signaling cascade in human skeletal muscles showed no significant differences in insulin receptor binding , tyrosine kinase activity , and insulin receptor substrate-1 content between pancreatic cancer patients and healthy controls . however , phosphatidylinositol 3-kinase ( pi3-k ) activity and glucose transport , which are located downstream to the initial insulin signaling steps , were impaired in pancreatic cancer patients . in addition , glycogen synthase activity was reduced in skeletal muscles of humans and rodents with pancreatic carcinoma and in isolated rat skeletal muscles exposed to human pancreatic tumor extracts in vitro . these data show that the insulin signaling cascade in skeletal muscle is impaired at multiple steps by pancreatic cancer . an italian group has performed a series of studies to investigate the effects of pancreatic cancer cells on hepatic insulin sensitivity . when mice were treated with culture medium conditioned by the human pancreatic cancer cell line mia paca2 , blood glucose was elevated compared to the control value seen in mice treated with unconditioned medium . in addition , isolated rat hepatocytes showed impaired glycolysis when incubated in culture media conditioned by four human pancreatic cancer cell lines . because the islet mass destroyed by the tumor is only a small proportion of the whole islet mass , the islet dysfunction is unlikely to be the result of decreased total islet volume . in fact , endocrine pancreatic function can be maintained even with a larger loss of pancreatic islets . insulin release was also reduced when isolated rat pancreatic islets were incubated in culture media conditioned by the human pancreatic cancer cell lines panc-1 and hpaf or co - cultured with panc-1 and hpaf cells . studies of chemically - induced pancreatic cancer in hamsters found that glucose - stimulated insulin release was impaired in vivo but not in isolated perfused pancreata . ishikawa et al . found an increase in proinsulin relative to insulin in pancreatic cancer patients , suggesting that the maturation of proinsulin may also be affected by the tumor . islet hormone profiles are changed in the circulation of pancreatic cancer patients , suggesting that secretion by different types of islet cells is disrupted by pancreatic cancer . changes in islet hormone concentrations in the circulation can also be seen in hamsters after induction of pancreatic cancer . human pancreatic islets adjacent to pancreatic carcinoma show morphological abnormalities characterized by abnormal co - localization of islet hormones in islet cells . the diabetogenic potential of islet amyloid polypeptide ( iapp or amylin ) has been investigated by several groups . iapp is normally produced in islet beta cells and co - released with insulin at a constant ratio . in 1994 , permert et al . the islets adjacent to human pancreatic carcinomas show reduced iapp staining . in contrast , the expression of iapp mrna in these islets is unchanged , suggesting normal production but increased release of iapp . the molar ratio of iapp / insulin was increased when rat pancreatic islets were co - cultured with panc-1 and hpaf cells or cultured in media conditioned by these cell lines . the ratio was normalized after the co - cultured cancer cells were removed . in a similar co - culture model , ding et al . found that culture media conditioned by human pancreatic cancer cells contained a soluble molecule that selectively enhanced iapp release from brin - bd11 beta cells . increased iapp / insulin ratios were also seen in rats with azaserine - induced acinar pancreatic tumors and in hamsters with ductular pancreatic tumors induced by carcinogen n - nitrosobis(2-oxopropyl)amine ( bop ) . however , exposure of isolated rat pancreatic islets to hamster pancreatic cancer cells did not change the secretion of insulin and iapp . a physiological study of isolated rat pancreatic islets has shown that endogenous iapp reduces arginine - stimulated insulin , glucagon , and somatostatin release . also , the improvement in glucose tolerance seen after tumor removal is associated with normalization of iapp levels in the circulation . therefore , the increased iapp release seen in pancreatic cancer patients may be responsible , at least in part , for the islet dysfunction seen in these individuals . however , when iapp is infused in rats to create circulating concentrations comparable to the circulating iapp levels in pancreatic cancer patients , the rats have normal glucose disposal . thus , the increased iapp secretion found in pancreatic cancer patients is unlikely to be responsible for their peripheral insulin resistance . everhart et al . examined 30 of the epidemiological studies that have looked at the association between diabetes and pancreatic cancer and used 20 of them in a meta - analysis . the pooled relative risk from these studies was 2.1 for diabetes with a duration of at least l year prior to cancer diagnosis or death and 2.0 for diabetes with a duration of at least 5 years . the authors concluded that pancreatic cancer could be added to the list of complications of diabetes . several epidemiological studies have analyzed relative risks associated with the different periods of time after the diagnosis of diabetes and have found a relatively modest but persistent increased risk of death from pancreatic cancer even when the diagnosis of diabetes preceded death by many years [ 32 - 37 ] . a population - based case - control study in the united states with 526 incident cases and 2,153 population controls showed a significant positive trend ( p = 0.016 ) in risk with increasing years prior to diagnosis of cancer . in other studies , the relative risk decreased with increasing follow - up time but remained significant . however , other epidemiological studies have concluded that diabetes is not a risk factor for pancreatic cancer or else that it is not a risk factor if recently - diagnosed cases are excluded [ 6,38 - 40 ] . studies of the relationship between diabetes and pancreatic cancer are complicated by the fact that diabetes has two major forms that are different entities in terms of pathophysiology . a number of studies have suggested that type i diabetes is not associated with an increased risk for pancreatic cancer [ 37 - 39 ] . most epidemiological studies , however , have not distinguished between type i and type ii diabetes . it is likely that the large majority of diabetics in the studies have type ii diabetes because this form of the disease constitutes 8090% of the cases and is typically found in older individuals . in patients with type ii diabetes ( non - insulin - dependent diabetes ) , the pancreas is generally exposed to substantial hyperinsulinemia for years , suggesting that insulin may be involved in the association between long - standing diabetes and pancreatic cancer . a number of experiments have tested the hypothesis that insulin may stimulate the growth of pancreatic cancers . binding studies have shown the presence of insulin receptors on pancreatic cancer cells [ 42 - 45 ] . in vitro studies have shown that insulin promotes growth of the hamster pancreatic cancer cell line h2 t , the rat acinar pancreatic cancer cell line ar42j , and numerous human pancreatic cancer cells lines [ 44,46 - 51 ] . however , the human pancreatic cancer cell line soj-6 was not stimulated by insulin , and one of the studies using panc-1 cells reported no response to exogenous insulin . in addition to hyperinsulinemia , the increased blood glucose and free fatty acids in diabetes may also promote the growth of pancreatic cancer . the genesis of the cancer is also influenced by the endocrine pancreas . in vivo studies concerning the effects of administration of exogenous insulin and/or induction of diabetes on pancreatic cancer have provided inconsistent data that reflect the complex interactions that may be involved in tumor growth [ 53 - 56 ] . exogenous insulin significantly reduced the induction of benign and malignant pancreatic lesions in hamsters when given 2 hours before bop , but the reduction in incidence was not significant when insulin was given simultaneously with bop or 2 hours after bop . cancer incidence in hamsters receiving insulin twice daily starting before bop administration and continuing through the experimental period did not differ significantly from that in controls that received bop only . when hamsters were given streptozotocin ( sz ) injection to diminish insulin cells and given insulin from the following day untill the end of the experiment , the inhibition of carcinogenesis in hamsters receiving sz+bop+insulin treatment was greater than that seen in the sz+bop group , compared to group treated by bop only . because insulin administration was associated with inhibition of beta cell regeneration and persistence of severe diabetes in hamsters treated with sz , the investigators in the sz / bop / insulin study concluded that intact islet cells , rather than the availability of insulin , are prerequisite for triggering the neoplastic effects of bop . the association of intact islets with pancreatic cancer induction is also shown in transplantation studies in which tumors develop in the submandibular gland after bop treatment if normal islets are transplanted to that site but not when pancreatic ductal cells , thyroid , heart muscle , or starch are introduced into the gland [ 58 - 60 ] . submandibular gland tumor incidence was not changed when hamsters were pre - treated with sz before islet transplantation . a study of pancreatic cancer in hamsters fed a high - fat diet that potentiated pancreatic cancer provided data suggesting that islet proliferation associated with insulin resistance enhances carcinogenesis . in that study , high - fat - fed hamsters had elevated insulin levels but normal glucose levels , which was consistent with a state of insulin resistance . the turn - over rate of cells in islets is significantly increased in the high - fat animals , suggesting a compensatory islet cell proliferation . administration of metformin , starting 2 weeks before the administration of bop and continuing throughout the experiment , normalized insulin concentrations and the rate of islet cell turnover . malignant pancreatic lesions were found in 50% of the high - fat / bop animals and none in the high - fat / bop / metformin group ( p < 0.05 ) . recent studies indicate that there is no simple answer to the question of which of the two hypotheses stated at the beginning of this review is right . however , it appears that these hypotheses are not mutually exclusive , since there is considerable experimental and epidemiological evidence in support of both of them . clearly , the relationships between pancreatic cancer and alterations in glucose metabolism are very complex . pi3-k : phosphatidylinositol 3-kinase , iapp : islet amyloid polypeptide , bop : n - nitrosobis(2-oxopropyl)amine , our research discussed in this article was supported by grants from the swedish research council , the swedish medical research council , and the swedish cancer society .

about 80% of pancreatic cancer patients have glucose intolerance or frank diabetes . this observation has led to the following two hypotheses : i. pancreatic cancer causes the associated diabetes and ii . the conditions associated with diabetes promote the development of pancreatic cancer . evidence supporting both hypotheses has been accumulated in previous studies . this article reviews these studies , especially those that have been conducted recently .

Review Evidence suggesting that pancreatic cancer causes diabetes Evidence for diabetes as a risk factor for pancreatic cancer Conclusion List of abbreviations used Authors' contributions Acknowledgements

the early symptoms of pancreatic cancer , such as abdominal pain , weight loss , fatigue , jaundice , and nausea , are nonspecific and may occur late in the course of the disease . as a result , pancreatic cancer is usually diagnosed at an advanced stage , frequently after the tumor has already metastasized . pancreatic cancer is insensitive to pharmacological and radiological intervention and often recurs after apparently curative surgery . this observation has led to the following two hypotheses : i. pancreatic cancer causes diabetes and ii . numerous studies have been performed in order to elucidate the relationship between these two diseases . the majority of diabetes associated with pancreatic cancer is diagnosed either concomitantly with the cancer or during the two years before the cancer is found ; 71% of the glucose intolerance found in pancreatic cancer patients is unknown before the cancer is diagnosed . these suggest that recently - developed glucose intolerance or diabetes may be a consequence of pancreatic cancer and that recent onset of glucose intolerance or diabetes may be an early sign of pancreatic cancer . several studies have demonstrated that diabetes in pancreatic cancer patients is characterized by peripheral insulin resistance . insulin resistance is also found in non - diabetic or glucose intolerant pancreatic cancer patients , though to a lesser degree . insulin sensitivity and overall diabetic state in pancreatic cancer patients who undergo tumor resection are markedly improved three months after the surgery . these data suggest that pancreatic tumors are causally related to the insulin resistance and diabetes seen in pancreatic cancer patients . in their study of sera from patients with pancreatic cancer and culture media conditioned by human pancreatic cancer cells , basso et al . found a 2030 mw peptide that they considered to be a putative pancreatic cancer associated diabetogenic factor . a number of investigators have studied insulin resistance at the organ , tissue , and cellular levels in pancreatic cancer [ 7 - 13 ] . studies of the initial steps in the insulin signaling cascade in human skeletal muscles showed no significant differences in insulin receptor binding , tyrosine kinase activity , and insulin receptor substrate-1 content between pancreatic cancer patients and healthy controls . however , phosphatidylinositol 3-kinase ( pi3-k ) activity and glucose transport , which are located downstream to the initial insulin signaling steps , were impaired in pancreatic cancer patients . in addition , glycogen synthase activity was reduced in skeletal muscles of humans and rodents with pancreatic carcinoma and in isolated rat skeletal muscles exposed to human pancreatic tumor extracts in vitro . these data show that the insulin signaling cascade in skeletal muscle is impaired at multiple steps by pancreatic cancer . an italian group has performed a series of studies to investigate the effects of pancreatic cancer cells on hepatic insulin sensitivity . when mice were treated with culture medium conditioned by the human pancreatic cancer cell line mia paca2 , blood glucose was elevated compared to the control value seen in mice treated with unconditioned medium . in addition , isolated rat hepatocytes showed impaired glycolysis when incubated in culture media conditioned by four human pancreatic cancer cell lines . because the islet mass destroyed by the tumor is only a small proportion of the whole islet mass , the islet dysfunction is unlikely to be the result of decreased total islet volume . in fact , endocrine pancreatic function can be maintained even with a larger loss of pancreatic islets . insulin release was also reduced when isolated rat pancreatic islets were incubated in culture media conditioned by the human pancreatic cancer cell lines panc-1 and hpaf or co - cultured with panc-1 and hpaf cells . studies of chemically - induced pancreatic cancer in hamsters found that glucose - stimulated insulin release was impaired in vivo but not in isolated perfused pancreata . found an increase in proinsulin relative to insulin in pancreatic cancer patients , suggesting that the maturation of proinsulin may also be affected by the tumor . islet hormone profiles are changed in the circulation of pancreatic cancer patients , suggesting that secretion by different types of islet cells is disrupted by pancreatic cancer . changes in islet hormone concentrations in the circulation can also be seen in hamsters after induction of pancreatic cancer . human pancreatic islets adjacent to pancreatic carcinoma show morphological abnormalities characterized by abnormal co - localization of islet hormones in islet cells . the diabetogenic potential of islet amyloid polypeptide ( iapp or amylin ) has been investigated by several groups . iapp is normally produced in islet beta cells and co - released with insulin at a constant ratio . found elevated circulating levels of iapp in patients with pancreatic cancer . found that culture media conditioned by human pancreatic cancer cells contained a soluble molecule that selectively enhanced iapp release from brin - bd11 beta cells . however , exposure of isolated rat pancreatic islets to hamster pancreatic cancer cells did not change the secretion of insulin and iapp . also , the improvement in glucose tolerance seen after tumor removal is associated with normalization of iapp levels in the circulation . therefore , the increased iapp release seen in pancreatic cancer patients may be responsible , at least in part , for the islet dysfunction seen in these individuals . however , when iapp is infused in rats to create circulating concentrations comparable to the circulating iapp levels in pancreatic cancer patients , the rats have normal glucose disposal . thus , the increased iapp secretion found in pancreatic cancer patients is unlikely to be responsible for their peripheral insulin resistance . examined 30 of the epidemiological studies that have looked at the association between diabetes and pancreatic cancer and used 20 of them in a meta - analysis . the pooled relative risk from these studies was 2.1 for diabetes with a duration of at least l year prior to cancer diagnosis or death and 2.0 for diabetes with a duration of at least 5 years . the authors concluded that pancreatic cancer could be added to the list of complications of diabetes . several epidemiological studies have analyzed relative risks associated with the different periods of time after the diagnosis of diabetes and have found a relatively modest but persistent increased risk of death from pancreatic cancer even when the diagnosis of diabetes preceded death by many years [ 32 - 37 ] . in other studies , the relative risk decreased with increasing follow - up time but remained significant . however , other epidemiological studies have concluded that diabetes is not a risk factor for pancreatic cancer or else that it is not a risk factor if recently - diagnosed cases are excluded [ 6,38 - 40 ] . studies of the relationship between diabetes and pancreatic cancer are complicated by the fact that diabetes has two major forms that are different entities in terms of pathophysiology . a number of studies have suggested that type i diabetes is not associated with an increased risk for pancreatic cancer [ 37 - 39 ] . most epidemiological studies , however , have not distinguished between type i and type ii diabetes . it is likely that the large majority of diabetics in the studies have type ii diabetes because this form of the disease constitutes 8090% of the cases and is typically found in older individuals . in patients with type ii diabetes ( non - insulin - dependent diabetes ) , the pancreas is generally exposed to substantial hyperinsulinemia for years , suggesting that insulin may be involved in the association between long - standing diabetes and pancreatic cancer . a number of experiments have tested the hypothesis that insulin may stimulate the growth of pancreatic cancers . binding studies have shown the presence of insulin receptors on pancreatic cancer cells [ 42 - 45 ] . in vitro studies have shown that insulin promotes growth of the hamster pancreatic cancer cell line h2 t , the rat acinar pancreatic cancer cell line ar42j , and numerous human pancreatic cancer cells lines [ 44,46 - 51 ] . however , the human pancreatic cancer cell line soj-6 was not stimulated by insulin , and one of the studies using panc-1 cells reported no response to exogenous insulin . in addition to hyperinsulinemia , the increased blood glucose and free fatty acids in diabetes may also promote the growth of pancreatic cancer . in vivo studies concerning the effects of administration of exogenous insulin and/or induction of diabetes on pancreatic cancer have provided inconsistent data that reflect the complex interactions that may be involved in tumor growth [ 53 - 56 ] . exogenous insulin significantly reduced the induction of benign and malignant pancreatic lesions in hamsters when given 2 hours before bop , but the reduction in incidence was not significant when insulin was given simultaneously with bop or 2 hours after bop . when hamsters were given streptozotocin ( sz ) injection to diminish insulin cells and given insulin from the following day untill the end of the experiment , the inhibition of carcinogenesis in hamsters receiving sz+bop+insulin treatment was greater than that seen in the sz+bop group , compared to group treated by bop only . because insulin administration was associated with inhibition of beta cell regeneration and persistence of severe diabetes in hamsters treated with sz , the investigators in the sz / bop / insulin study concluded that intact islet cells , rather than the availability of insulin , are prerequisite for triggering the neoplastic effects of bop . the association of intact islets with pancreatic cancer induction is also shown in transplantation studies in which tumors develop in the submandibular gland after bop treatment if normal islets are transplanted to that site but not when pancreatic ductal cells , thyroid , heart muscle , or starch are introduced into the gland [ 58 - 60 ] . submandibular gland tumor incidence was not changed when hamsters were pre - treated with sz before islet transplantation . a study of pancreatic cancer in hamsters fed a high - fat diet that potentiated pancreatic cancer provided data suggesting that islet proliferation associated with insulin resistance enhances carcinogenesis . the majority of diabetes associated with pancreatic cancer is diagnosed either concomitantly with the cancer or during the two years before the cancer is found ; 71% of the glucose intolerance found in pancreatic cancer patients is unknown before the cancer is diagnosed . these suggest that recently - developed glucose intolerance or diabetes may be a consequence of pancreatic cancer and that recent onset of glucose intolerance or diabetes may be an early sign of pancreatic cancer . several studies have demonstrated that diabetes in pancreatic cancer patients is characterized by peripheral insulin resistance . insulin resistance is also found in non - diabetic or glucose intolerant pancreatic cancer patients , though to a lesser degree . insulin sensitivity and overall diabetic state in pancreatic cancer patients who undergo tumor resection are markedly improved three months after the surgery . these data suggest that pancreatic tumors are causally related to the insulin resistance and diabetes seen in pancreatic cancer patients . in their study of sera from patients with pancreatic cancer and culture media conditioned by human pancreatic cancer cells , basso et al . found a 2030 mw peptide that they considered to be a putative pancreatic cancer associated diabetogenic factor . a number of investigators have studied insulin resistance at the organ , tissue , and cellular levels in pancreatic cancer [ 7 - 13 ] . studies of the initial steps in the insulin signaling cascade in human skeletal muscles showed no significant differences in insulin receptor binding , tyrosine kinase activity , and insulin receptor substrate-1 content between pancreatic cancer patients and healthy controls . however , phosphatidylinositol 3-kinase ( pi3-k ) activity and glucose transport , which are located downstream to the initial insulin signaling steps , were impaired in pancreatic cancer patients . these data show that the insulin signaling cascade in skeletal muscle is impaired at multiple steps by pancreatic cancer . an italian group has performed a series of studies to investigate the effects of pancreatic cancer cells on hepatic insulin sensitivity . when mice were treated with culture medium conditioned by the human pancreatic cancer cell line mia paca2 , blood glucose was elevated compared to the control value seen in mice treated with unconditioned medium . in addition , isolated rat hepatocytes showed impaired glycolysis when incubated in culture media conditioned by four human pancreatic cancer cell lines . because the islet mass destroyed by the tumor is only a small proportion of the whole islet mass , the islet dysfunction is unlikely to be the result of decreased total islet volume . in fact , endocrine pancreatic function can be maintained even with a larger loss of pancreatic islets . insulin release was also reduced when isolated rat pancreatic islets were incubated in culture media conditioned by the human pancreatic cancer cell lines panc-1 and hpaf or co - cultured with panc-1 and hpaf cells . studies of chemically - induced pancreatic cancer in hamsters found that glucose - stimulated insulin release was impaired in vivo but not in isolated perfused pancreata . found an increase in proinsulin relative to insulin in pancreatic cancer patients , suggesting that the maturation of proinsulin may also be affected by the tumor . islet hormone profiles are changed in the circulation of pancreatic cancer patients , suggesting that secretion by different types of islet cells is disrupted by pancreatic cancer . changes in islet hormone concentrations in the circulation can also be seen in hamsters after induction of pancreatic cancer . the diabetogenic potential of islet amyloid polypeptide ( iapp or amylin ) has been investigated by several groups . in contrast , the expression of iapp mrna in these islets is unchanged , suggesting normal production but increased release of iapp . the molar ratio of iapp / insulin was increased when rat pancreatic islets were co - cultured with panc-1 and hpaf cells or cultured in media conditioned by these cell lines . found that culture media conditioned by human pancreatic cancer cells contained a soluble molecule that selectively enhanced iapp release from brin - bd11 beta cells . increased iapp / insulin ratios were also seen in rats with azaserine - induced acinar pancreatic tumors and in hamsters with ductular pancreatic tumors induced by carcinogen n - nitrosobis(2-oxopropyl)amine ( bop ) . however , exposure of isolated rat pancreatic islets to hamster pancreatic cancer cells did not change the secretion of insulin and iapp . also , the improvement in glucose tolerance seen after tumor removal is associated with normalization of iapp levels in the circulation . therefore , the increased iapp release seen in pancreatic cancer patients may be responsible , at least in part , for the islet dysfunction seen in these individuals . however , when iapp is infused in rats to create circulating concentrations comparable to the circulating iapp levels in pancreatic cancer patients , the rats have normal glucose disposal . thus , the increased iapp secretion found in pancreatic cancer patients is unlikely to be responsible for their peripheral insulin resistance . examined 30 of the epidemiological studies that have looked at the association between diabetes and pancreatic cancer and used 20 of them in a meta - analysis . the pooled relative risk from these studies was 2.1 for diabetes with a duration of at least l year prior to cancer diagnosis or death and 2.0 for diabetes with a duration of at least 5 years . the authors concluded that pancreatic cancer could be added to the list of complications of diabetes . several epidemiological studies have analyzed relative risks associated with the different periods of time after the diagnosis of diabetes and have found a relatively modest but persistent increased risk of death from pancreatic cancer even when the diagnosis of diabetes preceded death by many years [ 32 - 37 ] . a population - based case - control study in the united states with 526 incident cases and 2,153 population controls showed a significant positive trend ( p = 0.016 ) in risk with increasing years prior to diagnosis of cancer . in other studies , the relative risk decreased with increasing follow - up time but remained significant . however , other epidemiological studies have concluded that diabetes is not a risk factor for pancreatic cancer or else that it is not a risk factor if recently - diagnosed cases are excluded [ 6,38 - 40 ] . studies of the relationship between diabetes and pancreatic cancer are complicated by the fact that diabetes has two major forms that are different entities in terms of pathophysiology . a number of studies have suggested that type i diabetes is not associated with an increased risk for pancreatic cancer [ 37 - 39 ] . most epidemiological studies , however , have not distinguished between type i and type ii diabetes . it is likely that the large majority of diabetics in the studies have type ii diabetes because this form of the disease constitutes 8090% of the cases and is typically found in older individuals . in patients with type ii diabetes ( non - insulin - dependent diabetes ) , the pancreas is generally exposed to substantial hyperinsulinemia for years , suggesting that insulin may be involved in the association between long - standing diabetes and pancreatic cancer . a number of experiments have tested the hypothesis that insulin may stimulate the growth of pancreatic cancers . binding studies have shown the presence of insulin receptors on pancreatic cancer cells [ 42 - 45 ] . in vitro studies have shown that insulin promotes growth of the hamster pancreatic cancer cell line h2 t , the rat acinar pancreatic cancer cell line ar42j , and numerous human pancreatic cancer cells lines [ 44,46 - 51 ] . however , the human pancreatic cancer cell line soj-6 was not stimulated by insulin , and one of the studies using panc-1 cells reported no response to exogenous insulin . in addition to hyperinsulinemia , the increased blood glucose and free fatty acids in diabetes may also promote the growth of pancreatic cancer . in vivo studies concerning the effects of administration of exogenous insulin and/or induction of diabetes on pancreatic cancer have provided inconsistent data that reflect the complex interactions that may be involved in tumor growth [ 53 - 56 ] . when hamsters were given streptozotocin ( sz ) injection to diminish insulin cells and given insulin from the following day untill the end of the experiment , the inhibition of carcinogenesis in hamsters receiving sz+bop+insulin treatment was greater than that seen in the sz+bop group , compared to group treated by bop only . because insulin administration was associated with inhibition of beta cell regeneration and persistence of severe diabetes in hamsters treated with sz , the investigators in the sz / bop / insulin study concluded that intact islet cells , rather than the availability of insulin , are prerequisite for triggering the neoplastic effects of bop . the association of intact islets with pancreatic cancer induction is also shown in transplantation studies in which tumors develop in the submandibular gland after bop treatment if normal islets are transplanted to that site but not when pancreatic ductal cells , thyroid , heart muscle , or starch are introduced into the gland [ 58 - 60 ] . a study of pancreatic cancer in hamsters fed a high - fat diet that potentiated pancreatic cancer provided data suggesting that islet proliferation associated with insulin resistance enhances carcinogenesis . administration of metformin , starting 2 weeks before the administration of bop and continuing throughout the experiment , normalized insulin concentrations and the rate of islet cell turnover . recent studies indicate that there is no simple answer to the question of which of the two hypotheses stated at the beginning of this review is right . clearly , the relationships between pancreatic cancer and alterations in glucose metabolism are very complex . pi3-k : phosphatidylinositol 3-kinase , iapp : islet amyloid polypeptide , bop : n - nitrosobis(2-oxopropyl)amine , our research discussed in this article was supported by grants from the swedish research council , the swedish medical research council , and the swedish cancer society .

the data derive from the baseline measurements of a large national thai cohort study ( tcs ) that began in 2005 , researching the health - risk transition in the adult thai population . the study began with a 20-page mailout questionnaire covering social demography , work , health and injuries , social networks and well - being , diet and physical activity , and tobacco , alcohol , and transport . there were 87,134 respondents aged 1587 years enrolled as distance learning students at sukhothai thammathirat open university ( stou ) . further details about the questionnaire data collection and the attributes of respondents are given in sleigh et al . the information on individual experiences of heat stress was derived from the question : during the last 12 months , how often have you experienced at work high temperatures which make you uncomfortable? , which 76,113 respondents answered on a four - point scale ( often, sometimes, rarely, and never ) . for analysis , poor overall health is based on the first question of the medical outcomes short form instrument ( sf8 ) overall how would you rate your health during the past four weeks ( excellent , very good , good , fair , poor , or very poor). for analysis , we combined the last two categories as poor overall health and the first four categories as not poor overall health. psychological distress is based on the three anxiety - oriented questions of the standard kessler 6 psychological distress questions : in the past 4 weeks , about how often did you feel ( nervous , restless or fidgety , or everything was an effort) with answers on a five - point scale ( 1=all of the time , 2=most of the time , 3=some of the time , 4=a little of the time , 5=none of the time ) . for our analysis , if the average response for the three questions was less than or equal to 2 , the respondent was classified as psychologically distressed . overall , most of those excluded did not answer the questions regarding work or were not working in paid employment for at least 40 hours per week . all respondents reported age , sex , education , income , location of job , job type , and five other hazards at work ( beside high temperature ) ; data on these variables were collected because they could confound or modify our estimates of the association between overall health , psychological distress , and occupational heat stress . selection process for the analyzed population from the thai cohort study data . for analysis , age in years was divided into three categories ( 1529 , 3044 , > 45 ) , highest education was divided into three categories ( high school , diploma , or university ) , monthly income ( in thai baht ) was divided into four categories ( up to 7,000, 7,00110,000, 10,00120,000, and 20,001 and more ) , and job location was divided into three categories ( bangkok , urban - outside bangkok , and rural ) . some analyses were restricted to the population providing information on job type categorized as office job or physical job ( those reporting as skilled workers were excluded because we could not categorize them further ) . complaints about five other hazards at work ( vibrations from tools or machines , loud noise , uncomfortably low temperatures , dangerous fumes ( vapors , chemicals , infectious agents ) , and touching dangerous substances ) were each also categorized as often / not often and the often results combined into three levels ( 0 other complaints , 12 other complaints , 35 other complaints ) . data were digitized using thai scandevet software and further processed using spss and stata statistical packages . the initial analysis group included 40,913 fulltime workers for overall heat stress analyses . final heat stress analyses were restricted to the 24,907 workers whose job type was known ( allowing assessment of this variable ) and who did not make non - thermal work hazard complaints ( preventing any influence on the results of adverse effects of multiple hazard complainers ) . summary statistics ( means and proportions by variable category and sex ) were prepared for heat stress , age , education , income , job , and work hazard complaints . all explanatory variables ( i.e. sex , age , job location , job type , education , income , other work hazards ) were assessed as potential confounders of our estimates of heat stress associations on health outcomes by investigating their association with the exposure of interest ( heat stress ) or the outcomes ( overall health or psychological distress ) . both outcomes were assessed graphically for association with heat stress and these analyses were broken down by age group ( 1529 , 3044 , > 45 years ) and sex . the association between heat stress and overall health or psychological distress was assessed by odds ratios ( ors ) and 95% confidence intervals . using multivariable logistic regression , these heat stress ors were adjusted for the influence of all confounding variables ( sex , age , job type , job location , education , income , and complaints of other work hazards ) . these variables were also evaluated as potential modifiers of heat stress associations by stratified analyses that estimated the ors separately for each category ( i.e. by sex , age group , education group , income group , complaint group , job location , job type and by the joint effect of job type and location ) . each stratified or was adjusted for the confounding influence of other explanatory variables ( e.g. sex , age group , etc . ) . finally , using stepwise logistic regression to include only significant confounders and heat stress interaction terms , models were prepared to show the fully adjusted heat stress association on overall health and on psychological distress . the final model for the 40,913 fulltime workers for overall health included heat stress , sex , age , education , other complaints , job location , and two interaction terms ( educationheat , and other complaintsheat ) . the final model for psychological distress included heat stress , sex , age , education , income , other complaints , job location , and an interaction term for other complaintsheat . when job type was offered for the stepwise models and these workers were potentially exposed to heat stress only ( restricted data set , n=24,907 ) , the final model for both poor health and psychological distress included heat stress , sex , age , and job location . there were no significant interaction terms in these final models linking heat stress to either health outcome . ethical approval was obtained from stou research committee and the australian national university human research ethics committee ( anu hrec ) . the data derive from the baseline measurements of a large national thai cohort study ( tcs ) that began in 2005 , researching the health - risk transition in the adult thai population . the study began with a 20-page mailout questionnaire covering social demography , work , health and injuries , social networks and well - being , diet and physical activity , and tobacco , alcohol , and transport . there were 87,134 respondents aged 1587 years enrolled as distance learning students at sukhothai thammathirat open university ( stou ) . further details about the questionnaire data collection and the attributes of respondents are given in sleigh et al . the information on individual experiences of heat stress was derived from the question : during the last 12 months , how often have you experienced at work high temperatures which make you uncomfortable? , which 76,113 respondents answered on a four - point scale ( often, sometimes, rarely, and never ) . for analysis , poor overall health is based on the first question of the medical outcomes short form instrument ( sf8 ) overall how would you rate your health during the past four weeks ( excellent , very good , good , fair , poor , or very poor). for analysis , we combined the last two categories as poor overall health and the first four categories as not poor overall health. psychological distress is based on the three anxiety - oriented questions of the standard kessler 6 psychological distress questions : in the past 4 weeks , about how often did you feel ( nervous , restless or fidgety , or everything was an effort) with answers on a five - point scale ( 1=all of the time , 2=most of the time , 3=some of the time , 4=a little of the time , 5=none of the time ) . for our analysis , if the average response for the three questions was less than or equal to 2 , the respondent was classified as psychologically distressed . 1 . overall , most of those excluded did not answer the questions regarding work or were not working in paid employment for at least 40 hours per week . all respondents reported age , sex , education , income , location of job , job type , and five other hazards at work ( beside high temperature ) ; data on these variables were collected because they could confound or modify our estimates of the association between overall health , psychological distress , and occupational heat stress . for analysis , age in years was divided into three categories ( 1529 , 3044 , > 45 ) , highest education was divided into three categories ( high school , diploma , or university ) , monthly income ( in thai baht ) was divided into four categories ( up to 7,000, 7,00110,000, 10,00120,000, and 20,001 and more ) , and job location was divided into three categories ( bangkok , urban - outside bangkok , and rural ) . some analyses were restricted to the population providing information on job type categorized as office job or physical job ( those reporting as skilled workers were excluded because we could not categorize them further ) . complaints about five other hazards at work ( vibrations from tools or machines , loud noise , uncomfortably low temperatures , dangerous fumes ( vapors , chemicals , infectious agents ) , and touching dangerous substances ) were each also categorized as often / not often and the often results combined into three levels ( 0 other complaints , 12 other complaints , 35 other complaints ) . data were digitized using thai scandevet software and further processed using spss and stata statistical packages . the initial analysis group included 40,913 fulltime workers for overall heat stress analyses . final heat stress analyses were restricted to the 24,907 workers whose job type was known ( allowing assessment of this variable ) and who did not make non - thermal work hazard complaints ( preventing any influence on the results of adverse effects of multiple hazard complainers ) . summary statistics ( means and proportions by variable category and sex ) were prepared for heat stress , age , education , income , job , and work hazard complaints . all explanatory variables ( i.e. sex , age , job location , job type , education , income , other work hazards ) were assessed as potential confounders of our estimates of heat stress associations on health outcomes by investigating their association with the exposure of interest ( heat stress ) or the outcomes ( overall health or psychological distress ) . both outcomes were assessed graphically for association with heat stress and these analyses were broken down by age group ( 1529 , 3044 , > 45 years ) and sex . the association between heat stress and overall health or psychological distress was assessed by odds ratios ( ors ) and 95% confidence intervals . using multivariable logistic regression , these heat stress ors were adjusted for the influence of all confounding variables ( sex , age , job type , job location , education , income , and complaints of other work hazards ) . these variables were also evaluated as potential modifiers of heat stress associations by stratified analyses that estimated the ors separately for each category ( i.e. by sex , age group , education group , income group , complaint group , job location , job type and by the joint effect of job type and location ) . each stratified or was adjusted for the confounding influence of other explanatory variables ( e.g. sex , age group , etc . ) . finally , using stepwise logistic regression to include only significant confounders and heat stress interaction terms , models were prepared to show the fully adjusted heat stress association on overall health and on psychological distress . the final model for the 40,913 fulltime workers for overall health included heat stress , sex , age , education , other complaints , job location , and two interaction terms ( educationheat , and other complaintsheat ) . the final model for psychological distress included heat stress , sex , age , education , income , other complaints , job location , and an interaction term for other complaintsheat . when job type was offered for the stepwise models and these workers were potentially exposed to heat stress only ( restricted data set , n=24,907 ) , the final model for both poor health and psychological distress included heat stress , sex , age , and job location . there were no significant interaction terms in these final models linking heat stress to either health outcome . ethical approval was obtained from stou research committee and the australian national university human research ethics committee ( anu hrec ) . tables 1 and 2 , and fig . 1 present the main characteristics of all 40,913 thai cohort members who were fulltime workers . more than half the cohort was young , aged between 15 and 29 years ( 52% ) . nearly 20% of workplaces were located in bangkok , 41% were in other urban areas ( outside bangkok ) , and 39% were in rural areas , 19% of classifiable jobs were physical and 81% were based in an office . compared with males , females were younger and worked in bangkok ( 22 vs 17% ) . on average , women had moderately higher educational achievements whereas men had substantially higher personal incomes . overall , 5% of fulltime workers reported often experiencing three to five other hazards beside high temperature at work , more for males ( 6% ) than females ( 3% ) . over 18% ( n=7,476 ) of the analyzed group this problem was worse for males ( 23% ) than females ( 15% ) ; also females more frequently than males reported never experiencing heat stress at work ( 27 vs 17% ) . prevalence of reported poor overall health by age group , sex , and reported occupational heat stress in 24,907 thai workers . socioeconomic and other attributes in a cohort of 40,913 fulltime workers in thailand job type is available for analysis with 24,907 respondents ( see section methods ) . reported heat stress at work in a cohort of 40,913 fulltime workers in thailand heat stress exposure together with health and psychological distress outcomes are summarized in table 3 . here we explore the associations of heat stress exposures and health outcomes with potential confounders of the heat stress effects . the prevalence of heat stress ( often experiencing high temperatures at work ( n=40,913 ) ) varied considerably among subgroups and was notably high for those with other complaints regarding hazards at work . nearly 5% of workers reported poor overall health and this figure rose to 9% for those reporting often being troubled by three to five other work hazards . psychological distress was more common ; overall , it affected 8% of workers and was reported by 20% of those with three to five other work hazards . heat stress , poor overall health , and psychological distress by potential confounding factors in a cohort of 40,913 fulltime workers associations with heat stress , poor overall health , and psychological distress each expressed as crude odds ratios ( ors ) . the potential confounders shown in table 3 ( sex , age group , education , income , job type , job location , and other work hazard complaints ) were significantly associated with both the exposure of interest ( heat stress ) and the two outcomes ( overall health and psychological distress ) . accordingly , our analyses of heat stress effects were adjusted for these confounding variables , or restricted to exclude all those respondents who had multiple work hazard complaints . 2 and 3 reveal the positive association between heat stress and poor health or psychological distress stratified by age group and sex . this analysis was restricted to the 24,907 workers who did not complain of multiple work hazards and whose job type was known . the association between overall health , psychological distress , and occupational heat stress are substantial and revealed no consistent pattern with age . generally , the adverse effects of heat stress on poor health were worse for females ; the opposite association was noted for heat stress and psychological distress , which was especially bad for males aged between 15 and 29 years . prevalence of reported psychological distress by age group , sex , and reported occupational heat stress in 24,907 thai workers . , the heat stress effects are increased for workers aged more than 45 years and for the better educated , and for work located in rural areas , but none of these interactions are strong . perhaps the most notable interaction is the attenuation of heat stress associations among those with multiple work hazard complaints ; for example , the association of heat stress and overall health ( or=1.75 ) compares to an or of 1.22 for those with three to five complaints . stratified odds ratios revealing interaction of demographic and work - related variables with the association between overall health , psychological distress , and occupational heat stress ( n=40,913 ) number of respondents who reported heat stress at work ( see section methods ) . odds ratios show within - strata estimates for heat stress associations adjusted for all other explanatory variables . table 5 shows crude and adjusted or estimates for associations between heat stress and poor health and psychological distress outcomes . the adjusted ors derive from models that include age , sex , income , education , job location , and other work hazard complaints . cohort members who experienced heat stress at work had higher odds of poor overall health ( crude or=1.67 , p - value < 0.001 ; adjusted or=1.49 , p - value < 0.001 ) . they also had higher odds of psychological distress ( crude or=2.22 , p<0.001 ; adjusted or=1.84 , p - value < 0.001 ) . for workers who did not complain of other work hazards and for whom job type was reported ( n=24,907 ) , the adjusted heat stress models ors for poor health ( 1.80 ) and psychological distress ( 2.19 ) differ little from previous estimates . the association between occupational heat stress , overall health , and psychological distress adjusted ors derived from heat stress models that included sex , age , income , education , job location , and complaints ( n=40,913 ) plus job type ( n=24,907 ) . final model for estimating the association between heat stress and poor overall health includes all statistically significant confounders ( sex , age , education , complaints , job location ) , and the two significant interaction terms ( educationheat and complaints heat ) . final model for estimating the association between heat stress and psychological distress includes all statistically significant confounders ( sex , age , education , income , complaints , job location ) and the one significant interaction term ( complaintsheat ) . final model in restricted group ( n=24,907 ) for association between heat stress and both poor overall health and psychological distress includes all statistically significant confounders ( sex , age , and job location ) . the final logistic regression models included all significant confounders and all significant interactors ( see section the final ors linking heat stress to poor overall health were 1.55 ( n=40,913 ) and 1.81 ( when restricted to those reporting job type and no other work hazard complaints , n=24,907 ) . for psychological distress , the corresponding final adjusted ors were similar for the two analytical groups ( 2.21 for n=40,913 and 2.17 for n=24,907 ) . accordingly , these final models show considerable and consistent strength of association between heat stress and health outcomes , and the confidence limits and p - values show results unlikely to be due to chance . tables 1 and 2 , and fig . 1 present the main characteristics of all 40,913 thai cohort members who were fulltime workers . more than half the cohort was young , aged between 15 and 29 years ( 52% ) . nearly 20% of workplaces were located in bangkok , 41% were in other urban areas ( outside bangkok ) , and 39% were in rural areas , 19% of classifiable jobs were physical and 81% were based in an office . compared with males , females were younger and worked in bangkok ( 22 vs 17% ) . on average , women had moderately higher educational achievements whereas men had substantially higher personal incomes . overall , 5% of fulltime workers reported often experiencing three to five other hazards beside high temperature at work , more for males ( 6% ) than females ( 3% ) . over 18% ( n=7,476 ) of the analyzed group this problem was worse for males ( 23% ) than females ( 15% ) ; also females more frequently than males reported never experiencing heat stress at work ( 27 vs 17% ) . prevalence of reported poor overall health by age group , sex , and reported occupational heat stress in 24,907 thai workers . socioeconomic and other attributes in a cohort of 40,913 fulltime workers in thailand job type is available for analysis with 24,907 respondents ( see section methods ) . reported heat stress at work in a cohort of 40,913 fulltime workers in thailand heat stress exposure together with health and psychological distress outcomes are summarized in table 3 . here we explore the associations of heat stress exposures and health outcomes with potential confounders of the heat stress effects . the prevalence of heat stress ( often experiencing high temperatures at work ( n=40,913 ) ) varied considerably among subgroups and was notably high for those with other complaints regarding hazards at work . nearly 5% of workers reported poor overall health and this figure rose to 9% for those reporting often being troubled by three to five other work hazards . psychological distress was more common ; overall , it affected 8% of workers and was reported by 20% of those with three to five other work hazards . heat stress , poor overall health , and psychological distress by potential confounding factors in a cohort of 40,913 fulltime workers associations with heat stress , poor overall health , and psychological distress each expressed as crude odds ratios ( ors ) . methods ) . in almost all instances the potential confounders shown in table 3 ( sex , age group , education , income , job type , job location , and other work hazard complaints ) were significantly associated with both the exposure of interest ( heat stress ) and the two outcomes ( overall health and psychological distress ) . accordingly , our analyses of heat stress effects were adjusted for these confounding variables , or restricted to exclude all those respondents who had multiple work hazard complaints . 2 and 3 reveal the positive association between heat stress and poor health or psychological distress stratified by age group and sex . this analysis was restricted to the 24,907 workers who did not complain of multiple work hazards and whose job type was known . the association between overall health , psychological distress , and occupational heat stress are substantial and revealed no consistent pattern with age . generally , the adverse effects of heat stress on poor health were worse for females ; the opposite association was noted for heat stress and psychological distress , which was especially bad for males aged between 15 and 29 years . prevalence of reported psychological distress by age group , sex , and reported occupational heat stress in 24,907 thai workers . modifications of the heat stress effects are summarized in table 4 . for both outcomes , the heat stress effects are increased for workers aged more than 45 years and for the better educated , and for work located in rural areas , but none of these interactions are strong . perhaps the most notable interaction is the attenuation of heat stress associations among those with multiple work hazard complaints ; for example , the association of heat stress and overall health ( or=1.75 ) compares to an or of 1.22 for those with three to five complaints . stratified odds ratios revealing interaction of demographic and work - related variables with the association between overall health , psychological distress , and occupational heat stress ( n=40,913 ) number of respondents who reported heat stress at work ( see section odds ratios show within - strata estimates for heat stress associations adjusted for all other explanatory variables . table 5 shows crude and adjusted or estimates for associations between heat stress and poor health and psychological distress outcomes . the adjusted ors derive from models that include age , sex , income , education , job location , and other work hazard complaints . cohort members who experienced heat stress at work had higher odds of poor overall health ( crude or=1.67 , p - value < 0.001 ; adjusted or=1.49 , p - value < 0.001 ) . they also had higher odds of psychological distress ( crude or=2.22 , p<0.001 ; adjusted or=1.84 , p - value < 0.001 ) . for workers who did not complain of other work hazards and for whom job type was reported ( n=24,907 ) , the adjusted heat stress models ors for poor health ( 1.80 ) and psychological distress ( 2.19 ) differ little from previous estimates . the association between occupational heat stress , overall health , and psychological distress adjusted ors derived from heat stress models that included sex , age , income , education , job location , and complaints ( n=40,913 ) plus job type ( n=24,907 ) . final model for estimating the association between heat stress and poor overall health includes all statistically significant confounders ( sex , age , education , complaints , job location ) , and the two significant interaction terms ( educationheat and complaints heat ) . final model for estimating the association between heat stress and psychological distress includes all statistically significant confounders ( sex , age , education , income , complaints , job location ) and the one significant interaction term ( complaintsheat ) . final model in restricted group ( n=24,907 ) for association between heat stress and both poor overall health and psychological distress includes all statistically significant confounders ( sex , age , and job location ) . the final logistic regression models included all significant confounders and all significant interactors ( see section the final ors linking heat stress to poor overall health were 1.55 ( n=40,913 ) and 1.81 ( when restricted to those reporting job type and no other work hazard complaints , n=24,907 ) . for psychological distress , the corresponding final adjusted ors were similar for the two analytical groups ( 2.21 for n=40,913 and 2.17 for n=24,907 ) . accordingly , these final models show considerable and consistent strength of association between heat stress and health outcomes , and the confidence limits and p - values show results unlikely to be due to chance . these findings add to the limited literature on the association between overall health , psychological distress , and occupational heat stress of thai workers ( 17 ) . we found heat stress exposure reported by nearly 20% of our large national cohort , more frequently among laborers and male workers , those with low incomes and low education , and declining with age possibly due to an age - related shift away from physical work . occupational heat stress was reported less frequently in bangkok , probably reflecting air conditioning in thai cities , especially in bangkok ( 9 ) . the proportion of male workers who reported poor health and psychological distress declined with age . this was especially noteworthy for psychological distress , possibly reflecting longer work experience and the ability to control emotions among older men . however , heat - stressed females did show a trend of worsening overall health with increasing age , which may reflect aging as well as more responsibilities and daily stress from the mix of professional work and homemaking once a family has formed . this finding is similar to lennon ( 1995 ) , who found that women have substantially higher psychological distress than men ( 18 ) . however , in our study , men aged between 15 and 29 years reported the highest prevalence of distress ( fig . 3 ) , which may be linked to the high rates of suicide and distress in young men in thailand ( 19 ) . our principal finding is that heat stress was strongly and significantly associated with both poor overall health and psychological distress , with adjusted ors ranging from 1.49 to 1.84 . these epidemiological associations remained substantial and highly significant statistically when extensively adjusted for confounding ( by age , sex , income , education , and job location ) and when restricted to those who did not have other ( non - thermal ) work hazard complaints ( i.e. non - complainers ) . the strength of association between overall health , psychological distress , and occupational heat stress interacts weakly with an array of other sociodemographic variables . access to a large national cohort of 40,913 thai workers is the main advantage of this study . it represents working - age thais reasonably well for geographic location , age , sex , and socioeconomic status ( 16 ) . cohort members are better educated than average thais of the same age and sex , but this difference enabled us to gather complex heat exposure and health outcomes on large numbers by questionnaire . the cohort shows a wide range of values for the variables of interest , allowing us to investigate the relationship between heat stress at work and health outcomes . furthermore , we can be reassured of our results by the restricted analyses of 24,907 workers with all the multi - complainers excluded and job types known . the results are similar to the overall analysis of 40,913 cohort members that was adjusted for confounding and interaction . however , this study could not directly establish that these overall health problems and psychological distress arose as a result of heat stress . there are some difficulties in interpreting these data on exposure patterns and associated health outcomes given that we can not be sure that heat stress preceded adverse outcomes . moreover , the source or nature of the heat stress or the work situations was not categorized in this study . indeed , we were not able to make a direct measurement of work environments or health outcomes , hence we must classify this study as preliminary in nature . therefore , there is need for more detailed direct observations in informative work settings to validate our findings and explore the underlying mechanisms . we were able to find only one other study ( an unpublished thesis ) of thermal environment and mental health in thailand , among a small population of 90 workers in a seafood factory ( 17 ) . this factory study did not document heat stress ( but did measure temperature ) ; nor did it find evidence of widespread psychological distress . although , there are reports on occupational heat exposure and comfort of workers , they do not usually mention psychological distress or mental health problems as a variable ( 11 , 14 ) . therefore , our study is one of the first to provide evidence on an association between heat stress and psychological distress of workers . this is the first large - scale study of occupational heat stress and adverse health outcomes in thailand . this is particularly important at present because we can expect that the existing problem will worsen if global warming continues and workplaces become even more thermally stressful . given the constrained resources in middle - income thailand , and the possibility that psychologically stressful work is becoming more widespread , our results on psychological distress are of particular concern . if workers exposed to excessive heat can not cool down , they can experience severe psychological distress caused by heat - related exhaustion or develop long - term conditions such as chronic depression or chronic anxiety disorders ( 11 , 20 ) . furthermore , ramsey ( 21 ) reported that heat stress diminished mental ability and increased injury risks , and others have noted increased suicide risk ( 15 , 22 , 23 ) . further studies are needed to accurately characterize workplace humidity , air movement , radiant temperature , health outcomes , and work performance in various settings in thailand , especially as the workforce structure changes with economic development ( 24 , 25 ) . these findings add to the limited literature on the association between overall health , psychological distress , and occupational heat stress of thai workers ( 17 ) . we found heat stress exposure reported by nearly 20% of our large national cohort , more frequently among laborers and male workers , those with low incomes and low education , and declining with age possibly due to an age - related shift away from physical work . occupational heat stress was reported less frequently in bangkok , probably reflecting air conditioning in thai cities , especially in bangkok ( 9 ) . the proportion of male workers who reported poor health and psychological distress declined with age . this was especially noteworthy for psychological distress , possibly reflecting longer work experience and the ability to control emotions among older men . however , heat - stressed females did show a trend of worsening overall health with increasing age , which may reflect aging as well as more responsibilities and daily stress from the mix of professional work and homemaking once a family has formed . this finding is similar to lennon ( 1995 ) , who found that women have substantially higher psychological distress than men ( 18 ) . however , in our study , men aged between 15 and 29 years reported the highest prevalence of distress ( fig . 3 ) , which may be linked to the high rates of suicide and distress in young men in thailand ( 19 ) . our principal finding is that heat stress was strongly and significantly associated with both poor overall health and psychological distress , with adjusted ors ranging from 1.49 to 1.84 . these epidemiological associations remained substantial and highly significant statistically when extensively adjusted for confounding ( by age , sex , income , education , and job location ) and when restricted to those who did not have other ( non - thermal ) work hazard complaints ( i.e. non - complainers ) . the strength of association between overall health , psychological distress , and occupational heat stress interacts weakly with an array of other sociodemographic variables . access to a large national cohort of 40,913 thai workers is the main advantage of this study . it represents working - age thais reasonably well for geographic location , age , sex , and socioeconomic status ( 16 ) . cohort members are better educated than average thais of the same age and sex , but this difference enabled us to gather complex heat exposure and health outcomes on large numbers by questionnaire . the cohort shows a wide range of values for the variables of interest , allowing us to investigate the relationship between heat stress at work and health outcomes . furthermore , we can be reassured of our results by the restricted analyses of 24,907 workers with all the multi - complainers excluded and job types known . the results are similar to the overall analysis of 40,913 cohort members that was adjusted for confounding and interaction . however , this study could not directly establish that these overall health problems and psychological distress arose as a result of heat stress . there are some difficulties in interpreting these data on exposure patterns and associated health outcomes given that we can not be sure that heat stress preceded adverse outcomes . moreover , the source or nature of the heat stress or the work situations was not categorized in this study . indeed , we were not able to make a direct measurement of work environments or health outcomes , hence we must classify this study as preliminary in nature . therefore , there is need for more detailed direct observations in informative work settings to validate our findings and explore the underlying mechanisms . we were able to find only one other study ( an unpublished thesis ) of thermal environment and mental health in thailand , among a small population of 90 workers in a seafood factory ( 17 ) . this factory study did not document heat stress ( but did measure temperature ) ; nor did it find evidence of widespread psychological distress . although , there are reports on occupational heat exposure and comfort of workers , they do not usually mention psychological distress or mental health problems as a variable ( 11 , 14 ) . therefore , our study is one of the first to provide evidence on an association between heat stress and psychological distress of workers . this is the first large - scale study of occupational heat stress and adverse health outcomes in thailand . the findings of widespread heat stress and associated ill - health are disturbing . this is particularly important at present because we can expect that the existing problem will worsen if global warming continues and workplaces become even more thermally stressful . given the constrained resources in middle - income thailand , and the possibility that psychologically stressful work is becoming more widespread , our results on psychological distress are of particular concern . if workers exposed to excessive heat can not cool down , they can experience severe psychological distress caused by heat - related exhaustion or develop long - term conditions such as chronic depression or chronic anxiety disorders ( 11 , 20 ) . furthermore , ramsey ( 21 ) reported that heat stress diminished mental ability and increased injury risks , and others have noted increased suicide risk ( 15 , 22 , 23 ) . further studies are needed to accurately characterize workplace humidity , air movement , radiant temperature , health outcomes , and work performance in various settings in thailand , especially as the workforce structure changes with economic development ( 24 , 25 ) . this study was supported by the international collaborative research grants scheme with joint grants from the wellcome trust uk ( gr0587ma ) and the australian nhmrc ( 268055 ) .

backgroundoccupational heat stress is a well - known problem , particularly in tropical countries , affecting workers , health and well - being . there are very few recent studies that have reported on the effect of heat stress on mental health , or overall health in workers , although socioeconomic development and rapid urbanization in tropical developing countries like thailand create working conditions in which heat stress is likely.objectivethis study is aimed at identifying the relationship between self - reported heat stress and psychological distress , and overall health status in thai workers.results18% of our large national cohort ( > 40,000 subjects ) often works under heat stress conditions and males are exposed to heat stress more often than females . furthermore , working under heat stress conditions is associated with both worse overall health and psychological distress ( adjusted odds ratios ranging from 1.49 to 1.84).conclusionsthis association between occupational heat stress and worse health needs more public health attention and further development on occupational health interventions as climate change increases thailand 's temperatures .

Methodology Data Measurements Data analysis Ethical issues Results Characteristics of the cohort Associations of cohort attributes with heat stress, overall health, and psychological distress Effect of heat stress on overall health and psychological distress Discussion Statement of principal findings Strengths and weaknesses of the study Significance of this study Future research Conflict of interest and funding

the study began with a 20-page mailout questionnaire covering social demography , work , health and injuries , social networks and well - being , diet and physical activity , and tobacco , alcohol , and transport . the information on individual experiences of heat stress was derived from the question : during the last 12 months , how often have you experienced at work high temperatures which make you uncomfortable? for analysis , poor overall health is based on the first question of the medical outcomes short form instrument ( sf8 ) overall how would you rate your health during the past four weeks ( excellent , very good , good , fair , poor , or very poor). psychological distress is based on the three anxiety - oriented questions of the standard kessler 6 psychological distress questions : in the past 4 weeks , about how often did you feel ( nervous , restless or fidgety , or everything was an effort) with answers on a five - point scale ( 1=all of the time , 2=most of the time , 3=some of the time , 4=a little of the time , 5=none of the time ) . all respondents reported age , sex , education , income , location of job , job type , and five other hazards at work ( beside high temperature ) ; data on these variables were collected because they could confound or modify our estimates of the association between overall health , psychological distress , and occupational heat stress . for analysis , age in years was divided into three categories ( 1529 , 3044 , > 45 ) , highest education was divided into three categories ( high school , diploma , or university ) , monthly income ( in thai baht ) was divided into four categories ( up to 7,000, 7,00110,000, 10,00120,000, and 20,001 and more ) , and job location was divided into three categories ( bangkok , urban - outside bangkok , and rural ) . sex , age , job location , job type , education , income , other work hazards ) were assessed as potential confounders of our estimates of heat stress associations on health outcomes by investigating their association with the exposure of interest ( heat stress ) or the outcomes ( overall health or psychological distress ) . both outcomes were assessed graphically for association with heat stress and these analyses were broken down by age group ( 1529 , 3044 , > 45 years ) and sex . the association between heat stress and overall health or psychological distress was assessed by odds ratios ( ors ) and 95% confidence intervals . using multivariable logistic regression , these heat stress ors were adjusted for the influence of all confounding variables ( sex , age , job type , job location , education , income , and complaints of other work hazards ) . finally , using stepwise logistic regression to include only significant confounders and heat stress interaction terms , models were prepared to show the fully adjusted heat stress association on overall health and on psychological distress . the final model for the 40,913 fulltime workers for overall health included heat stress , sex , age , education , other complaints , job location , and two interaction terms ( educationheat , and other complaintsheat ) . the final model for psychological distress included heat stress , sex , age , education , income , other complaints , job location , and an interaction term for other complaintsheat . when job type was offered for the stepwise models and these workers were potentially exposed to heat stress only ( restricted data set , n=24,907 ) , the final model for both poor health and psychological distress included heat stress , sex , age , and job location . the study began with a 20-page mailout questionnaire covering social demography , work , health and injuries , social networks and well - being , diet and physical activity , and tobacco , alcohol , and transport . the information on individual experiences of heat stress was derived from the question : during the last 12 months , how often have you experienced at work high temperatures which make you uncomfortable? for analysis , poor overall health is based on the first question of the medical outcomes short form instrument ( sf8 ) overall how would you rate your health during the past four weeks ( excellent , very good , good , fair , poor , or very poor). psychological distress is based on the three anxiety - oriented questions of the standard kessler 6 psychological distress questions : in the past 4 weeks , about how often did you feel ( nervous , restless or fidgety , or everything was an effort) with answers on a five - point scale ( 1=all of the time , 2=most of the time , 3=some of the time , 4=a little of the time , 5=none of the time ) . all respondents reported age , sex , education , income , location of job , job type , and five other hazards at work ( beside high temperature ) ; data on these variables were collected because they could confound or modify our estimates of the association between overall health , psychological distress , and occupational heat stress . for analysis , age in years was divided into three categories ( 1529 , 3044 , > 45 ) , highest education was divided into three categories ( high school , diploma , or university ) , monthly income ( in thai baht ) was divided into four categories ( up to 7,000, 7,00110,000, 10,00120,000, and 20,001 and more ) , and job location was divided into three categories ( bangkok , urban - outside bangkok , and rural ) . sex , age , job location , job type , education , income , other work hazards ) were assessed as potential confounders of our estimates of heat stress associations on health outcomes by investigating their association with the exposure of interest ( heat stress ) or the outcomes ( overall health or psychological distress ) . both outcomes were assessed graphically for association with heat stress and these analyses were broken down by age group ( 1529 , 3044 , > 45 years ) and sex . the association between heat stress and overall health or psychological distress was assessed by odds ratios ( ors ) and 95% confidence intervals . finally , using stepwise logistic regression to include only significant confounders and heat stress interaction terms , models were prepared to show the fully adjusted heat stress association on overall health and on psychological distress . the final model for the 40,913 fulltime workers for overall health included heat stress , sex , age , education , other complaints , job location , and two interaction terms ( educationheat , and other complaintsheat ) . the final model for psychological distress included heat stress , sex , age , education , income , other complaints , job location , and an interaction term for other complaintsheat . when job type was offered for the stepwise models and these workers were potentially exposed to heat stress only ( restricted data set , n=24,907 ) , the final model for both poor health and psychological distress included heat stress , sex , age , and job location . over 18% ( n=7,476 ) of the analyzed group this problem was worse for males ( 23% ) than females ( 15% ) ; also females more frequently than males reported never experiencing heat stress at work ( 27 vs 17% ) . prevalence of reported poor overall health by age group , sex , and reported occupational heat stress in 24,907 thai workers . reported heat stress at work in a cohort of 40,913 fulltime workers in thailand heat stress exposure together with health and psychological distress outcomes are summarized in table 3 . the prevalence of heat stress ( often experiencing high temperatures at work ( n=40,913 ) ) varied considerably among subgroups and was notably high for those with other complaints regarding hazards at work . nearly 5% of workers reported poor overall health and this figure rose to 9% for those reporting often being troubled by three to five other work hazards . heat stress , poor overall health , and psychological distress by potential confounding factors in a cohort of 40,913 fulltime workers associations with heat stress , poor overall health , and psychological distress each expressed as crude odds ratios ( ors ) . the potential confounders shown in table 3 ( sex , age group , education , income , job type , job location , and other work hazard complaints ) were significantly associated with both the exposure of interest ( heat stress ) and the two outcomes ( overall health and psychological distress ) . accordingly , our analyses of heat stress effects were adjusted for these confounding variables , or restricted to exclude all those respondents who had multiple work hazard complaints . 2 and 3 reveal the positive association between heat stress and poor health or psychological distress stratified by age group and sex . the association between overall health , psychological distress , and occupational heat stress are substantial and revealed no consistent pattern with age . generally , the adverse effects of heat stress on poor health were worse for females ; the opposite association was noted for heat stress and psychological distress , which was especially bad for males aged between 15 and 29 years . prevalence of reported psychological distress by age group , sex , and reported occupational heat stress in 24,907 thai workers . perhaps the most notable interaction is the attenuation of heat stress associations among those with multiple work hazard complaints ; for example , the association of heat stress and overall health ( or=1.75 ) compares to an or of 1.22 for those with three to five complaints . stratified odds ratios revealing interaction of demographic and work - related variables with the association between overall health , psychological distress , and occupational heat stress ( n=40,913 ) number of respondents who reported heat stress at work ( see section methods ) . table 5 shows crude and adjusted or estimates for associations between heat stress and poor health and psychological distress outcomes . for workers who did not complain of other work hazards and for whom job type was reported ( n=24,907 ) , the adjusted heat stress models ors for poor health ( 1.80 ) and psychological distress ( 2.19 ) differ little from previous estimates . the association between occupational heat stress , overall health , and psychological distress adjusted ors derived from heat stress models that included sex , age , income , education , job location , and complaints ( n=40,913 ) plus job type ( n=24,907 ) . final model for estimating the association between heat stress and poor overall health includes all statistically significant confounders ( sex , age , education , complaints , job location ) , and the two significant interaction terms ( educationheat and complaints heat ) . final model for estimating the association between heat stress and psychological distress includes all statistically significant confounders ( sex , age , education , income , complaints , job location ) and the one significant interaction term ( complaintsheat ) . final model in restricted group ( n=24,907 ) for association between heat stress and both poor overall health and psychological distress includes all statistically significant confounders ( sex , age , and job location ) . accordingly , these final models show considerable and consistent strength of association between heat stress and health outcomes , and the confidence limits and p - values show results unlikely to be due to chance . prevalence of reported poor overall health by age group , sex , and reported occupational heat stress in 24,907 thai workers . reported heat stress at work in a cohort of 40,913 fulltime workers in thailand heat stress exposure together with health and psychological distress outcomes are summarized in table 3 . nearly 5% of workers reported poor overall health and this figure rose to 9% for those reporting often being troubled by three to five other work hazards . heat stress , poor overall health , and psychological distress by potential confounding factors in a cohort of 40,913 fulltime workers associations with heat stress , poor overall health , and psychological distress each expressed as crude odds ratios ( ors ) . in almost all instances the potential confounders shown in table 3 ( sex , age group , education , income , job type , job location , and other work hazard complaints ) were significantly associated with both the exposure of interest ( heat stress ) and the two outcomes ( overall health and psychological distress ) . accordingly , our analyses of heat stress effects were adjusted for these confounding variables , or restricted to exclude all those respondents who had multiple work hazard complaints . 2 and 3 reveal the positive association between heat stress and poor health or psychological distress stratified by age group and sex . the association between overall health , psychological distress , and occupational heat stress are substantial and revealed no consistent pattern with age . generally , the adverse effects of heat stress on poor health were worse for females ; the opposite association was noted for heat stress and psychological distress , which was especially bad for males aged between 15 and 29 years . prevalence of reported psychological distress by age group , sex , and reported occupational heat stress in 24,907 thai workers . for both outcomes , the heat stress effects are increased for workers aged more than 45 years and for the better educated , and for work located in rural areas , but none of these interactions are strong . perhaps the most notable interaction is the attenuation of heat stress associations among those with multiple work hazard complaints ; for example , the association of heat stress and overall health ( or=1.75 ) compares to an or of 1.22 for those with three to five complaints . stratified odds ratios revealing interaction of demographic and work - related variables with the association between overall health , psychological distress , and occupational heat stress ( n=40,913 ) number of respondents who reported heat stress at work ( see section odds ratios show within - strata estimates for heat stress associations adjusted for all other explanatory variables . table 5 shows crude and adjusted or estimates for associations between heat stress and poor health and psychological distress outcomes . for workers who did not complain of other work hazards and for whom job type was reported ( n=24,907 ) , the adjusted heat stress models ors for poor health ( 1.80 ) and psychological distress ( 2.19 ) differ little from previous estimates . the association between occupational heat stress , overall health , and psychological distress adjusted ors derived from heat stress models that included sex , age , income , education , job location , and complaints ( n=40,913 ) plus job type ( n=24,907 ) . final model for estimating the association between heat stress and poor overall health includes all statistically significant confounders ( sex , age , education , complaints , job location ) , and the two significant interaction terms ( educationheat and complaints heat ) . final model for estimating the association between heat stress and psychological distress includes all statistically significant confounders ( sex , age , education , income , complaints , job location ) and the one significant interaction term ( complaintsheat ) . final model in restricted group ( n=24,907 ) for association between heat stress and both poor overall health and psychological distress includes all statistically significant confounders ( sex , age , and job location ) . the final logistic regression models included all significant confounders and all significant interactors ( see section the final ors linking heat stress to poor overall health were 1.55 ( n=40,913 ) and 1.81 ( when restricted to those reporting job type and no other work hazard complaints , n=24,907 ) . accordingly , these final models show considerable and consistent strength of association between heat stress and health outcomes , and the confidence limits and p - values show results unlikely to be due to chance . these findings add to the limited literature on the association between overall health , psychological distress , and occupational heat stress of thai workers ( 17 ) . we found heat stress exposure reported by nearly 20% of our large national cohort , more frequently among laborers and male workers , those with low incomes and low education , and declining with age possibly due to an age - related shift away from physical work . occupational heat stress was reported less frequently in bangkok , probably reflecting air conditioning in thai cities , especially in bangkok ( 9 ) . the proportion of male workers who reported poor health and psychological distress declined with age . this was especially noteworthy for psychological distress , possibly reflecting longer work experience and the ability to control emotions among older men . our principal finding is that heat stress was strongly and significantly associated with both poor overall health and psychological distress , with adjusted ors ranging from 1.49 to 1.84 . the strength of association between overall health , psychological distress , and occupational heat stress interacts weakly with an array of other sociodemographic variables . access to a large national cohort of 40,913 thai workers is the main advantage of this study . the cohort shows a wide range of values for the variables of interest , allowing us to investigate the relationship between heat stress at work and health outcomes . however , this study could not directly establish that these overall health problems and psychological distress arose as a result of heat stress . we were able to find only one other study ( an unpublished thesis ) of thermal environment and mental health in thailand , among a small population of 90 workers in a seafood factory ( 17 ) . although , there are reports on occupational heat exposure and comfort of workers , they do not usually mention psychological distress or mental health problems as a variable ( 11 , 14 ) . therefore , our study is one of the first to provide evidence on an association between heat stress and psychological distress of workers . this is the first large - scale study of occupational heat stress and adverse health outcomes in thailand . given the constrained resources in middle - income thailand , and the possibility that psychologically stressful work is becoming more widespread , our results on psychological distress are of particular concern . furthermore , ramsey ( 21 ) reported that heat stress diminished mental ability and increased injury risks , and others have noted increased suicide risk ( 15 , 22 , 23 ) . these findings add to the limited literature on the association between overall health , psychological distress , and occupational heat stress of thai workers ( 17 ) . we found heat stress exposure reported by nearly 20% of our large national cohort , more frequently among laborers and male workers , those with low incomes and low education , and declining with age possibly due to an age - related shift away from physical work . occupational heat stress was reported less frequently in bangkok , probably reflecting air conditioning in thai cities , especially in bangkok ( 9 ) . the proportion of male workers who reported poor health and psychological distress declined with age . our principal finding is that heat stress was strongly and significantly associated with both poor overall health and psychological distress , with adjusted ors ranging from 1.49 to 1.84 . the strength of association between overall health , psychological distress , and occupational heat stress interacts weakly with an array of other sociodemographic variables . access to a large national cohort of 40,913 thai workers is the main advantage of this study . the cohort shows a wide range of values for the variables of interest , allowing us to investigate the relationship between heat stress at work and health outcomes . however , this study could not directly establish that these overall health problems and psychological distress arose as a result of heat stress . this factory study did not document heat stress ( but did measure temperature ) ; nor did it find evidence of widespread psychological distress . although , there are reports on occupational heat exposure and comfort of workers , they do not usually mention psychological distress or mental health problems as a variable ( 11 , 14 ) . therefore , our study is one of the first to provide evidence on an association between heat stress and psychological distress of workers . this is the first large - scale study of occupational heat stress and adverse health outcomes in thailand . the findings of widespread heat stress and associated ill - health are disturbing . if workers exposed to excessive heat can not cool down , they can experience severe psychological distress caused by heat - related exhaustion or develop long - term conditions such as chronic depression or chronic anxiety disorders ( 11 , 20 ) . furthermore , ramsey ( 21 ) reported that heat stress diminished mental ability and increased injury risks , and others have noted increased suicide risk ( 15 , 22 , 23 ) .

functional connectivity ( fc ) , broadly defined , is correlations between remote neurophysiological events . there are a number of approaches for the detection of fc from blood - oxygen level dependent ( bold ) signals measured by functional magnetic resonance imaging ( fmri ) of the brain . a commonly implemented approach is a seed - based approach that can be applied with a general linear model ( glm ) using time course regressors derived from selected brain regions to find other brain regions having correlated bold signal activity patterns [ 2 , 3 ] . another commonly used data - driven approach is independent component analysis ( ica ) , which derives spatiotemporal components ( pairs of spatial maps and associated time courses ) through blind signal source separation and linear decomposition of fmri data [ 4 , 5 ] . independent component ( ic ) spatial maps represent constellations of brain regions that are partially synchronized with the corresponding component time courses . ica in this article is synonymous with spatial ica , the most common implementation of ica used with fmri data , which allows components to be temporally correlated , but restricts them to be spatially independent . both the seed - based approach and ica have produced similar fc maps in experiments where no experimentally - derived time course was available ( e.g. , resting - state ) [ 68 ] . both approaches have produced pairs of spatial maps and associated time courses comparable to those produced by standard glm techniques that regress fmri data against experimentally derived a priori hemodynamic response functions , in experiments driven by task activities with known time courses [ 917 ] . the results derived with ica are sometimes more consistent with expected bold response patterns than those derived with the a priori , task - related approach , especially for transient task - related bold responses or responses whose time courses are difficult to predict beforehand [ 4 , 1821 ] . ica does not require an a priori time course or choice of seed region , making it ideal for exploratory studies . in some cases , ica can be more sensitive , specific , and accurate in showing functionally connected regions than seed - based approaches [ 22 , 23 ] , as we demonstrate with an example ( experiment 1 ) . despite potential advantages of ica over task - related or seed - based glm approaches in visualizing correlated brain activity , ica reproducibility issues may complicate or limit ica 's use in the generation of fc maps to be used for group comparison . temporally correlated ics can sometimes be expressed as a single component [ 4 , 24 , 25 ] , and vice versa . iterative ica algorithms that use a random seed can produce different sets of components on separate analyses performed on exactly the same data [ 27 , 28 ] . from one ica to the next such components can appear to be modified , split into two components , or to disappear such variations in ics also occur during bootstrapping , where individual icas are performed on separate portions of a dataset involving similar tasks [ 27 , 28 ] , and when analyzing separate subjects performing the same tasks . ica 's variability in parsing fmri data variance into separate ics can complicate comparison of an ic spatial map generated from one subject 's ( or session 's ) data to similar ic spatial maps generated from other subjects ' ( or sessions ' ) data , because the signal sources represented by each ic in any group of relatively homologous ics ( ics with closely matching spatial patterns across subjects / sessions ) may not exactly match the signal sources represented by the other ics in the group . addressing these ica reproducibility issues is a subject of ongoing research [ 12 , 16 , 31 , 32 ] . some of these reproducibility issues may be avoided by comparing fc spatial maps generated through seed - based methods . such methods usually select seed brain regions based on information derived from theoretical considerations , task - related glm studies [ 3436 ] , or ica studies . deriving seed information from ica can be considered a hybrid ica - seed - based approach where the exploratory power of ica is utilized to select a seed from a huge number of possible choices . literature on the subject of the potential benefits and limitations of such an approach is scarce . however , other ica - based approaches for addressing these reproducibility issues have been described [ 12 , 26 , 31 , 37 , 38 ] . two of the most widely implemented of these approaches that can be applied in a data - driven fashion , without a priori time courses , or any other prior knowledge from an experimental paradigm , are the back - reconstruction [ 12 , 37 ] and dual - regression [ 31 , 39 ] methods . both methods use data from a group ica performed on the temporally concatenated fmri datasets of all subjects / sessions in a group as the source of data to derive individual subject / session spatial maps that can then be compared to draw inferences about individual or subgroup differences . here , we report the results of comparing five hybrid ica - seed - based methods to each other and to these two methods , using standards of comparison derived from knowledge of visuomotor experimental paradigms involved during collection of fmri data , and from results of fmri analyses with task - related glm methods ( i.e. , glm applied using time course regressors derived from the experimental paradigms ) . the glm is often expressed in matrix notation as [ 3 , 20 , 4043 ] ( 1)x = g+ , where x is a t v matrix ( of fmri data after preprocessing ) containing one row for each point in time ( volume acquisition ) and one column for each point in space ( voxel ) . each row represents the spatial map ( bold signal at each voxel ) of a volume of data ( t = number of volumes ) , and each column represents the time course for a single voxel ( v = number of voxels ) . g is a t s design matrix where each column contains the hypothesized time course of the bold response for a signal of interest ( s = number of signals of interest ) or for a covariate of no interest . is an s v matrix where each row contains a spatial map of parameters to be estimated ( one parameter for each voxel ) corresponding to one of the time courses ( the ith row of corresponds to the ith column of g ) . the parameters in can be estimated through standard , least - square linear regression techniques , minimizing the sums ( across all time points ) of the squared residual error terms for each voxel . these maps of parameter estimates ( with other information , such as the parameter estimate standard errors ) can be used for hypothesis testing of each time course of g in various regions of the brain . they can also provide the basis for group comparison statistics , derived from comparing the spatial maps for different conditions or different groups of subjects . the spatial maps of in ( 1 ) are uniquely determined by least - square linear regression if the time courses in the design matrix g are completely specified and none of the time courses are colinear . similarly , the time courses in g can be uniquely determined through glm by regressing the fmri data against completely specified , non - colinear spatial maps in . this relationship is readily apparent if we convert ( 1 ) to an equivalent form by transposing matrices on both sides of the equation and apply the identities ( a + b ) = a + b and ( ab ) = ba to yield ( 2)xt=tgt+t . least - square linear regression to determine the time courses in g proceeds in the same manner as for determination of the spatial maps of in ( 1 ) , except that the sums ( across all voxels ) of the squared residual error terms for each time point are minimized to determine g. ica , like glm , is based on a linear model . for a general case , one may consider including an error term in a standard ica model , like the model used in probabilistic independent component analysis ( pica ) . in this study , we use this form of ica model , where the relationships among the preprocessed fmri data , spatial maps , and time courses can be expressed as ( 3)x = mc+e . x here is defined in the same manner as for ( 1 ) and represents the same data , while m , c , and e are matrices that represent time courses , spatial maps , and error terms in the same format as for g , , and , but that generally do not contain the same matrix values , and the number of time courses and associated spatial maps in m and c may differ from the number in g and . the entries in e are error terms that represent predominately random , gaussian noise . they are derived using principle component analysis ( pca ) with dimensionality reduction to separate the data into e and a portion that represents predominately neural signals and structured , nonrandom noise ( mc ) . ica then solves for both m and c simultaneously using only the fmri data ( minus e ) and some assumptions concerning the form that the solution should take ( e.g. , nonrandom components whose spatial maps are statistically independent ) . in this article we broadly define seed - based fc as the result of a two - step process . the first step derives one or more time courses from one or more seeds ( spatial maps or portions thereof ) and the fmri data being analyzed . the second step regresses ( glm ) the fmri data against the time courses to derive corresponding fc spatial maps . a simple case of seed - based fc is region - of - interest - based ( roi - based ) fc derived by averaging the fmri data time courses of each voxel within a predefined roi ( the seed ) to produce a time course against which the fmri data is regressed to yield a single fc spatial map . a more complex case of seed - based fc involves dual regression , a process where fmri data is regressed against a set of predefined spatial maps to derive a set of time courses against which the fmri data is regressed once more to produce a corresponding set of fc spatial maps . if the predefined spatial maps consist of some spatial maps of interest and some nuisance covariate maps of no interest , then the corresponding time courses and fc maps can also be divided into those of interest and those of no interest . five experiments were performed to evaluate the performance of the five hybrid ica - seed - based fc methods . experiment 1 illustrated the potential advantages of a hybrid ica - seed - based method over a seed - based method that does not utilize information from ica , using natural data from an fmri run , with artificial data added . experiment 2 tested the performance of the five hybrid methods , comparing results to those obtained from task - related glm , using six runs of data from a single participant . experiment 3 tested the performance of the five hybrid methods , comparing results to those obtained from task - related glm and two methods for extracting information about individual subjects , based on group ica . finally , in experiments 4 and 5 the effects that ica dimensionality might have on the hybrid methods were evaluated by repeating experiment 3 using a smaller ( 5 ) and larger ( 30 ) number of ics ( instead of 14 ) . a right - handed adult male volunteer was recruited after informed consent , from among students in an fmri lab course at columbia university . handedness was determined by the laterality quotient ( + 100 ) from the edinburgh handedness inventory [ 44 , 45 ] . 14 healthy , right - handed adults were recruited in accordance with institutional guidelines approved by the medical college of wisconsin and the university of new mexico school of medicine . fmri data were collected from an experiment originally intended as a pilot study of attachment theory [ 4648 ] . the participant provided digitized ( electronic ) photos of a male friend , a female friend , photos of his parents taken during his childhood , and recent parental photos . to these six photos were added a photo of a famous person similar in appearance to each of the young parental images and a photo of a complete stranger similar to each of the older parental photos . the participant was briefly shown the images in advance of the experiment to ensure recognition of the famous faces and lack of recognition of the strangers . the photo images were processed using adobe photoshop elements ( version 2.0 ) to make them as similar as possible in appearance apart from the forms of the faces . background scenes were eliminated and replaced with a dark grey background , making a smooth transition in brightness toward the edges of the face image to avoid sharp boundaries at the periphery of the faces . photos were converted from color to grayscale and adjusted for brightness , contrast , head size , and blurriness of image ( making sharper photos grainier and blurrier ) . the facial images were cropped from the chin up , leaving enough room at the top to ensure that the bridges of the nose were vertically at the center of each image . the width of each image was cropped to whatever length was necessary to show the face from ear to ear . strangers were represented by the words a woman or a man . word images of the names were generated by presentation software ( version 10.2 ) , adjusted in size so that the longest names ( rita hayworth and frank sinatra ) took up half of the full width of the image display , the same as for the widest photos . the heights of capital letters were approximately 10 times smaller vertically than the photos . the word images were colored either blue or green and shown on a medium - gray background . the photos were shown on the same background and colored either blue - gray or green - gray using 30% opacity . the 10 photos and names in two different colors yielded a total of 40 images ( figure 1 ) , which were each shown in pseudorandomized order exactly once during each of the six runs . the pictures were each presented for an average of 9.0 seconds ( random durations of 7.510.5 seconds ) , with a blank , medium - gray screen shown between images for 0.5 seconds , yielding an average run time of 380 seconds . he was given ear plugs , had his forehead taped down , and was equipped with a button - box while in the scanner . in order to maximize attention and brain activity and minimize sources of data noise , the subject was instructed to be as still as possible ( avoiding swallowing ) during fmri runs , focusing eyes on the center of the screen at all times ( trying to avoid blinking during image presentation ) , and to press one button for blue images and another for green images as soon as he was sure of the color of the image . he was asked to concentrate as continuously and exclusively as possible on the person corresponding to each image as it was being shown , and to imagine that he was greeting the person in a usual setting where he might see the individual ( e.g. , for famous people , on television ) . fmri data were used from a study of the association between brain hemispheric lateralization patterns and motor task complexity . the publicly available data was downloaded from the fmri data center ( http://www.fmridc.org/ , accession number 2 - 2003 - 114e5 ) . eight runs were collected in that study , with each run involving either a complex or a simple motor task , and either the left or right hand ; two runs were collected for each of these four cases . each block involved 12 seconds rest followed by 4 , 3-second trials where the participants were shown sequences of five digits ( 1 , 2 , or 3 ) and responded by pressing buttons corresponding to the sequence shown ( 1 = index finger , 2 = middle finger , and 3 = ring finger ) . the complex condition consisted of heterogeneous sequences , which always used three fingers and four transitions , whereas the simple condition involved repetition of the same digit in each trial . the digits were displayed for 2.5 seconds , and approximately 2 seconds were required for participants to manually enter the digits . we only used data from the first run of the complex condition performed with the right hand , for the following reasons . first , we chose only one run in order to minimize memory requirements on our 32-bit architecture computers . second , we chose the complex condition rather than the simple condition because the complex condition involved use of each of the three fingers in every trial , thereby providing a more even use of the corresponding brain regions during the latter half of each block . third , we chose the first run rather than the second run , because we reasoned that the effects of fatigue would probably make the brain 's response to the task weaker during the second run , but this choice was somewhat arbitrary . finally , we chose the right hand rather than left , because a quick scan and processing of the data with ica and glm for the first two subjects revealed that the activation in response to use of the right hand appeared to be stronger than in response to use of the left hand . images were acquired using a 1.5 t general electric ( ge ) twinspeed mri scanner . functional scans were performed using epi - bold ( tr = 2000 ms ; te = 38 ms ; flip angle = 90 degrees ; fov = 19.2 cm ; 64 64 matrix ; 29 oblique - axial slices 4.5 mm thick ; skip 0 mm ; interleaved acquisition ; voxel size = 3 3 4.5 mm ) . immediately after the functional scans , a 10-minute , high resolution , t1-weighted structural mri image was acquired using the 3d spgr sequence ( 186 slices ; 256 256 ; fov = 256 mm ; voxel size = 1 1 1 mm ) . echo - planar ( ep ) images were collected using a single - shot , blipped , gradient - echo ep pulse sequence ( tr = 4000 ms ; te = 40 msec ; fov = 24 cm ; 64 64 matrix ; 22 contiguous sagittal 6-mm thick slices ; voxel size = 3.75 3.75 6 mm ) . prior to functional imaging , high - resolution 3d spoiled gradient - recalled at steady - state anatomic images were collected : tr = 24 msec , te = 5 msec , flip angle = 40 degrees , number of excitations = 1 , slice thickness = 1.2 mm , fov = 24 cm , and resolution = 256 128 . fmri image preprocessing for all experiments was carried out using fsl ( version 4.1 ) , fmrib 's software library ( http://www.fmrib.ox.ac.uk/fsl ) [ 50 , 51 ] , involving nonbrain removal using bet ; motion correction with mcflirt ; slice - timing correction for interleaved acquisitions using fourier - space time - series phase - shifting ; highpass temporal filtering using gaussian - weighted least - squares straight line fitting ( with = 27.5 seconds for experiments 1 and 2 , and = 50 seconds for experiments 35 ) ; spatial smoothing using a gaussian kernel with full - width half - maximum 8 mm ; coregistration to high - resolution t1-weighted images ; and normalization of all images to standard space ( mni , montreal neurological institute atlas , using resolutions of 3 3 3 mm for experiments 1 and 2 , and 4 4 4 mm for experiments 35 , which were the smallest voxel sizes that did not result in memory overflow errors while performing group icas ) with affine registration , fsl flirt [ 53 , 54 ] . icas were performed using pica as implemented in fsl 's melodic ( multivariate exploratory linear decomposition into independent components ) version 3.09 , which can be considered a variant of fastica where components are estimated simultaneously rather than one at a time . this process involved masking of nonbrain voxels , voxelwise demeaning of the data , normalization of the voxel - wise variance , whitening , projection into an n - dimensional subspace using pca , and decomposition into n time courses and corresponding spatial maps ( spatiotemporal ica components ) by optimizing for non - gaussian spatial source distributions using a fixed - point iteration technique . n was estimated using the fsl default , laplace approximation [ 11 , 56 ] . estimated component maps were divided by the standard deviation of the residual noise and thresholded by fitting a mixture model to the histogram of intensity values . for experiment 1 , ic spatial maps were thresholded conservatively , using a mixture model and alternative hypothesis testing approach , with the mmthresh parameter set to 0.95 . for experiment 2 , the fsl default value of mmthresh = 0.5 was used . ics produced with pica can vary , even if repeated with identical fmri data , because pica is an iterative approach based on a random seed . for this reason , in experiments 13 we repeated pica 10 times every time it was used , so that we could arrive at a best estimate by averaging the 10 sets of component spatial maps and time courses in the series . however , we found that for experiments 13 , in all 10 cases pica produced component spatial maps and time courses that from visual inspection appeared to be identical . an examination of the exact time course values revealed that in most cases they were identical , and when they were not , in all cases they differed from the first pica results in the series by less than 3 10 ( for time courses normalized to zero mean and unit variance ) , a figure comparable to rounding error . for this reason , rather than averaging , we used the results from the first pica in each series of 10 . the reason we encountered negligible differences among members of each series of 10 may have been that we chose a very small value ( 10 ) of epsilon , the minimum error change parameter used to determine at what point the ica algorithm has converged on a single solution . at this setting of epsilon , convergence generally required relatively few steps ( less than 300 ) , and allowing many steps did not help in cases of nonconvergence ; so we used the default maximum number of iterations before restart ( 500 ) . group ica was performed with melodic , using parameters as for experiments 1 and 2 , with the multisubject temporal concatenation option , which performed pica on the normalized data from all 14 participants , temporally concatenated into a single group fmri run . some or all of the group ics were the seed source for the five hybrid ica - seed - based approaches . in addition , a group ica was performed using the group ica of fmri toolbox ( gift , v1.3 g ) implemented in matlab ( http://icatb.sourceforge.net/ ) , using two data - reduction steps . this group ica approach , described in , like pica began with subjectwise , temporal concatenation of the fmri data , followed by whitening and projection into an n - dimensional subspace using pca . n was specified to be the same as the number of components that were estimated for the pica group ica ( derived using the laplace method ) . group ics were then generated using infomax ( the gift default ) , a neural network algorithm that attempts to minimize the mutual information of the network outputs [ 12 , 30 , 57 ] . finally , individual - subject spatial maps and time courses corresponding to each group ic were derived using the regular back - reconstruction method . z - score scaling was applied to the group and individual - subject spatial maps , normalizing them to zero mean and unit variance . these maps were thresholded for figure display at z > 3.1 ( one - tailed p < .001 ) as in [ 12 , 58 ] . individual - subject spatial maps not derived directly from ica were derived with glm by regressing fmri data against one or more time courses using fsl feat with film local autocorrelation correction to generate gaussianised , z - statistic images reflecting parameter estimates in relation to their standard errors and degrees of freedom . the time course for each task - related glm analysis was generated by convolving an experimentally derived time course against a gaussian function with peak lag = 5 seconds and = 2.8 seconds . time courses derived from seed - based approaches were not modified . for experiments 1 , 3 , 4 , and 5 , z - statistic images were processed by maximum - height thresholding voxels based on gaussian random field theory ( grft ) to a corrected , one - tailed significance of p .05 . for experiment 2 , z - statistic images were thresholded by cluster size determined by counting contiguous ( 26-neighbor ) voxels with p < .01 ( one - tailed , z > 2.3 ) and applying grft to determine a corrected cluster size threshold corresponding to one - tailed significance of p .05 . we chose the more conservative voxel - thresholding approach for experiments 1 , 3 , 4 , and 5 because we judged that tightly controlling false positives would be more suitable for the visual - inspection - based comparisons of experiment 1 and the assessments of receiver operating characteristics of experiments 35 . we chose the more sensitive cluster - based thresholding for experiment 2 in order to better visualize activation in the right temporal occipital fusiform cortex ( rtof ) , where the fusiform face area ( ffa ) is located , a region commonly involved during experimental tasks with imagined or perceived images of faces [ 6163 ] . such activation was present in the rtof for task - related glm analyses in 5/6 runs with cluster - based thresholding , compared with only 3/6 runs with voxel - based thresholding . task - related glm was applied to the preprocessed fmri data from run 1 ( figure 2(a ) ) , and the location ( mni coordinates 21 , 93 , 9 ) of the voxel with the highest z - score ( 10.6 ) was determined . artificial signal was added to the axial slice containing that voxel , creating an artificial component whose variance was comparable to the variance of the natural occipital component ( figure 2(b ) ) . the artificial signal was added to the voxel at coordinates 21 , 93 , 9 in addition to all voxels in three large triangular regions outside of the occipital cortex ( figure 2(c ) ) . the artificial signal consisted of a square wave with a period of 60 seconds ( 30 volumes ) and amplitude at each voxel corresponding to 0.5% of the mean mr signal intensity for the voxel over time . adding the artificial signal before fmri preprocessing would have better simulated neural signal , but we added it after preprocessing to sharpen image boundaries for the illustrative purposes of this experiment . the voxel at coordinates 21 , 93 , 9 was chosen as the seed to produce an roi - based fc map containing both the natural and artificial components ( figure 2(d ) ) . we repeated this seed - based fc analysis twice more ( figures 2(e ) and 2(f ) ) , choosing the voxel in the z = 9 slice with the highest z - score from the natural ( coordinates 12 , 99 , 9 ) and artificial ( coordinates 30 , 9 , 9 ) ic spatial maps . results were compared with visual inspection , and statistics were gathered on sensitivity and specificity for correctly identifying voxels where artificial signal had been added . we compared the results of task - based glm to the results of five hybrid ica - seed - based fc methods using preprocessed fmri data without added artificial signal . the seed source for each method was the collection of ic spatial maps ( 13 in all ) derived from ica of run 3 's fmri data . the spatial map and time course of the last ( ranked in descending order of explained variance ) of these components ( ic13 ) resembled the spatial map and time course derived from task - related glm ( figure 3(a ) ) . run 3 was chosen because it was the run whose task - related glm analysis had the highest z - score ( 11.9 ) in any voxel : we hypothesized that run 3 might therefore be most representative of the activation pattern we were studying . the first hybrid method ( single - voxel seed , sv ) used the voxel ( coordinates 12 , 102 , 3 ) in ic13 with the highest z - score ( 18.27 ) as the seed for roi - based fc . thus , fc maps were derived for each of the five target runs ( i.e. , nonseed runs 1 , 2 , 4 , 5 , and 6 ) by regressing their fmri data against their seed voxel time course ( i.e. , the time course of the voxel at coordinates 12 , 102 , 3 for the run being analyzed ) . the second hybrid method ( few - voxel seed , fv ) derived roi - based fc using the same procedure , but the regressor for each run was the average time course over a few seed voxels . those were the voxels ( five in all ) whose z - score was higher than 17.27 , one less than the highest z - score . we thresholded by z - score rather than arbitrarily choosing the number of voxels to be used . the third hybrid method ( many - voxel seed , mv ) derived roi - based fc using as the regressor the average time course over many seed voxels , chosen as close to 100 in number as possible by thresholding with whole z - scores . a threshold of z > 13 yielded 118 voxels . the fourth hybrid method ( dual regression with single - ic seed , drs ) used ic13 's spatial map as the seed for dual - regression - based fc . the fmri data for each of the target runs was regressed against ic13 's spatial map using fsl 's dual_regression program ( beta version 0.3 ) to yield a time course , against which the fmri data was regressed to produce an fc map for each run . the fifth hybrid method ( dual regression with all ics as the seed source , dra ) used all 13 ics as the seed for dual - regression - based fc , using the same procedure as for the fourth method , to yield time courses against which the fmri data was regressed to produce corresponding fc maps for each target run . each hybrid method was evaluated qualitatively through visual inspection of all spatial maps and time courses generated in this experiment and quantitatively according to the following criteria . fc map correlation with task - related glm spatial map from same run > 0 : we did not assume that a successful hybrid method fc map would correlate highly with the task - related glm map from the same run because the correlations of the glm maps from the six runs with each other were in the range 0.130.45 , and differences in latency of hemodynamic responses [ 64 , 65 ] could potentially result in considerable differences between the fc maps and task - related glm maps . however , we did expect the spatial maps to be similar in the occipital cortex , the rtof , and possibly other brain regions ; so we expected the maps to be at least somewhat correlated . fsl 's fslcc utility was used to calculate the correlation coefficient between the fc spatial map and task - related glm spatial map for each of the five target runs and the mean correlation was compared to zero with a two - tailed , one - sample t - test . all correlation values in this study were fisher z - transformed before statistical testing and calculation of confidence intervals . fc map correlation with task - related glm spatial map from same run > fc map correlation with task - related glm spatial map from seed run : for hybrid ica - seed - based methods to be considered successful , the resulting fc maps should be unique to the fmri data being regressed rather than merely a reflection of the seed source spatial maps or fmri data . this criterion was a test of this expectation . however , we expected the hybrid fc maps from target runs to correlate with the task - related glm spatial map from the seed run because the latter was correlated with the task - related glm spatial maps from target runs ( correlation coefficients ranging from 0.25 to 0.44 ) . nonetheless , we expected the hybrid fc maps to be more strongly correlated with the task - related glm maps from the same run than from the seed run . we tested this criterion for each hybrid method with a two - tailed , paired two - sample t - test , comparing the mean correlation between hybrid fc maps and the task - related glm spatial map from the corresponding run to the mean correlation between fc maps and the task - related glm spatial map from the seed run . fc map correlation with task - related glm spatial map from same run a comparison of each method 's mean correlation with that of the other four hybrid methods : depending upon the results of a one - way repeated - measures anova omnibus test of differences in mean correlations ( = 0.05 ) , paired t - tests ( two - tailed ) were performed to test for individual differences in means between methods . a bonferroni correction for multiple comparisons with = 0.05 was applied ( significance at p < .005 for each of the 10 method pairs compared ) . overlap of thresholded voxels from fc map with task - related glm spatial map from same subject a comparison of each method 's overlap across runs with the other four methods of deriving individual - subject fc maps : we examined the percentage of the thresholded voxels in the task - related glm map that were also thresholded in the corresponding hybrid fc map . before making this comparison , we eliminated the effects that the thresholding level might have in making this comparison by adjusting the thresholding z - scores of the fc maps so that each map had the same number of thresholded voxels as the task - related glm map from the same run . this adjustment was equivalent to making the number of false negatives ( negative voxels falling within the thresholded task - related glm map ) equal to the number of false positives ( positive voxels falling outside of the thresholded task - related glm map ) . we then compared the percentage overlap for each method to the other four methods , across the six runs , using the wilcoxon signed rank test ( two - tailed ) . this test was not sufficiently powered after correction for multiple comparisons , but we included it to provide a qualitative comparison with the results of experiments 35 . comparable sensitivity to task - related glm in detecting activation in the rtof : the rtof was the target area for this sensitivity criterion for two reasons . the first reason was that an increase in activity of portions of the rtof ( and some adjacent brain regions ) , the ffa , has been reported in response to a wide variety of stimuli involving real or imagined images of faces [ 6163 , 66 , 67 ] . the exact locations of these portions of the rtof have varied across subjects and have varied depending upon the context of facial presentation ( e.g. , unfamiliar versus famous faces ) [ 61 , 6870 ] . we did not know the exact location of the ffa in our data because no ffa localizer scan had been performed , but we expected some activation in the rtof in response to viewing faces . for this reason we liberally defined our criterion for success to be any thresholded voxels found within the rtof , defined as voxels whose probability of belonging to the rtof was 0.5 according to the harvard - oxford cortical structural atlas ( hocsa ) provided with fsl . by this criterion , the task - related glm method was successful in 4/5 target runs ( no activation was found for run 5 , not shown ) . the low level of activation in the rtof compared with other portions of the occipital cortex was the second reason for targeting the rtof : we wanted a test that could distinguish task - related glm from methods with poorer sensitivity . we expected that less sensitive imaging methods would not show any activation . in the rtof because such activation was barely detectable with task - related glm . we also reasoned that this test might reveal potential weaknesses with the hybrid approaches because the rtof was not in close proximity to the regions with the highest z - scores in the glm- and ica - derived spatial maps , and therefore the hybrid approaches could not benefit from the effects of smoothing or a tendency for activation patterns to be clustered , making voxel activation patterns similar to those of neighboring voxels . in order to test for statistically significant differences , we compared task - related glm results with those from each of the hybrid methods using a two - tailed wilcoxon signed - rank test with = 0.05 . due to the small sample size , the only condition for which a statistically significant difference between task - related glm and hybrid methods could have been detected would have been the case where a hybrid method did not find rtof activation in any of the five target runs , and only if we did not correct for multiple comparisons ( exploratory study ) . we compared the results of task - based glm to the results of seven methods of deriving individual - subject fc maps from a group ica . the group ics were derived with pica ( fsl ) for the first five methods , and with infomax ( gift ) for the other two . the group ic of interest for each of these methods was selected with visual inspection by comparing group ic spatial maps with the group task - related spatial map ( figure 6(a ) ) derived from multilevel linear modeling with fsl flame ( fmrib 's local analysis of mixed effects ) [ 71 , 72 ] performed on the lower - level , task - related glm results from all 14 participants ( i.e. , a random - effects analysis ) . for pica , the second of 14 ics ranked by ic variance ( pica ic2 , figure 6(b ) ) was chosen ; for infomax , the fourteenth ( infomax ic14 , figure 6(c ) ) . the spatial maps of these ics were similar in appearance and highly correlated ( r = 0.61 ) . the first five methods were the same as the hybrid ica - seed - based methods tested in experiment 2 ( i.e. , roi - based : single - voxel , few - voxels , and many voxels ; dual - regression - based : single ic map and all ic maps ) , using the pica group ics as the seed source . for the first method ( sv ) , the seed voxel was located in a region that according to the hocsa probably corresponded to the left precentral ( 40% probability ) or postcentral gyrus ( 19% ) , at mni coordinates 38 , 22 , 60 . for the second hybrid method ( fv ) , thresholding at z = 12.69 yielded 8 more voxels , contiguous with the first voxel . for the third hybrid method ( mv ) , we used the target seed volume from experiment 2 ( 100 voxels 27 l ) rather than the target number of voxels ( 100 ) because we did not wish the seed volume to become disproportionately large . thresholding at z = 11 yielded 40 voxels ( closest to the target of 42 ) , which were also contiguous . the fourth hybrid method ( drs ) used pica ic2 's spatial map as the seed for dual - regression - based fc . the fifth hybrid method ( dra ) used all 14 pica group ics as the seed for dual - regression - based fc , choosing pica ic2 's spatial map as the fc map of interest . the fifth method as implemented in this experiment is the same as the multisubject ica and dual regression approach described in , which can be considered a special case of dra where the seed ics are taken from a group ica performed on the same data being analyzed with the hybrid method . the sixth method ( back - reconstruction with infomax , br - i ) derived the individual - subject ics using the back - reconstruction method . the seventh method ( drs with infomax , drs - i ) was added to evaluate what effect the choice of method for derivation of group ics might have on the resulting fc spatial maps and time courses . this method used dual regression based on a single ic map selected from the group ics derived with infomax . we chose the dual - regression , single - map method because overall the fourth method produced spatial maps that most closely resembled the task - based glm - derived spatial maps . each of the seven methods for deriving fc maps from group ics was evaluated qualitatively through visual inspection of all spatial maps and time courses generated in this experiment and quantitatively according to the following criteria . fc map correlation with task - related glm spatial map from same subject > 0 : the same test as described for experiment 2 , but performed for each method across the 14 subjects . fc map correlation with task - related glm spatial map from same subject > fc map correlation with group ic map of interest ( spatial prior ) : the same test as described for experiment 2 , but performed for each method across the 14 subjects , using the group ic map ( from pica for the first five methods , and infomax for the other two methods ) . fc map correlation with task - related glm spatial map from same subject a comparison of each method 's mean correlation with that of the other six fc methods : depending upon the results of a one - way repeated - measures anova omnibus test of differences in mean correlations ( = 0.05 ) , two - tailed , paired t - tests were performed to test for individual differences in means between methods . a bonferroni correction for multiple comparisons with = 0.05 was applied ( significance at p < .0024 for each of the 21 method pairs compared ) . overlap of thresholded voxels from fc map with task - related glm spatial map from same subject a comparison of each method 's overlap across subjects with the other six methods of deriving individual - subject fc maps : the same test as described for experiment 2 , but performed for each method across the 14 subjects . a bonferroni correction for multiple comparisons with = 0.05 was applied ( significance at p < .0024 for each of the 21 method pairs compared ) . roc pauc from fc maps , using task - related glm spatial maps as the standards of comparison : for each subject and method evaluated , the partial area under the curve ( pauc ) of the receiver operating characteristic ( roc ) of the fc maps was calculated with fsl 's fslmaths program . the task - related , glm - derived map for each subject was used as the true standard of comparison . we chose for our pauc the area between 0 and the largest false positive rate ( lfpr ) that we could accept , as in , because we did not wish to include area under portions of the roc curve that would not be utilized in practice . we accepted the default suggested value for lfpr from the fslmaths program because false positive rates above this number ( 0.05 ) corresponded to the ( unacceptable ) situation where the number of false positives exceeded the total number of thresholded voxels in the reference maps ( i.e. , the number of thresholded voxels in the reference task - related glm spatial maps in almost all cases was below 5% of the total number of voxels in the brain ) . we compared the pauc values for each method to the other 6 methods , across the 14 runs , using the wilcoxon signed rank test ( two - tailed ) . we wanted to evaluate how the hybrid methods might perform using a considerably different number of ics . to do so , we repeated the 5 evaluations from experiment 3 for the 5 hybrid methods only , using seed group ics generated with pica that was set to produce only 5 ics from the group data instead of 14 ( the second of which was selected as the component of interest ) . we used the same number of seed voxels for the sv , fv , and mv methods as were used for experiment 3 . there is no consensus on how to select the optimal number of components , but some suggested methods for specifying dimensionality include the laplace method , bayesian information criterion ( bic ) , minimum description length ( mdl ) , and akaike 's information criterion ( aic ) [ 11 , 12 , 16 ] . we used each of these methods to estimate the number of components ( through options in fsl melodic ) , resulting in dimensionalities of 11 ( laplace ) , 11 ( aic ) , 6 ( mdl ) , and 5 ( bic ) . we chose the smallest dimensionality because we had already explored the highest number of components in using the laplace option . the reason we ended up with 14 components instead of 11 is because melodic only rarely converged on a solution using a dimension of 11 ; so we accepted the melodic default alternative suggestion of 14 . we repeated experiment 4 using a much higher ica dimensionality than was used in experiments 3 and 4 , to explore those effects that high dimensionality might have on hybrid method results . ica generated 17 components from run 1 . upon visual inspection , one spatial map ( figure 2(b ) ) resembled the natural visual cortex component from glm and one ( figure 2(c ) ) resembled the artificial component . the time course of the natural component ( figure 2(b ) ) resembled the time course of the voxel with the highest z - score from the task - related glm time course ( figure 2(a ) ) , and the square wave pattern of the artificial signal was evident in the time course of the artificial component ( figure 2(c ) ) , despite the obvious presence of a considerable amount of noise . the natural component represented 2.46% of the total variance in the fmri data ( ranked 13th among components ) , and the artificial component represented 2.81% of the total variance ( ranked 8th ) . thresholded voxels of the artificial component spatial map corresponded to the voxels where signal had been added , with 100% sensitivity and specificity . the roi - based fc map ( figure 2(d ) ) derived using the voxel at mni coordinates 21 , 93 , 9 as the seed contained thresholded voxels corresponding to both the natural and artificial components . only 19 of the 984 voxels where artificial signal had been added were not represented in the map , corresponding to a sensitivity of 98.1% . the corresponding time course appeared to be somewhat similar to the time courses of both the natural and artificial components . it was not possible to discern the unique relationship that existed among the voxels involved with the natural component , or the unique relationship that existed among the voxels involved with the artificial component , because the spatial maps and time courses corresponding to the natural and artificial components were represented together in a single spatial map and time course . the roi - based fc map ( figure 2(e ) ) derived using the voxel with the highest z - score in the natural component spatial map showed robust activation in the occipital cortex without any activation in the voxels from the artificial component , except for the voxel at coordinates 21 , 93 , 9 . the corresponding time course appeared very similar to the time course of both the voxel with the highest z - score from the task - related glm ( figure 2(a ) ) and the natural component ( figure 2(b ) ) . similarly , the roi - based fc map ( figure 2(f ) ) derived using the voxel with the highest z - score in the artificial component spatial map identified all of the voxels where artificial signal had been added ( 100% sensitivity ) , while only 10 of the whole brain 's remaining 90,295 voxels were also thresholded ( 99.99% specificity ) . the corresponding time course was similar to the square wave pattern of the artificial signal . thus , for these two cases the hybrid ica - seed - based approaches resulted in spatial maps and time courses that were very similar to the seed ic spatial maps . it was possible to discern the two components as separate entities from these spatial maps and time courses . figure 3(a ) shows the spatial map and time course derived from task - related glm for run 3 . figures 3(b)3(e ) show the fc spatial maps and time courses derived from the first four hybrid methods using run 3 's ic13 as the seed source and fmri data from run 3 . figure 3(f ) shows the fc spatial map and time course derived from the fifth hybrid method using all of run 3 's ics as the seed source , with fmri data from run 3 . figure 3(g ) shows ic13 's spatial map and time course , and figure 3(h ) shows the spatial map that resulted from regressing the fmri data against ic13 's time course . visual inspection revealed that the spatial maps and time courses in figures 3(a)3(h ) were very similar to each other . some thresholded voxels in the rtof were found in all seven of these spatial maps . figure 4(a ) shows the spatial map and time course derived from task - related glm for run 2 , which was the run with the second - highest z - score ( 11.5 ) in any voxel , derived from task - related glm in response to viewing faces . figures 4(b)4(e ) show the fc spatial maps and time courses derived from the first four hybrid methods using run 3 's ic13 as the seed source , with fmri data from run 2 . figure 4(f ) shows the fc spatial map and time course derived from the fifth hybrid method using all of run 3 's ics as the seed source , with fmri data from run 2 . visual inspection revealed that the spatial maps and time courses in figures 4(a) 4(f ) were very similar to each other . each spatial map showed an arm of a cluster extending rostrally from the right lateral occipital cortex into the rtof , visible in the axial slice at z = 21 in figures 4(a)4(f ) . figure 5(a ) shows the spatial map and time course derived from task - related glm for run 4 , which was the run whose highest z - score ( 10.4 ) in any voxel was the lowest among the six runs . figures 5(b)5(e ) show the fc spatial maps and time courses derived from the first four hybrid methods using run 3 's ic13 as the seed source , with fmri data from run 4 . figure 5(f ) shows the fc spatial map and time course derived from the fifth hybrid method using all of run 3 's ics as the seed source , with fmri data from run 4 . visual inspection revealed that the spatial maps and time courses in figures 5(a)5(f ) were similar to each other . however , no thresholded voxels were found in the rtof for the first two hybrid methods ( the single - voxel and few - voxel hybrid methods ) . other than these two cases and the task - related glm map for run 5 , at least one thresholded voxel was found in the rtof in all task - related and fc spatial maps . criterionall mean correlations ( same column in table 1 ) were significantly greater than zero ( p < .001 ) . in addition , the range of mean correlations ( 0.710.79 ) compared favorably with the range of correlations of the task - related glm maps with each other ( 0.130.45 ) . all mean correlations ( same column in table 1 ) were significantly greater than zero ( p < .001 ) . in addition , the range of mean correlations ( 0.710.79 ) compared favorably with the range of correlations of the task - related glm maps with each other ( 0.130.45 ) . criterionfor each hybrid method , the mean correlation between hybrid fc maps and the task - related glm spatial map from the same run was greater than the mean correlation between hybrid fc maps and the task - related glm spatial map from the seed run , with p .001 ( table 1 ) . for each hybrid method , the mean correlation between hybrid fc maps and the task - related glm spatial map from the same run was greater than the mean correlation between hybrid fc maps and the task - related glm spatial map from the seed run , with p .001 ( table 1 ) . criterionthe omnibus test of differences in mean correlations ( same column , table 1 ) was nonsignificant ( f = 2.1 , p = .13 ) . the omnibus test of differences in mean correlations ( same column , table 1 ) was nonsignificant ( f = 2.1 , p = .13 ) . criterionthe threshold - adjusted overlap percentages were comparable for each hybrid method ( table 2 ) . the mv approach had better coverage of its task - related glm maps for all five target runs than the sv , fv , and dra approaches ( p = .043 ) , but this result was nonsignificant after correction for multiple comparisons . the threshold - adjusted overlap percentages were comparable for each hybrid method ( table 2 ) . the mv approach had better coverage of its task - related glm maps for all five target runs than the sv , fv , and dra approaches ( p = .043 ) , but this result was nonsignificant after correction for multiple comparisons . criterionno statistically significant differences were found between the five target - run , task - related glm maps and the five target - run fc maps for each of the five hybrid methods : 4/5 of the task - related glm maps contained at least one thresholded voxel in the rtof compared with 4/5 of the fc spatial maps for the single - voxel and few - voxel hybrid methods and 5/5 of the fc spatial maps for the many - voxel hybrid method and for both dual regression hybrid methods . no statistically significant differences were found between the five target - run , task - related glm maps and the five target - run fc maps for each of the five hybrid methods : 4/5 of the task - related glm maps contained at least one thresholded voxel in the rtof compared with 4/5 of the fc spatial maps for the single - voxel and few - voxel hybrid methods and 5/5 of the fc spatial maps for the many - voxel hybrid method and for both dual regression hybrid methods . criterionall mean correlations ( task - related column in table 3 ) were significantly greater than zero ( p < .001 ) . all mean correlations ( task - related column in table 3 ) were significantly greater than zero ( p < .001 ) . criterionexcept for dra ( nonsignificant ) , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 3 ) . in contrast , fc maps generated with br - i were significantly less correlated with the corresponding task - related glm maps than with the infomax group ic of interest ( ic14 ) . except for dra ( nonsignificant ) , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 3 ) . in contrast , fc maps generated with br - i were significantly less correlated with the corresponding task - related glm maps than with the infomax group ic of interest ( ic14 ) . criterionthe omnibus test showed that the mean correlations ( task - related column in table 3 ) differed significantly across methods ( f = 80.3 , p < .001 ) . post hoc , paired t - test analyses with bonferroni correction revealed that the mean correlation for br - i was significantly lower than the mean correlations for the other six methods ; the mean correlation for dra was significantly lower than the mean correlations for the remaining five methods ; and the mean correlation for drs - i was lower than the mean correlation for drs . the omnibus test showed that the mean correlations ( task - related column in table 3 ) differed significantly across methods ( f = 80.3 , p < .001 ) . post hoc , paired t - test analyses with bonferroni correction revealed that the mean correlation for br - i was significantly lower than the mean correlations for the other six methods ; the mean correlation for dra was significantly lower than the mean correlations for the remaining five methods ; and the mean correlation for drs - i was lower than the mean correlation for drs . criterionthe threshold - adjusted overlap percentages were significantly lower for br - i than those for the other six methods and significantly greater for drs than for dra and drs - i ( table 4 ) . the threshold - adjusted overlap percentages were significantly lower for br - i than those for the other six methods and significantly greater for drs than for dra and drs - i ( table 4 ) . criterionthe roc paucs were significantly lower for br - i than for the other six methods ; significantly greater for drs than those for dra and drs - i ; and significantly greater for mv than for drs - i ( data not shown ) . the roc paucs were significantly lower for br - i than for the other six methods ; significantly greater for drs than those for dra and drs - i ; and significantly greater for mv than for drs - i ( data not shown ) . qualitative analyseswe examined the data qualitatively to understand why these results were better ( more like the task - related glm results ) for some of the subjects than for others . an examination of the roc curves and paucs by subject ( across methods ) revealed that 13 of the subjects showed highly similar patterns and ranged in mean adjusted pauc ( scaled to a maximum possible area of 1 ) from 0.52 to 0.79 . in contrast , one of the subjects ( subject 8) had a mean adjusted pauc of 0.24 , and the data for this subject appeared to deviate in many respects from the others . therefore , we selected subject 8 's data for further study and compared this data to that from subject 1 ( highest mean pauc ) . the roc curves for subject 1 and subject 8 are shown in figures 7 and 8 . we examined the data qualitatively to understand why these results were better ( more like the task - related glm results ) for some of the subjects than for others . an examination of the roc curves and paucs by subject ( across methods ) revealed that 13 of the subjects showed highly similar patterns and ranged in mean adjusted pauc ( scaled to a maximum possible area of 1 ) from 0.52 to 0.79 . in contrast , one of the subjects ( subject 8) had a mean adjusted pauc of 0.24 , and the data for this subject appeared to deviate in many respects from the others . therefore , we selected subject 8 's data for further study and compared this data to that from subject 1 ( highest mean pauc ) . the roc curves for subject 1 and subject 8 are shown in figures 7 and 8 . for subject 1 , the fc maps derived with each method showed a fairly robust activation in sensorimotor ( primarily left ) and visual cortices ( figures 9(b) 9(h ) ) , and the corresponding time courses showed the expected cyclical time course of brain activity in response to the 10 repeated 24-second blocks of the experimental paradigm ( figures 10(b)10(h ) ) . these spatial maps and time courses resembled subject 1 's task - related glm spatial map ( figure 9(a ) ) and time course ( figure 10(a ) ) . for subject 8 we also saw activation in sensorimotor and visual cortices ( figures 11(b)11(h ) ) but the regions of activation were smaller and not as clearly lateralized to the left as for subject 1 . the activation pattern for subject 8 's task - related glm map ( figure 11(a ) ) was smaller and missing posterior portions of the sensorimotor cortex that were visible for subject 1 . subject 8 's fc maps bore some resemblance to the task - related glm map , except in the case of br - i , which appeared not to bear any resemblance ( spatial correlation 0.10 and threshold - adjusted activation overlap 0.3% ) ; however , the spatial correlation of br - i 's fc map ( figure 11(g ) ) with infomax ic14 ( figure 6(c ) ) was 0.59 . the fc time courses for subject 8 ( figures 12(b)12(h ) ) resembled subject 8 's task - related glm time course ( a priori time course in blue , figure 12(a ) ) with 10 equidistant peaks roughly discernable , but these were not as close to the task - related glm time course as for subject 1 . we also examined the data to understand why the results were better for some of the fc methods than for others . the drs and dra methods differed greatly in how well their fc maps matched the corresponding task - related glm maps , yet the only difference between the two methods was that 13 nuisance regressors were added to the linear model for dra . some of those regressors had a very similar time course to that of pica ic2 , and all but one of the 14 ics had a peak in its time course power spectrum at 0.042 hz ( the frequency corresponding to the block period of the experiment ) , with most of those peaks being the highest peak for the component . also , most of the ic spatial maps included some subthreshold or barely suprathreshold activation in the left sensorimotor brain region . inspection of the infomax ics revealed similar relationships between infomax ic14 and the other infomax group ics . criterionall mean correlations ( task - related column in table 5 ) were significantly greater than zero ( p < .001 ) . all mean correlations ( task - related column in table 5 ) were significantly greater than zero ( p < .001 ) . criterionexcept for the sv and dra methods , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 5 ) . except for the sv and dra methods , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 5 ) . criterionthe omnibus test showed that the mean correlations ( task - related column in table 5 ) differed significantly across methods ( f = 19.1 , p < .001 ) . posthoc , paired t - test analyses with bonferroni correction revealed that the mean correlations for fv , mv , and drs were significantly greater than those for sv and dra . the omnibus test showed that the mean correlations ( task - related column in table 5 ) differed significantly across methods ( f = 19.1 , p < .001 ) . posthoc , paired t - test analyses with bonferroni correction revealed that the mean correlations for fv , mv , and drs were significantly greater than those for sv and dra . criterionthe threshold - adjusted overlap percentages were significantly lower for sv than those for fv , mv , and drs ; and significantly lower for dra than those for mv ( table 6 ) . the threshold - adjusted overlap percentages were significantly lower for sv than those for fv , mv , and drs ; and significantly lower for dra than those for mv ( table 6 ) . criterionthe roc paucs were significantly lower for sv than those for fv and mv and significantly lower for dra than those for mv ( data not shown ) . the roc paucs were significantly lower for sv than those for fv and mv and significantly lower for dra than those for mv ( data not shown ) . qualitative analyseswe examined the data qualitatively to understand why the results in experiment 4 were better for the fv , mv , and drs methods than those for sv , which had not been the case in experiment 3 . examination of the pica group ics from experiments 3 and 4 showed that the spatial maps and time courses for the group ics of interest ( ic2 , in both cases ) were highly similar ( r = 0.70 ) for both experiments , but while experiment 3 's ic2 had the highest z - scores in a large contiguous region in the left sensorimotor cortex , experiment 4 's ic2 had its highest z - scores distributed to voxels in the same region of the sensorimotor cortex and to smaller regions in caudal and ventral portions of the left visual cortex , in peripheral brain regions . for this reason , in experiment 4 the sv method chose a seed voxel in the visual cortex rather than in the sensorimotor cortex . in contrast , the fv and mv methods chose seed voxels that were split between the visual and sensorimotor cortices , with most voxels in the latter . we examined the data qualitatively to understand why the results in experiment 4 were better for the fv , mv , and drs methods than those for sv , which had not been the case in experiment 3 . examination of the pica group ics from experiments 3 and 4 showed that the spatial maps and time courses for the group ics of interest ( ic2 , in both cases ) were highly similar ( r = 0.70 ) for both experiments , but while experiment 3 's ic2 had the highest z - scores in a large contiguous region in the left sensorimotor cortex , experiment 4 's ic2 had its highest z - scores distributed to voxels in the same region of the sensorimotor cortex and to smaller regions in caudal and ventral portions of the left visual cortex , in peripheral brain regions . for this reason , in experiment 4 the sv method chose a seed voxel in the visual cortex rather than in the sensorimotor cortex . in contrast , the fv and mv methods chose seed voxels that were split between the visual and sensorimotor cortices , with most voxels in the latter . criterionall mean correlations ( task - related column in table 7 ) were significantly greater than zero ( p < .001 ) . all mean correlations ( task - related column in table 7 ) were significantly greater than zero ( p < .001 ) . criterionfc maps generated with the dra method were significantly less correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 7 ) . fc maps generated with the other four hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources . fc maps generated with the dra method were significantly less correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 7 ) . fc maps generated with the other four hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources . criterionthe omnibus test showed that the mean correlations ( task - related column in table 7 ) differed significantly across methods ( f = 62.4 , p < .001 ) . posthoc , paired t - test analyses with bonferroni correction revealed that the mean correlations for sv , fv , mv , and drs were significantly greater than those for dra and the mean correlations for drs were significantly greater than for those fv ( p = .004 , uncorrected ) and mv ( p = .004 ) , but not sv ( p = .006 ) . the omnibus test showed that the mean correlations ( task - related column in table 7 ) differed significantly across methods ( f = 62.4 , p < .001 ) . posthoc , paired t - test analyses with bonferroni correction revealed that the mean correlations for sv , fv , mv , and drs were significantly greater than those for dra and the mean correlations for drs were significantly greater than for those fv ( p = .004 , uncorrected ) and mv ( p = .004 ) , but not sv ( p = .006 ) . criterionthe threshold - adjusted overlap percentages were significantly lower for dra than those for sv , fv , mv , and drs ( table 8) . the threshold - adjusted overlap percentages were significantly lower for dra than those for sv , fv , mv , and drs ( table 8) . criterionthe roc paucs were significantly lower for dra than those for sv , fv , mv , and drs ( data not shown ) . in experiments 35 , the p - values for the comparisons using roc pauc were very similar to those obtained from using threshold - adjusted overlap . differences in reported statistical significance only occurred for cases where p - values fell close to the threshold for statistical significance . the roc paucs were significantly lower for dra than those for sv , fv , mv , and drs ( data not shown ) . in experiments 35 , the p - values for the comparisons using roc pauc were very similar to those obtained from using threshold - adjusted overlap . differences in reported statistical significance only occurred for cases where p - values fell close to the threshold for statistical significance . qualitative analyseswe examined the data qualitatively to understand why the results in experiment 5 were similar to those from experiment 3 , but not experiment 4 . the group ic spatial maps for experiments 3 and 5 were very similar in appearance ( figures 6(b ) and 6(e ) ) and highly correlated ( 0.87 ) . this similarity apparently resulted in very similar locations for the seed voxels for the sv , fv , and mv methods . the main difference in the locations was that the seed voxels for experiment 5 appeared to be shifted posteriorly by one or two voxels compared with those from experiment 3 . we examined the data qualitatively to understand why the results in experiment 5 were similar to those from experiment 3 , but not experiment 4 . the group ic spatial maps for experiments 3 and 5 were very similar in appearance ( figures 6(b ) and 6(e ) ) and highly correlated ( 0.87 ) . this similarity apparently resulted in very similar locations for the seed voxels for the sv , fv , and mv methods . the main difference in the locations was that the seed voxels for experiment 5 appeared to be shifted posteriorly by one or two voxels compared with those from experiment 3 . the main purpose of this study was to perform a small - scale comparison of several hybrid ica - seed - based methods of fc assessment to determine which , if any , of these methods might merit further evaluation in larger - scale studies , to determine their potential usefulness compared with other , standard methods of assessing fc . all five hybrid methods performed well in each of four experiments ( experiments 25 ) , producing spatial maps that were consistently significantly and highly spatially correlated ( > 0.7 for fv , mv , and drs ; > 0.5 for sv ; and > 0.4 for dra ) with the corresponding task - related glm maps . these correlations were significantly higher than the corresponding correlations with the seed sources , except for the sv method in experiment 4 and the dra method in experiments 35 , indicating that the spatial maps produced were a reflection of the individual subject / session 's data rather than possibly being unduly influenced by the seed source . also , in experiment 2 the hybrid methods were not inferior to task - related glm in their capacity to detect activation in the rtof from fmri data collected during multiple runs where images of faces were viewed . these results suggest that , at least for some applications , it is possible to derive fc maps with glm , based on seeds from an exploratory ica , resulting in fc maps that resemble the seed ic(s ) ; yet the fc maps are unique to the fmri data being analyzed . this study also demonstrates that the ica and fc analyses need not be derived from the same data . the main motivation for exploration of hybrid ica - seed - based fc methods is the possibility that such methods will allow production of fc spatial maps and time courses without the limitations of each method performed separately . hybrid ica - seed - based fc methods utilize the exploratory power of ica in the generation of one or more seed ics , while providing a rationale for choice of seed . such a rationale may be helpful in cases where a choice of seed based on theoretical considerations might seem arbitrary and chosen from a large number of possibilities , although some theoretical considerations might still be needed to select the seed ic or ic of interest . hybrid ica - seed - based fc methods might have the discerning power of ica to provide accurate information about special relationships that exist among subpopulations of voxels in fc maps , as demonstrated in experiment 1 . this capacity can be particularly helpful in cases where glm seed - based fc maps would otherwise be generated from seed voxels where multiple components overlap . finally , hybrid ica - seed - based fc methods allow circumvention of statistical complications arising from reproducibility issues associated with group comparison of spatial maps produced by ica , because the last step of such methods involves glm rather than ica . for the sv , fv , and mv hybrid methods this last step is a standard roi , seed - based assessment of fc , where ica has provided the choice of seed roi . the drs hybrid method is not much different conceptually , because rather than averaging the voxel time courses within an roi , a weighted average is performed through least - square linear regression , which heavily weights the values of the derived time course toward those of the voxels with highest z - scores in the seed ic . previous investigations into the potential benefits of hybrid ica - seed - based fc approaches have not been reported in the literature , to the authors ' knowledge . however , a number of approaches designed to utilize the exploratory power of ica while addressing reproducibility issues involved with group comparison of ica - derived spatial maps have been described [ 12 , 26 , 31 , 37 , 38 , 75 , 76 ] . some of these require prior temporal information [ 37 , 38 , 76 ] and are therefore not suitable when such information is unavailable , as in resting - state studies . among methods suitable for resting state studies , one of the first proposed methods is br - i , compared in this study . it makes intuitive sense that restricting ics for all subjects to be based on a single group ica would limit the intersubject variability for spatial maps corresponding to each component , but it remains unclear whether this results in truly homologous groups of ics to be compared ( i.e. , ics based on signals having exactly the same neural origins ) across subjects . approach is reproducibility across studies : the stochastic nature of ica means that group ics derived in one study might not exactly match the ics from another study with different participants . for example , even consistently reproducible ics such as the default mode component [ 8 , 74 ] can sometimes be split into two components [ 77 , 78 ] . these issues are also potentially problematic with the multisubject ica and dual regression approach . seed - based approaches can address this last issue by utilizing fixed spatial priors to facilitate reproducibility across studies . such maps can be derived from ica or task - related glm or artificially constructed based on theoretical considerations . instead of deriving seed spatial maps from all subjects being compared , it may be advantageous in some cases to derive seed spatial maps from an outside reference group of subjects . for example , it might be desirable to compare fc results from a current study with those of a previous study by deriving fc from exactly the same seed maps that were used in the previous study , particularly if the previous study involves a much larger sample size . when healthy and diseased samples of subjects are compared , it might be advantageous in some cases to use seed maps derived from exclusively healthy or exclusively diseased subjects , rather than seed maps derived from a mixed sample . finally , in cases where a group ic of interest appears ( by chance ) to be fragmented into multiple ics , rather than attempting to piece together the fragments or to use them individually as seeds for dual - regression - based fc analysis , it might be better to choose seeds from a previously derived ic or set of ics that are deemed prototypical of the spatial patterns of interest . another approach that utilizes fixed spatial priors is spatial semiblind spatial ica ( sss - ica ) . sss - ica uses predetermined spatial maps to guide ica toward more correct solutions and was demonstrated to yield higher signal - to - noise ratios than infomax ica and ica - with - reference algorithms ( ica - r ) , particularly with an accurate spatial prior under low - noise conditions . it would be useful to know to what extent sss - ica might unduly influence the resulting spatial maps to resemble the spatial priors . sss - ica was reported not to be influenced by an incorrect spatial prior in the absence of noise , but further exploration of this question would be helpful . another reported ica method involving spatial priors that can be used without a priori temporal information is fixed - average spatial ica ( fas - ica ) , which is similar to hybrid ica - seed - based fc methods because it involves regression of fmri data against spatial maps generated with ica . with fas - ica , the spatial maps used as seeds are generated by averaging the ic spatial maps derived from performing br - i on multiple fmri runs for the same subject under one condition ; then the fmri data from another condition is regressed against the complete set of averaged ic spatial maps ( averaged across homologous ics from different runs ) to generate corresponding time courses . if one were to add , as a final step to fas - ica , regression of the individual - subject fmri data against those time courses to generate corresponding spatial maps , this would be equivalent to the dra approach described here ( using an averaged set of ic spatial maps derived with br - i as the seed spatial maps ) . the hybrid ica - seed - based fc methods fv , mv , and drs performed the best overall in experiments 25 . the high correlations , overlaps , and roc paucs with the hypothesis - driven , reference task - related glm spatial maps serve as confirmation that those hybrid methods produced meaningful and valid results that reflected brain neural activity . however , the relatively poor results obtained in some cases by the sv and dra methods and in every case by the br - i method do not serve as a disconfirmation of the validity of those methods . for example , it is possible that the results from both br - i and task - related glm were highly valid , accurately reflecting brain activity during the experiment described in . these methods may have reflected somewhat separate elements of the total brain activity that was occurring during the experiment . from the qualitative analyses for experiment 3 it appeared as if the spatial and temporal properties of the task - related glm were split among several infomax group ic 's , and the same was true for the pica group ics , which helps to explain why the dra spatial maps were not as closely related to the task - related glm maps as were the spatial maps of the other hybrid methods . although these findings do not indicate that the dra and br - i methods are inferior to the first four hybrid methods , they do not provide support for their use either ; and a reason to be discouraged from using these two methods and any method that directly generates with ica the spatial maps to be used for groupwise comparisons is the risk of not making a valid comparison across homologous - looking components , due to a seemingly random ( because we do not fully understand this process ) distribution of fmri variance into the various components . until we know more about the conditions under which ica will split variance from one component into separate , temporally related components , it may be safer to use seed - based or hybrid seed - based methods to derive fc maps used for group inferences . the hybrid ( and seed - based ) fc methods are also affected by factors that can decrease the validity of group inferences , but these are issues that are more easily understood and addressed and that affect nearly all types of fmri data analyses . for example , factors that might have contributed to the somewhat different appearance of subject 8 's task - related glm and fc maps compared with the other subjects include differing activities during the experiment , differing intensities of responses , differing brain hemodynamic responses or response times , differing brain anatomy , differing localization of areas of brain function , and differing specialization of functions used to perform the same task . although the latter four factors can be problematic and invalidate or reduce the sensitivity of results of fmri analyses , these are relatively easier to understand and are amenable to being addressed with experimental measures . for example , a brain localizer scan or improved methods of coregistration of different brains to each other and to a standard brain can help to reduce the effect of such factors [ 81 , 82 ] . which among the hybrid ica - seed - based fc methods sv , fv , mv , and drs is best is unclear and may depend upon the fc application in question . the sv method has the advantage of being the easiest to report and reproduce , because the only information needed is the coordinates of a single voxel . the sv method should in most cases be comparable to the fv and mv methods ( as in experiments 2 , 3 , and 5 ) , because after spatial smoothing , the voxel 's time course and variance will become more similar to its neighbors . however , experiment 4 underscored a potential weakness of the sv method . in order for the sv method to work well , the single voxel should preferably be placed in the middle of a large area of activation , typical for the type of activity being studied . if the voxel is placed in the periphery , on the border between brain areas with widely differing functions , or in a brain area not well representative of the activity being studied , then use of the fc maps that result may not be suitable for groupwise comparison due to heterogeneity of the spatial maps and time courses that may result . thus , if we use the sv method , it is important to inspect the data to determine the appropriateness of the location of the single voxel . if automated methods are used it may be safer to use the drs method , which includes information from the entire seed spatial map and which performed the best overall in experiments 25 . first , different thresholding levels and scaling used for spatial maps derived with pica and infomax may have caused the pica - derived maps to appear more robust than the infomax - derived maps , even though we were using a relatively conservative thresholding level for pica - derived maps . however , the primary measures we used for statistical testing ( spatial correlation , threshold - adjusted overlap , and pauc ) were not influenced by spatial map scaling and threshold cutoffs . second , the dimensionalities used in experiments 3 and 4 were both lower than those typically reported in the literature for group ica , where the number of components has often been set manually to a round number such as 20 or 30 [ 39 , 58 , 75 , 83 , 84 ] , and when estimated automatically has typically been greater than 15 [ 6 , 85 ] . one possible explanation for the low estimates in our study could be that they accurately reflected a relatively low amount of structured noise in the fmri data , but the most appropriate choice of dimensionality is not clear and therefore remains somewhat arbitrary . however , the hybrid methods as a group performed well for the wide range of dimensionalities tested in this study . third , although we have demonstrated that the hybrid methods can work on task - related data , we have not demonstrated that they can work on resting - state data , for which such hybrid methods are primarily intended . we chose experimental paradigms associated with activation of visual and sensorimotor brain regions because such paradigms generally result in strong and predictable fmri activation patterns , which facilitated confirmation of the validity of the fc spatial maps and time courses derived with hybrid methods . also , the use of such paradigms is commonly reported in the literature for testing of fc- and ica - related methods and to compare fc- and ica - related spatial maps to those derived based on hypothesis testing with a priori time courses related to the experimental paradigm [ 12 , 26 , 35 , 37 , 75 , 86 ] . we believe that the comparisons here can be extrapolated to resting - state studies because a number of studies have demonstrated the parallels between resting - state activity and task - related activity . for example , group ica studies have demonstrated that 810 group ics ( depending upon the group ica method used ) can consistently be found across subjects , resting - state sessions , and within sessions ( with bootstrapping ) with bold signal changes of up to 3% , comparable to those found for task - related studies [ 6 , 87 , 88 ] . in , single - subject ics exhibited an extremely high degree of consistency in spatial , temporal , and frequency parameters within and between subjects . reproducibility across subjects and sessions was also found for resting - state , roi - based fc , and although such roi - based fc was found to frequently correspond to structural connections as measured with diffusion spectrum imaging tractography , some fc was not explained by direct structural connections . in resting state , the activity of such functionally connected regions is frequently correlated with functionally related areas [ 35 , 78 ] and such activity levels are modulated by task activity , resulting in increased fc in functionally relevant areas being exercised by the experimental paradigm . fourth , another possible limitation in the conclusions that may be drawn from this study concerns the question of whether it is appropriate to measure task - related brain activity using seed - based fc measures , which are fundamentally different than measures based on a priori time courses , because the time courses of the former are based upon the temporal variations at one or more brain regions , while the time courses of the latter are based upon the temporal variations that are expected in response to an experimental paradigm . the differences between these approaches should not differ by much provided that the seed location corresponds to a brain location whose activity is sufficiently synchronous with the hemodynamic response to the experimental paradigm . as long as the chosen location is functionally related to the experimental task , this should not be a problem ( unlike the case for the sv method in experiment 4 ) . in conclusion , our experiments demonstrate that at least four of the hybrid ica - seed - based fc methods tested ( sv , fv , mv , and drs ) can produce spatial maps and time courses that correspond closely to what would be expected from knowledge of the experimental paradigm in play during fmri data collection and from examination of spatial maps and time courses generated with task - related glm , based on hypothesis testing with a priori time courses . we hope that further empirical and theoretical studies will help to elucidate what methods for data - driven extraction of components ( spatial maps and time courses ) are most likely to improve the validity of groupwise comparisons . our findings suggest that one or more hybrid ica - seed - based methods should be included in such studies . until we know more about factors that govern the seemingly stochastic nature of how ica parses fmri variance into temporally related components , it may be safer to use hybrid ica - seed - based fc methods to generate ica - related spatial maps and time courses used for groupwise comparison . the use of such methods effectively channels exploratory information from ica into the realm of seed - based fc , which for years has been widely accepted , understood , and implemented by the imaging community [ 1 , 2 , 91 , 92 ] .

brain functional connectivity ( fc ) is often assessed from fmri data using seed - based methods , such as those of detecting temporal correlation between a predefined region ( seed ) and all other regions in the brain ; or using multivariate methods , such as independent component analysis ( ica ) . ica is a useful data - driven tool , but reproducibility issues complicate group inferences based on fc maps derived with ica . these reproducibility issues can be circumvented with hybrid methods that use information from ica - derived spatial maps as seeds to produce seed - based fc maps . we report results from five experiments to demonstrate the potential advantages of hybrid ica - seed - based fc methods , comparing results from regressing fmri data against task - related a priori time courses , with back - reconstruction from a group ica , and with five hybrid ica - seed - based fc methods : roi - based with ( 1 ) single - voxel , ( 2 ) few - voxel , and ( 3 ) many - voxel seed ; and dual - regression - based with ( 4 ) single ica map and ( 5 ) multiple ica map seed .

1. Introduction 2. Theory 3. Materials and Methods 4. Results 5. Discussion

another commonly used data - driven approach is independent component analysis ( ica ) , which derives spatiotemporal components ( pairs of spatial maps and associated time courses ) through blind signal source separation and linear decomposition of fmri data [ 4 , 5 ] . both approaches have produced pairs of spatial maps and associated time courses comparable to those produced by standard glm techniques that regress fmri data against experimentally derived a priori hemodynamic response functions , in experiments driven by task activities with known time courses [ 917 ] . the results derived with ica are sometimes more consistent with expected bold response patterns than those derived with the a priori , task - related approach , especially for transient task - related bold responses or responses whose time courses are difficult to predict beforehand [ 4 , 1821 ] . despite potential advantages of ica over task - related or seed - based glm approaches in visualizing correlated brain activity , ica reproducibility issues may complicate or limit ica 's use in the generation of fc maps to be used for group comparison . some of these reproducibility issues may be avoided by comparing fc spatial maps generated through seed - based methods . deriving seed information from ica can be considered a hybrid ica - seed - based approach where the exploratory power of ica is utilized to select a seed from a huge number of possible choices . two of the most widely implemented of these approaches that can be applied in a data - driven fashion , without a priori time courses , or any other prior knowledge from an experimental paradigm , are the back - reconstruction [ 12 , 37 ] and dual - regression [ 31 , 39 ] methods . here , we report the results of comparing five hybrid ica - seed - based methods to each other and to these two methods , using standards of comparison derived from knowledge of visuomotor experimental paradigms involved during collection of fmri data , and from results of fmri analyses with task - related glm methods ( i.e. similarly , the time courses in g can be uniquely determined through glm by regressing the fmri data against completely specified , non - colinear spatial maps in . least - square linear regression to determine the time courses in g proceeds in the same manner as for determination of the spatial maps of in ( 1 ) , except that the sums ( across all voxels ) of the squared residual error terms for each time point are minimized to determine g. ica , like glm , is based on a linear model . x here is defined in the same manner as for ( 1 ) and represents the same data , while m , c , and e are matrices that represent time courses , spatial maps , and error terms in the same format as for g , , and , but that generally do not contain the same matrix values , and the number of time courses and associated spatial maps in m and c may differ from the number in g and . a simple case of seed - based fc is region - of - interest - based ( roi - based ) fc derived by averaging the fmri data time courses of each voxel within a predefined roi ( the seed ) to produce a time course against which the fmri data is regressed to yield a single fc spatial map . a more complex case of seed - based fc involves dual regression , a process where fmri data is regressed against a set of predefined spatial maps to derive a set of time courses against which the fmri data is regressed once more to produce a corresponding set of fc spatial maps . five experiments were performed to evaluate the performance of the five hybrid ica - seed - based fc methods . experiment 1 illustrated the potential advantages of a hybrid ica - seed - based method over a seed - based method that does not utilize information from ica , using natural data from an fmri run , with artificial data added . experiment 2 tested the performance of the five hybrid methods , comparing results to those obtained from task - related glm , using six runs of data from a single participant . experiment 3 tested the performance of the five hybrid methods , comparing results to those obtained from task - related glm and two methods for extracting information about individual subjects , based on group ica . some or all of the group ics were the seed source for the five hybrid ica - seed - based approaches . individual - subject spatial maps not derived directly from ica were derived with glm by regressing fmri data against one or more time courses using fsl feat with film local autocorrelation correction to generate gaussianised , z - statistic images reflecting parameter estimates in relation to their standard errors and degrees of freedom . we repeated this seed - based fc analysis twice more ( figures 2(e ) and 2(f ) ) , choosing the voxel in the z = 9 slice with the highest z - score from the natural ( coordinates 12 , 99 , 9 ) and artificial ( coordinates 30 , 9 , 9 ) ic spatial maps . we compared the results of task - based glm to the results of five hybrid ica - seed - based fc methods using preprocessed fmri data without added artificial signal . the first hybrid method ( single - voxel seed , sv ) used the voxel ( coordinates 12 , 102 , 3 ) in ic13 with the highest z - score ( 18.27 ) as the seed for roi - based fc . the second hybrid method ( few - voxel seed , fv ) derived roi - based fc using the same procedure , but the regressor for each run was the average time course over a few seed voxels . the third hybrid method ( many - voxel seed , mv ) derived roi - based fc using as the regressor the average time course over many seed voxels , chosen as close to 100 in number as possible by thresholding with whole z - scores . the fourth hybrid method ( dual regression with single - ic seed , drs ) used ic13 's spatial map as the seed for dual - regression - based fc . the fifth hybrid method ( dual regression with all ics as the seed source , dra ) used all 13 ics as the seed for dual - regression - based fc , using the same procedure as for the fourth method , to yield time courses against which the fmri data was regressed to produce corresponding fc maps for each target run . fc map correlation with task - related glm spatial map from same run > 0 : we did not assume that a successful hybrid method fc map would correlate highly with the task - related glm map from the same run because the correlations of the glm maps from the six runs with each other were in the range 0.130.45 , and differences in latency of hemodynamic responses [ 64 , 65 ] could potentially result in considerable differences between the fc maps and task - related glm maps . fc map correlation with task - related glm spatial map from same run > fc map correlation with task - related glm spatial map from seed run : for hybrid ica - seed - based methods to be considered successful , the resulting fc maps should be unique to the fmri data being regressed rather than merely a reflection of the seed source spatial maps or fmri data . we also reasoned that this test might reveal potential weaknesses with the hybrid approaches because the rtof was not in close proximity to the regions with the highest z - scores in the glm- and ica - derived spatial maps , and therefore the hybrid approaches could not benefit from the effects of smoothing or a tendency for activation patterns to be clustered , making voxel activation patterns similar to those of neighboring voxels . we compared the results of task - based glm to the results of seven methods of deriving individual - subject fc maps from a group ica . the first five methods were the same as the hybrid ica - seed - based methods tested in experiment 2 ( i.e. , roi - based : single - voxel , few - voxels , and many voxels ; dual - regression - based : single ic map and all ic maps ) , using the pica group ics as the seed source . we chose the dual - regression , single - map method because overall the fourth method produced spatial maps that most closely resembled the task - based glm - derived spatial maps . roc pauc from fc maps , using task - related glm spatial maps as the standards of comparison : for each subject and method evaluated , the partial area under the curve ( pauc ) of the receiver operating characteristic ( roc ) of the fc maps was calculated with fsl 's fslmaths program . thus , for these two cases the hybrid ica - seed - based approaches resulted in spatial maps and time courses that were very similar to the seed ic spatial maps . figures 4(b)4(e ) show the fc spatial maps and time courses derived from the first four hybrid methods using run 3 's ic13 as the seed source , with fmri data from run 2 . criterionfor each hybrid method , the mean correlation between hybrid fc maps and the task - related glm spatial map from the same run was greater than the mean correlation between hybrid fc maps and the task - related glm spatial map from the seed run , with p .001 ( table 1 ) . for each hybrid method , the mean correlation between hybrid fc maps and the task - related glm spatial map from the same run was greater than the mean correlation between hybrid fc maps and the task - related glm spatial map from the seed run , with p .001 ( table 1 ) . criterionno statistically significant differences were found between the five target - run , task - related glm maps and the five target - run fc maps for each of the five hybrid methods : 4/5 of the task - related glm maps contained at least one thresholded voxel in the rtof compared with 4/5 of the fc spatial maps for the single - voxel and few - voxel hybrid methods and 5/5 of the fc spatial maps for the many - voxel hybrid method and for both dual regression hybrid methods . no statistically significant differences were found between the five target - run , task - related glm maps and the five target - run fc maps for each of the five hybrid methods : 4/5 of the task - related glm maps contained at least one thresholded voxel in the rtof compared with 4/5 of the fc spatial maps for the single - voxel and few - voxel hybrid methods and 5/5 of the fc spatial maps for the many - voxel hybrid method and for both dual regression hybrid methods . for subject 1 , the fc maps derived with each method showed a fairly robust activation in sensorimotor ( primarily left ) and visual cortices ( figures 9(b) 9(h ) ) , and the corresponding time courses showed the expected cyclical time course of brain activity in response to the 10 repeated 24-second blocks of the experimental paradigm ( figures 10(b)10(h ) ) . the fc time courses for subject 8 ( figures 12(b)12(h ) ) resembled subject 8 's task - related glm time course ( a priori time course in blue , figure 12(a ) ) with 10 equidistant peaks roughly discernable , but these were not as close to the task - related glm time course as for subject 1 . criterionexcept for the sv and dra methods , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 5 ) . except for the sv and dra methods , fc maps generated with the hybrid methods were significantly more correlated with the corresponding task - related glm maps than with the group ic seed sources ( table 5 ) . examination of the pica group ics from experiments 3 and 4 showed that the spatial maps and time courses for the group ics of interest ( ic2 , in both cases ) were highly similar ( r = 0.70 ) for both experiments , but while experiment 3 's ic2 had the highest z - scores in a large contiguous region in the left sensorimotor cortex , experiment 4 's ic2 had its highest z - scores distributed to voxels in the same region of the sensorimotor cortex and to smaller regions in caudal and ventral portions of the left visual cortex , in peripheral brain regions . the main purpose of this study was to perform a small - scale comparison of several hybrid ica - seed - based methods of fc assessment to determine which , if any , of these methods might merit further evaluation in larger - scale studies , to determine their potential usefulness compared with other , standard methods of assessing fc . all five hybrid methods performed well in each of four experiments ( experiments 25 ) , producing spatial maps that were consistently significantly and highly spatially correlated ( > 0.7 for fv , mv , and drs ; > 0.5 for sv ; and > 0.4 for dra ) with the corresponding task - related glm maps . also , in experiment 2 the hybrid methods were not inferior to task - related glm in their capacity to detect activation in the rtof from fmri data collected during multiple runs where images of faces were viewed . the main motivation for exploration of hybrid ica - seed - based fc methods is the possibility that such methods will allow production of fc spatial maps and time courses without the limitations of each method performed separately . hybrid ica - seed - based fc methods utilize the exploratory power of ica in the generation of one or more seed ics , while providing a rationale for choice of seed . hybrid ica - seed - based fc methods might have the discerning power of ica to provide accurate information about special relationships that exist among subpopulations of voxels in fc maps , as demonstrated in experiment 1 . finally , hybrid ica - seed - based fc methods allow circumvention of statistical complications arising from reproducibility issues associated with group comparison of spatial maps produced by ica , because the last step of such methods involves glm rather than ica . previous investigations into the potential benefits of hybrid ica - seed - based fc approaches have not been reported in the literature , to the authors ' knowledge . however , a number of approaches designed to utilize the exploratory power of ica while addressing reproducibility issues involved with group comparison of ica - derived spatial maps have been described [ 12 , 26 , 31 , 37 , 38 , 75 , 76 ] . finally , in cases where a group ic of interest appears ( by chance ) to be fragmented into multiple ics , rather than attempting to piece together the fragments or to use them individually as seeds for dual - regression - based fc analysis , it might be better to choose seeds from a previously derived ic or set of ics that are deemed prototypical of the spatial patterns of interest . another reported ica method involving spatial priors that can be used without a priori temporal information is fixed - average spatial ica ( fas - ica ) , which is similar to hybrid ica - seed - based fc methods because it involves regression of fmri data against spatial maps generated with ica . with fas - ica , the spatial maps used as seeds are generated by averaging the ic spatial maps derived from performing br - i on multiple fmri runs for the same subject under one condition ; then the fmri data from another condition is regressed against the complete set of averaged ic spatial maps ( averaged across homologous ics from different runs ) to generate corresponding time courses . if one were to add , as a final step to fas - ica , regression of the individual - subject fmri data against those time courses to generate corresponding spatial maps , this would be equivalent to the dra approach described here ( using an averaged set of ic spatial maps derived with br - i as the seed spatial maps ) . the hybrid ica - seed - based fc methods fv , mv , and drs performed the best overall in experiments 25 . the high correlations , overlaps , and roc paucs with the hypothesis - driven , reference task - related glm spatial maps serve as confirmation that those hybrid methods produced meaningful and valid results that reflected brain neural activity . from the qualitative analyses for experiment 3 it appeared as if the spatial and temporal properties of the task - related glm were split among several infomax group ic 's , and the same was true for the pica group ics , which helps to explain why the dra spatial maps were not as closely related to the task - related glm maps as were the spatial maps of the other hybrid methods . until we know more about the conditions under which ica will split variance from one component into separate , temporally related components , it may be safer to use seed - based or hybrid seed - based methods to derive fc maps used for group inferences . the hybrid ( and seed - based ) fc methods are also affected by factors that can decrease the validity of group inferences , but these are issues that are more easily understood and addressed and that affect nearly all types of fmri data analyses . which among the hybrid ica - seed - based fc methods sv , fv , mv , and drs is best is unclear and may depend upon the fc application in question . we chose experimental paradigms associated with activation of visual and sensorimotor brain regions because such paradigms generally result in strong and predictable fmri activation patterns , which facilitated confirmation of the validity of the fc spatial maps and time courses derived with hybrid methods . also , the use of such paradigms is commonly reported in the literature for testing of fc- and ica - related methods and to compare fc- and ica - related spatial maps to those derived based on hypothesis testing with a priori time courses related to the experimental paradigm [ 12 , 26 , 35 , 37 , 75 , 86 ] . fourth , another possible limitation in the conclusions that may be drawn from this study concerns the question of whether it is appropriate to measure task - related brain activity using seed - based fc measures , which are fundamentally different than measures based on a priori time courses , because the time courses of the former are based upon the temporal variations at one or more brain regions , while the time courses of the latter are based upon the temporal variations that are expected in response to an experimental paradigm . in conclusion , our experiments demonstrate that at least four of the hybrid ica - seed - based fc methods tested ( sv , fv , mv , and drs ) can produce spatial maps and time courses that correspond closely to what would be expected from knowledge of the experimental paradigm in play during fmri data collection and from examination of spatial maps and time courses generated with task - related glm , based on hypothesis testing with a priori time courses . our findings suggest that one or more hybrid ica - seed - based methods should be included in such studies . until we know more about factors that govern the seemingly stochastic nature of how ica parses fmri variance into temporally related components , it may be safer to use hybrid ica - seed - based fc methods to generate ica - related spatial maps and time courses used for groupwise comparison . the use of such methods effectively channels exploratory information from ica into the realm of seed - based fc , which for years has been widely accepted , understood , and implemented by the imaging community [ 1 , 2 , 91 , 92 ] .

small molecule kinase inhibitors are cornerstones of advanced chemotherapy . in their ideal embodiment , they may selectively target a validated signaling pathway that is uniquely dysregulated in cancer cells ; in reality , multiple factors complicate this strategy . first , the structural and functional homology of kinases combined with the abundance of atp - binding folds throughout the cell make off - target binding events unavoidable . second , compensatory or alternate modes of pathway activation can lead to the development of resistance , requiring additional antineoplastic drugs or a different chemotherapy regimen . ideally the observation of compensatory shifts in signaling on a systemic and live cell level could facilitate the early development of second - generation drugs with larger barriers or at least anticipated paths to adaptation . in this context , assaying changes in receptor kinase states ( i.e. , level of expression , oligomerization , localization , conformation , phosphorylation ) remains a challenge , particularly in live cells or tissue preparations . in an effort to develop molecular probes capable of interrogating kinase signaling pathways , we have introduced the concept of turn - on fluorescent ligands that target the atp - binding pocket of the egfr / erbb family of receptor tyrosine kinases . these molecular probes are built upon the 4-aminoquinazoline scaffold , which is an established egfr / erbb pharmacophore ; examples include gefitinib or erlotinib ( figure 1a , c ) , so - called type i inhibitors that preferentially binding the active kinase conformation , and lapatinib ( figure 1b , d ) , a type ii inhibitor targeting the inactive conformation . the preference for active and inactive conformations is addressed through substitution at the 4-amino position ( figure 1e ) . while the quinazoline core conveys binding to the nucleotide pocket , but with limited specificity for erbb - type receptors , the 4-amino aryl arm further increases specificity and contributes discrimination between activation states . this selectivity reflects increased access to the hydrophobic pocket adjacent to the nucleotide binding site in the inactive conformation . together , the quinazoline core and n - aryl arm constitute the pharmacophore . crystal structures of the kinase domain of egfr with either erlotinib ( 1m17 ) or lapatinib ( 1xkk ) ( figure 1a , b ) show that while the pharmacophore arm is oriented deep in the binding pocket , the 6-position is amenable to chemical modification without perturbing the key conserved contacts of the binding pocket . the structure of lapatinib demonstrates this point as the aromatic core is extended by the addition of a furan ring . thus , a potential strategy for synthesizing fluorescent kinase inhibitors is to modify the 4-aminoquinazoline core with fluorphore arms ( figure 1e ) at the 6-position . in principle , probes targeting specific kinases ( i.e. , igf1r vs egfr ) or activation states could be encoded with unique optical outputs by tuning the excitation and emission energies through the fluorophore arm . crystal structures of the egfr atp - binding pocket with ( a ) erlotinib ( pdb i d : 1m17 ) and ( b ) lapatinib ( pdb i d : 1xkk ) reveal the inhibitor binding modes . the arms at the 6-position of the quinazoline core ( in blue ; c , d ) may be replaced by fluorophore arms without disturbing the key binding contacts . herein , we investigate the effect of conjugation length and auxochrome substitution on the optical properties of a family of n - phenyl-4-aminoquinazoline probes . we find that extension of the framework effectively lowers the excitation and emission energies , yielding fluorescent probes that compare favorably with other fluorescent adenosine analogues . the introduction of strong electron donors or strong electron - withdrawing groups generates donor acceptor systems that are highly sensitive to solvent polarity . as a result , several probes exhibit high fluorescence on / off ratios , a feature that is key to their performance as self - reporting fluorescent kinase inhibitors . despite the modifications to the quinazoline core , we also found that several of these probes inhibit erbb2 phosphorylation in a live cell setting , demonstrating that binding to the atp pocket and cell permeability are preserved . overall , these fluorescent adenosine mimics compare favorably with other nucleobase analogues as they possess tunable optical properties and high fluorescence turn - on ratios and compete effectively with the native substrate to inhibit tyrosine phosphorylation . the optical properties of kinase - binding probes of general structure i can be addressed through the fluorophore arm depicted in figure 1e . we envisaged tuning two parameters : ( 1 ) the electron - donating ability of the substituent on the phenyl ring and ( 2 ) the conjugation length between the phenyl ring and the quinazoline core . td - dft calculations , vide infra , guided the selection of the electron - donating and electron - withdrawing substituents that span the range from the strongly donating dimethylamino group ( e.g. , 1a3a ) to strongly electron - withdrawing nitro substitution ( 1e3e ) . the 15-membered family of quinazolines , 1a3e ( figure 1 ) , was synthesized from a common intermediate , 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , by suzuki coupling of the appropriate arylboronic acid ( 1a1e , scheme 1 , route a ) or by heck coupling ( scheme1 , route b ) of the styryl ( 2a2e ) and phenyl butadiene ( 3a3e ) arms . following reaction workup , flash chromatography and crystallization from 2-propanol / ethyl acetate mixtures , the products were isolated in moderate yields as microcrystals that appeared colorless ( e.g. , 1c and 3c ) to bright orange yellow ( e.g. , 1a and 3e ) . two photophysical properties , the fluorescence on / off ratio and the optical band gap , of the kinase - binding probes can simultaneously be addressed by varying the donor or acceptor substitution and the conjugation length . ideally , the probes should be nonemissive or off in solution , but upon binding in the solvent excluding and geometrically confined atp pocket , emission should be enhanced or turned on . to achieve this emission behavior , a probe should possess an excited state with a high degree of charge - transfer ( ct ) character and possibly access at twisted intramolecular charge - transfer ( tict ) excited state . td - dft calculations ( 6 - 31 g * , chcl3 , smd solvent model ) show that both strong electron donors and strong electron acceptors will lead to an s1 state with ct character . the s1 state is accessible via the allowed one - electron transition between the homo and lumo for all 15 members , with energies ranging from 2.5 ev ( 500 nm ) in the case of 3e to 3.6 ev ( 345 nm ) in the case of 1c ( table 1 ) . the addition of each vinylene bridge lowers the s1 transition energy by approximately 0.25 ev . the introduction of a moderately electron - donating or -withdrawing group ( i.e. , ome or cn ) lowers the energy of the s0 s1 transition energy by 0.1 to 0.2 ev when compared to that of isostructural members of the unsubstituted series ( i.e. , 1c3c ) . the presence of the strong electron - donating dimethylamino group more effectively lowers the homo lumo band gap by 0.50.3 ev when comparing 1a3a and 1a3c , whereas the strongly electron - withdrawing nitro group has the greatest effect , with differences of 0.8 to 0.5 ev when comparing 1a3c and 1a3e . in addition to manipulating the optical band gap , the presence of an electron - donating or electron - withdrawing substituent on the pendant phenyl arm influences the ct character of the excited state ; the polarization and spatial segregation of the homo and lumo is directly linked to the identity of the substituent . in the series of dimethylamino - substituted compounds ( e.g. , 2a ; figure 2 ) , the homo is largely localized to the fluorophore arm , whereas the lumo is polarized toward the quinazoline core . the presence of a strong electron - withdrawing group ( i.e. , no2 or cn ) on the fluorophore arm reverses the frontier molecular orbital distribution . in the case of 2e ( figure 2 ) , the homo is polarized toward the quinazoline core , whereas the lumo is largely localized to the styryl arm ; an excited state with high ct character still results from the promotion of an electron from the homo to the lumo . compounds 2b2d show a gradual redistribution of the homo and lumo densities between the two extreme cases , 2a and 2e . similar polarization of the frontier molecular orbitals is also observed in the 1a1e ( figure s16 ) and 3a3e series ( figure s17 ) . frontier molecular orbitals of 2a2e calculated at the cam - b3lyp/6 - 31 g * level : the polarization of the homo and lumo shifts across the series ( compare 2a and 2e ) ; strong ct character is expected for both 2a and 2e , although the localization of charge should be reversed . probes 1a3c were designed as turn - on fluorescent ligands and thus were expected to be nonemissive in solvents of high polarity ( e.g. , water and methanol ) and emissive in less polar solvents . this behavior can be qualitatively observed by eye as in chloroform ( et(30 ) = 39.1 kcal / mol ) solutions ; most of the probes appear bright , with emissions ranging from blue ( 2b and 3c ) to green ( 1a and 2a ) to yellow or orange ( 3a and 3e ) , figure 3 , whereas in aqueous solutions , weak or no emission was observed . chloroform solutions of selected probes ( 5 m ) under uv illumination ( 354 nm ) ; aqueous solutions showed weak or no emission ( vials not shown ) . to investigate their optical properties quantitatively , we obtained their uv vis and fluorescence spectra in chloroform . the absorption maxima ( figure 4 and table 2 ) correlate very well with the predicted values both in terms of transition energies as well as oscillator strength . the transition energies progress in a clear trend , as max , abs increases with increasing conjugation length . the presence of an auxochrome also serves to modulate the absorption wavelengths , as is evident in the lower transition energies for compounds possessing the dimethylamino ( 1a3a ) or nitro substituent ( 1e3e ) ; these modifications enhance ct character and lead to a longer wavelength absorption band lacking vibronic structure . some vibronic structure is visible in the absorption spectra of compounds lacking an auxochrome or possessing the methoxy or cyano groups ; however , these compounds possess some degree of ct in the excited state , as their emission spectra are largely devoid of vibronic progressions . despite the presence of ct character , the allowability of the s0 s1 transition is relatively high for most compounds , as is evident from the good to moderate molar absorptivities . increasing the conjugation length increases the extinction coefficient in a stepwise fashion : compounds 2a2e possess molar absorptivities roughly twice the values observed for 1a1e , whereas compounds 3a3c have molar absorptivities approximately three times greater than those of 1a1e this trend , which reflects the relative magnitudes of transition dipoles , can be directly linked to the spatial overlap of the contributing molecular orbitals , in this case the homo and lumo . in terms of optical compatibility , all of the synthesized probes are compatible with dapi , hoechst 33342 , or blue fluorescent protein filter sets for epifluorescence microscopy , whereas 2a , 2e , and 3a3e are optimally matched to the 405 nm diode laser . absorption ( solid lines ) and emission ( dashed lines ) spectra of ( a ) 1a1e , ( b ) 2a2e , and ( c ) 3a3e in chcl3 . emission intensities are given relative to 3d , which has the highest quantum yield ( see table 2 ) . the emission spectra of 1a3c in chloroform are shown in figure 4 ; most of the probes exhibit good to moderate quantum yields ( table 2 ) . emission intensities are enhanced in less polar solvents such as toluene and are reduced in more polar solvents such as acetonitrile , supporting the existence of excited states with significant ct character ( see supporting information , figure s18 ) . although many of the probes exhibit emission on the blue end of the visible spectrum , several probes show longer - wavelength emission owing to their longer conjugation length and/or the presence of strong electron - donating or -withdrawing groups . of the probes exhibiting strong emission , 3c has the bluest emission maximum ( 42 nm ) , whereas 3e has the reddest emission maximum ( 561 nm ) . surprisingly , strong ct character does not necessarily equate to poor quantum yields , as seen for probes 1a and 2a . indeed , 1a is the only member of the 4-phenylquinazoline series ( 1a1e ) of compounds that exhibits an appreciable degree of fluorescence ( em = 0.37 ) . in the 4-styrylquinazoline ( 2a2e ) series , compounds possessing electron - donating groups ( 2a and 2b ) also exhibit the highest quantum yields . with longer conjugation lengths , quantum yields of fluorescence are markedly higher and the identity of the auxochrome influences em to a lesser degree . the overall brightness ( em ) is one important parameter when considering the utility of the probes as optical reporters . an additional parameter , the turn - on ratio , should also be considered as a measure of the probes responsiveness to changes in their chemical microenvironment . water ( et(30 ) = 63.1 kcal / mol , = 0.89 mpas ) and octanol ( et(30 ) = 48.3 kcal / mol , = 7.24 mpas ) represent two distinct environments that can be used to assess the physical properties and distribution of organic molecules . the ratios of emission intensities obtained in octanol and water are shown in figure 5 . compounds 1a , 2a , and 3e showed the largest on / off ratios with enhancements greater than 50-fold ; moderate ratios , between 20 and 40 were observed for 2d , 3a , 3b , and 3d . moderate enhancements were found for the remaining members of the 2 and 3 series , whereas the remaining 6-arylquinazolines ( series 1 ) showed essentially no emission enhancements . emission intensities obtained in octanol and water reveal that several probes are highly responsive to changes in their chemical microenvironment and possess high on / off ratios . the extension of the aromatic system via the 6-position of the quinazoline core was not anticipated to affect the key binding contacts between the probes and the atp - binding fold of the erbb family . the crystallographically determined binding modes of gefitinib , erlotinib , and lapatinib show that the 6-position is amenable to substitution and , in some cases , decreases koff of inhibitors . to test this assumption , we evaluated selected probes as inhibitors of erbb2 phosphorylation in mcf7 cells initially using two probe concentrations , 10 m and 100 nm ( figure 6 ) . mcf7 cells are a well - established model system for the ligand - induced activation of erbb2erbb3 heterocomplexes by ligands of the neuregulin family ( nrg1 in this case ) . compounds 1a , 2a , 2d , 3d , and 3e were selected because they showed the highest turn - on ratios of their respective series . all five probes showed inhibition of nrg1-induced phosphorylation of erbb2 at 10 m ; however , little to no inhibitory action was observed at 100 nm . these results demonstrate two key features of these fluorescent quinazoline probes : first , despite chemical modification , the probes remain membrane permeable and are able to access the intracellular kinase binding domain of erbb2 . second , the pharmacophore remains an inhibitor of erbb2 phosphorylation despite the presence of bulky extensions of the aromatic system . ( a ) comparative inhibitory properties of selected probes : the inhibition of nrg1-induced erbb2 tyrosine phosphorylation in mcf7 cells was evaluated at 0.1 and 10 m concentrations of the indicated compounds . for comparison ( b ) compounds 3d and 1a show comparable affinity in inhibition studies but a difference in the maximum achievable inhibition . we next obtained the inhibition curves of 1a , which possesses the smallest modification at the 6-position , and 3d , which possesses one of the longest arms . the ki for these probes was similar with values of 10 m for 1a and 9 m for 3d . a direct comparison with actual binding constants for a type i inhibitor such as gefitinib in a cellular setting are difficult to obtain . recombinant egfr kinase domains have yielded an in vitro kd of approximately 1 nm . equilibrium models in live cells treated with 10 m gefitinib have yielded kd estimates of 2 to 3 nm after competition with cellular atp was taken into account . by this standard , the derivatives used in our study show comparable potency . although 1a exhibited a typical inhibition curve profile , with complete inhibition of erbb2 phosphorylation at approximately 20 m , higher concentrations of 3d do not lead to complete inhibition . while 3d is a larger molecule , hydrophobicity does not appear to play a role , as 1a and 3d have similar octanol / water partition coefficients , with log poctanol / water = 1.50 and 1.55 , respectively . the lack of complete inhibition may reflect alternative binding modes for the inhibitors , as similar partial inhibition resulting from competing cellular atp has been described for gefitinib when compared to the complete inhibition by the type ii inhibitor , lapatinib . we are presently evaluating the binding kinetics of these probes and will report their binding modes separately . emission spectroscopy identified several probes ( 1a , 2a , and 3e ) that exhibit large turn - on ratios , and the phosphorylation inhibition studies demonstrate that the modified n - phenyl quinazolines are capable of accessing the atp - binding pocket of erbb2 . to determine if turn - on emission is observed upon binding to erbb2 , we obtained the fluorescence spectra of 1a , 2a , and 3e in pbs in the presence and absence of the soluble erbb2 kinase domain . in pbs alone , the emission of the probes is largely quenched , whereas the addition of the erbb2 kinase domain produced substantial emission enhancements for both of the dimethylamino - substituted probes . in the case of 1a , the emission enhancement was 4-fold when comparing all emission wavelengths , with a maximum enhancement of 10 at 435 nm ( figure 7 ) . for 2a , the emission was increased by a factor of 8 when comparing all emission wavelengths , with a maximum enhancement of 12 at 560 nm . no emission enhancement was observed for 3e despite the fact that it also exhibits solvent sensitivity and was shown to inhibit atp binding to erbb2 . the lack of binding - induced emission enhancement may be a result of the longer conjugated arm projecting beyond the binding pocket and being exposed to the polar solvent environment . ( a ) 1a and ( b ) 2a exhibit turn - on emission upon binding to soluble erbb2 kinase domain . in pbs alone , emission ( in black ) is largely quenched , whereas in the presence of erbb2 kinase , emission ( in green ) is significantly enhanced ; conditions : [ 1a ] = [ 2a ] = 1 m ; [ erbb2 ] = 100 nm , ex = 370 nm . we have synthesized a family fluorescent of quinazoline probes targeting the atp - binding pocket of erbb2 and evaluated the influence of extended conjugation as well as substituent effects through the 6-position of the quinazoline core . our results demonstrate that the optical band gap can be manipulated by varying the conjugation length and auxochrome substitution . depending on the auxochrome identity , the quinazoline core can function either as an electron acceptor or an electron donor to achieve polar ct excited states . the strongest electron - donating and -withdrawing groups ( e.g. , dimethylamine , cyano , and nitro ) yield high on / off ratios , suggesting that they are the best candidates for designing future turn - on probes . importantly , the presence of a fluorophore arm at the 6-position of the quinazoline does not significantly attenuate the ability of the probes to function as inhibitors of erbb2 . two probes , 1a and 2a , were successfully demonstrated as turn - on probes that can report binding to the erbb2 kinase domain in solution . one limitation of the current family of fluorescent inhibitors is their relatively low solubility in aqueous solutions , although this is not unique to their modified structure , as lapatinib shows significant aggregation at physiologically relevant concentrations . our future work will focus on improving the solubility of the probes through chemical modification at either the 7-position of the core ( figure 8) or on the fluorophore arm . r1 , r2 , and r3 are possible sites for chemical modification of 1a and 2a to enhance aqueous solubility . fluorescence studies were performed using probe concentrations of 1 m . for determination of em , solutions were prepared to an optical density of 0.05 or less in order to minimize inner filter effects . quantum chemical calculations were carried out utilizing the gaussian 09 suite of electronic structure modeling software . ground - state geometries of the dyes were optimized by dft with the b3lyp/6 - 31g(d ) method using truhlar s smd solvation model . vertical transition energies were obtained by td - dft calculations with the b3lyp/6 - 31g(d ) with the smd model . mcf cells were seeded with equal quantity ( 200 000/well ) in six - well plates . after 48 h , cells were pretreated with small molecule inhibitors of various concentrations for 1 h before induction by neuregulin ( nrg1 , 20 nm , 15 min ) . erbb2 phosphorylation was evaluated for tyr1239 located close to the extreme cytoplasmic c - terminus of the receptor ( validated by pan - tyrp detection ( 4g10 ) ) . the signal obtained for ptyr1239 relative to the erbb2 receptor levels was determined as the relative receptor phosphorylation . three hundred forty seven milligrams ( 1.0 mmol ) of 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , 248 mg ( 1.5 mmol ) of boronic acid , 425 mg of csco3 ( 1.3 mmol ) , and 3 ml of dmf were placed in a 25 ml schlenk flask with a stirrer bar under argon purge . the reaction mixture was degassed for a further 20 min under a slow stream of argon , at which point 25 mg of a 1:2 mixture of pdcl2(pph3)2 and pph3 was added . the reaction was heated at 80 c for 24 h , cooled , poured into 100 ml of h2o , and extracted with etoac ( 3 50 ml ) . the organic layer was concentrated and purified over silica ( 100% ch2cl2 to 100% etoac ) followed by crystallization from 2-propanol / etoac to afford 1a ( 47 mg , 14% ) ; mp 257259 c ( dec ) ; ir max ( cm ) : 2957.1 , 1934.3 , 1600.9 , 1568.8 , 1523.8 , 1499.1 , 1446.5 , 1403.8 , 1357.4 , 1327.4 , 1290.7 , 1228.1 , 810.1 , 744.4 , 692.5 ; h nmr ( 500 mhz , dmso - d6 ) : 2.98 ( s , 6h ) , 6.87 ( d , 2h , j = 8.9 hz ) , 7.15 ( t , 1h , j = 7.4 hz ) , 7.41 ( t , 2h , j = 8.3 hz ) , 7.78 ( t , 3h 8.4 hz ) , 7.86 ( d , 2h , j = 8.4 hz ) , 8.14 ( dd , 1h , j = 8.7 , 1.8 hz ) , 8.53 ( s , 1h ) , 8.73 ( s , 1h ) , 9.88 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 40.4 , 112.9 , 116 , 118.6 , 123 , 124.1 , 126.8 , 128 , 128.6 , 128.9 , 131.4 , 138.7 , 139.6 , 148.7 , 150.5,.154.2 , 158.1 ; hr - esi ( q - tof ) m / z : calcd for c22h21n4 [ m + h ] , 341.1766 ; found , 341.1770 . five hundred milligrams ( 1.44 mmol ) of 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , 289 mg ( 2.2 mmol ) of 4-vinylanisole , 0.38 ml of et3n ( 2.87 mmol ) , and 5 ml of dmf were placed in a 50 ml schlenk flask with a stirrer bar under argon purge . the reaction mixture was degassed for a further 20 min under a slow stream of argon , at which point 48 mg of pd(oac)2 and 56 mg of pph3 and the subsequent mixture was further degassed and heated at 8085 c for 24 h , cooled , poured into 100 ml of h2o , and extracted with etoac ( 3 50 ml ) . the organic layer was concentrated and purified over silica ( 100% ch2cl2 to 100% etoac ) followed by crystallization from 2-propanol to afford 2b ( 111 mg , 22% ) ; mp : 238241 c ( dec ) ; ir max ( cm ) : 3034.4 , 1600.9 , 1568.2 , 1524.6 , 1512.3 , 1495.0 , 1445.8 , 1409.0 , 1358.4 , 1272.8 , 1250.3 , 1174.0 , 837.0 , 751.3 , 688.5 ; h nmr ( 500 mhz , dmso - d6 ) : 3.80 ( s , 3h ) , 7.00 ( d , 2h , j = 8.7 hz ) , 7.15 ( t , 1h , j = 7.3 hz ) , 7.24 ( d , 1h , j = 16.4 hz ) , 7.407.45 ( m , 3h ) , 7.60 ( d , 2h , j = 8.6 hz ) , 7.76 ( d , 1h , j = 8.6 hz ) , 7.86 ( d , 2h , j = 7.6 hz ) , 8.10 ( d , 1h , j = 8.0 hz ) , 8.55 ( s , 1h ) , 8.67 ( s , 1h ) , 9.79 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 55.6 , 114.8 , 115.9 , 120.2 , 122.8 , 124.2 , 125.7 , 128.3 , 128.5 , 128.9 , 129.9 , 130.1 , 131.6 , 136.0 , 139.6 , 149.6 , 154.5 , 158.0 , 159.7 ; hr - esi ( q - tof ) m / z : calcd for c23h21n3o [ m + h ] , 354.1606 ; found , : 354.1621 . yield : 52 mg , 16% ; mp 245247 c ; ir max ( cm ) : 2999.8 , 2835.4 , 1937.5 , 1600.6 , 1570.2 , 1522.6 , 1496.5 , 1445.4 , 1400.9 , 1358.3 , 1270.5 , 1247.2 , 828.7 , 818.3 753.3 ; h nmr ( 500 mhz , dmso - d6 ) : 3.83 ( s , 3h ) , 7.11 ( d , 1h , j = 8.7 hz ) , 7.15 ( t , 2h , j = 7.4 hz ) , 7.42 ( t , 2h , j = 8.1 hz ) , 7.817.86 ( m , 5h ) , 8.15 ( dd , 1h , j = 8.7 , 1.5 hz ) , 8.57 ( s , 1h ) , 8.79 ( s , 1h ) , 9.93 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 , tfa ) 55.7 , 114.6 , 115.0 , 120.8 , 122.1 , 124.9 , 126.8 , 128.8 , 129.2 , 130.7 , 134.3 , 137.4 , 139.7 , 140.1 , 151.7 , 159.8 , 160.2 ; hrms hr - esi ( q - tof ) m / z : calcd for c21h18n3o [ m + h ] , 328.1449 ; found , : 328.1453 . yield : 41 mg , 14% ; mp 258262 c ; ir max ( cm ) : 3067.3 , 1939.0 , 1600.5 1567.8 , 1528.9 , 1488.6 , 1445.3 , 1405.6 , 1359.8 , 1325.9 , 1295.0 , 840.7 , 748.5 , 689.2 ; h nmr ( 500 mhz , dmso - d6 ) : 7.16 ( t , 1h , j = 7.3 hz ) , 7.407.46 ( m , 3h ) , 7.56 ( t , 2h , j = 7.6 hz ) , 7.857.91 ( m , 5h ) , 8.20 ( d , 1h , j = 8.7 hz ) , 8.59 ( s , 1h ) , 8.86 ( s , 1h ) , 9.96 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) 115.8 , 120.9 , 123.1 , 124.3 , 127.6 , 128.4 , 128.8 , 128.9 , 129.5 , 132.2 , 138.4 , 139.5 , 139.6 , 149.5 , 155.0 , 158.3 ; hr - esi ( q - tof ) m / z : calcd for c c20h16n3 [ m + h ] , 298.1344 ; found , 298.1352 . yield : 48 mg , 15% ; mp 242245 c ; ir max ( cm ) : 3107.2 , 2228.4 , 1624.1 , 1596.7 , 1571.4 , 1527.8 , 1496.9 , 1443.8 , 1403.4 , 1326.6 , 1255.8 , 1106.2 , 829.1 , 747.3 , 690.4 ; h nmr ( 500 mhz , dmso - d6 ) : 7.31 ( t , 1h , j = 7.4 hz ) , 7.57 ( t , 2h , j = 8.3 hz ) , 7.98 ( d , 2h , j = 7.8 hz ) , 8.02 ( d , 1h , j = 8.7 hz ) , 8.19 ( d , 2h , j = 8.5 hz ) , 8.26 ( d , 2h , j = 8.4 hz ) , 8.41 ( dd , 1h , j = 8.7 , 1.9 hz ) , 8.75 ( s , 1h ) , 9.09 ( s , 1h ) , 10.15 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 110.8 , 115.8 , 118.4 , 119.2 , 121.9 , 123.2 , 124.5 , 128.3 , 129.0 , 132.0 , 133.3 , 136.3 , 139.3 , 144.0 , 150.2 , 155.5 , 158.4 ; hr - esi ( q - tof ) m / z : calcd for c21h15n4 [ m + h ] , 323.1296 ; found , 323.1305 . yield : 55 mg , 16% ; mp 262264 c ; ir max ( cm ) : 3089.6 , 1934.3 , 1597.5 , 1569.0 , 1531.1 , 1514.6 , 1488.0 , 1449.5 , 1401.4 , 1417.5 , 1341.8 , 1271.0 , 834.7 , 752.1 , 693.2 ; h nmr ( 500 mhz , dmso - d6 ) : 7.32 ( t , 1h , j = 7.4 hz ) , 7.57 ( t , 2h , j = 8.1 hz ) , 7.98 ( d , 2h , j = 7.6 hz ) , 8.05 ( d , 1h , j = 8.6 hz ) , 8.33 ( d , 2h , j = 8.8 hz ) , 8.44 ( dd , 1h , j = 8.7 , 1.7 hz ) , 8.55 ( d , 2h , j = 8.8 hz ) , 8.76 ( s , 1h ) , 9.14 ( d , 1h , j = 1.3 hz ) , 10.20 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 115.8 , 122.3 , 123.2 , 124.5 , 128.6 , 128.9 , 129.1 , 132.1 , 135.8 , 139.3 , 145.9 , 147.3 , 150.3 , 155.7 , 158.5 ; hr - esi ( q - tof ) m / z : calcd for c20h15n4o2 [ m + h ] , 343.1195 ; found , 343.1194 . yield : 130 mg , 25% ; mp 220 c ( dec ) ; ir max ( cm ) : 3206.0 , 2940.8 , 1646.0 , 1599.4 , 1564.6 , 1521.6 , 1493.6 , 1443.9 , 1403.9 , 1382.3 , 1363.2 , 1254.4 , 957.2 , 752.1 , 691.3 ; h nmr ( 400 mhz , dmso - d6 ) : 2.94 ( s , 6h ) , 6.76 ( d , 2h , j = 8.7 hz ) , 7.107.14 ( m , 2h ) , 7.347.42 ( m , 3h ) , 7.48 ( d , 2h , j = 8.7 hz ) , 7.73 ( d , 1h , j = 8.7 hz ) , 7.87 ( d , 2h , j = 7.8 hz ) , 8.07 ( d , 1h , j = 8.7 hz ) , 8.52 ( s , 1h ) , 8.60 ( s , 1h ) , 9.78 ( s , 1h ) ; c nmr ( 125 mhz , dmso ) : 112.3 , 115.5 , 119.1 , 122.4 , 123.8 , 124.6 , 127.7 , 128.0 , 128.5 , 130.5 , 131.1 , 136.2 , 139.3 , 148.7 , 150.3 , 153.8 , 157.6 ; hr - esi ( q - tof ) m / z : calcd for c24h23n4 [ m + h ] , 367.1923 ; found , 367.1929 . yield : 74 mg , 16% ; mp 248252 c ( dec ) ; ir max ( cm ) : 3048.8 , 1949.9 , 1622.7 , 1600.8 , 1570.5 , 1523.4 , 1494.9 , 1446.5 , 1409.5 , 1385.9 , 1360.5 , 1332.6 , 1254.5 , 827.7 , 747.8 , 687.8 ; h nmr ( 500 mhz , dmso - d6 ) : 7.16 ( t , 1h , j = 7.2 hz ) , 7.32 ( t , 1h , j = 7.2 hz ) , 7.387.51 ( m , 6h ) , 7.66 ( d , 2h , j = 7.4 hz ) , 7.78 ( d , 1h , j = 8.6 hz ) , 7.87 ( d , 2h , j = 7.6 hz ) , 8.14 ( d , 1h , j = 8.3 hz ) , 8.56 ( s , 1h ) , 8.72 ( s , 1h ) , 9.81 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 115.8 , 120.8 , 122.9 , 124.2 , 127.0 , 128.0 , 128.4 , 128.6 , 128.9 , 129.3 , 130.3 , 131.7 , 135.6 , 137.2 , 139.6 , 149.8 , 154.8 , 158.1 ; hr - esi ( q - tof ) m / z : calcd for c22h18n3 [ m + h ] , 324.1500 ; found , 324.1516 . yield : 98 mg , 20% ; mp 250254 c ; ir max ( cm ) : 3071.4 , 2221.6 , 1599.8 , 1571.8 , 1498.4 , 1441.7 , 1403.6 , 1362.3 , 964.8 , 838.4 , 752.4 , 693.8 ; h nmr ( 500 mhz , dmso - d6 ) : 7.15 ( t , 1h , j = 7.4 hz ) , 7.42 ( t , 2 h , j = 8.3 hz ) , 7.56 ( d , 1h , j = 16.4 hz ) , 7.59 ( d , 1h , j = 16.4 hz ) , 7.797 . 89 ( br m , 7h ) , 8.17 ( dd , 1h , j = 8.7 , 1.7 hz ) , 8.57 ( s , 1h ) , 8.76 ( s , 1h ) , 9.88 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 110.2 , 115.8 , 119.3 , 121.8 , 122.9 , 124.2 , 127.5 , 128.5 , 129.1 , 129.2 , 131.9 133.1 , 134.7 , 139.5 , 141.9 , 150.2 , 155.1 , 158.2 ; hr - esi ( q - tof ) m / z : calcd for c23h17n4 [ m + h ] , 349.1453 ; found , 349.1453 . yield : 128 mg , 24% ; mp 240 c ( dec ) ; ir max(cm ) : 3085.1 , 1932.2 , 1602.1 , 1591.5 , 1571.4 , 1527.7 , 1501.7 , 1328.8 , 1404.1 , 1443.2 , 1365.5 , 1255.4 , 1105.2 , 968.0 , 749.5 , 684.1 , 694.9 ; h nmr ( 500 mhz , dmso - d6 ) : 7.30 ( t , 1h , j = 7.3 hz ) , 7.56 ( t , 2h , j = 7.8 hz ) , 7.72 ( d , 1h , j = 16.5 hz ) , 7.76 ( d , 1h , j = 16.5 hz ) , 7.93 ( d , 1h , j = 8.6 hz ) , 8.01 ( t , 4h , j = 7.8 hz ) , 8.30 , ( d , 1h , j = 8.6 hz ) , 8.40 ( d , 2h , j = 8.4 hz ) , 8.71 ( s , 1h ) , 8.91(s , 1h ) , 9.98 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 115.8 , 122.1 , 122.9 , 124.3 , 124.6 , 127.8 , 128.1 , 128.7 , 128.9 , 131.9 , 132.7 , 134.7 , 139.4 , 144.1 , 146.8 , 150.3 , 155.2 , 158.2 ; hr - esi ( q - tof ) m / z : calcd for c22h17n4o2 [ m + h ] , 369.1351 ; found , 369.1346 . yield : 90 mg , 16% ; mp 255258 c ( dec ) ; ir max ( cm ) : 2980.6 , 1596.3 , 1570.0 , 1521.7 , 1496.4 , 1445.6 , 1409.3 , 1385.4 , 1354.0 , 1186.4 , 987.4 , 801.0 , 746.0 , 689.8 ; h nmr ( 500 mhz , dmso - d6 ) : 2.94 ( s , 6h ) , 6.71 ( d , 2h , j = 8.7 hz ) , 6.77 ( d , 2h , j = 15.6 hz ) , 6.95 ( dd , 1h , j = 15.2 , 11.7 hz ) , 7.15 ( t , 1h , j = 7.0 hz ) , 7.27 ( dd , 1h , j = 15.0 , 11.1 hz ) , 7.387.43 ( m , 4h ) , 7.72 ( d , 1h , j = 8.4 hz ) , 7.87 ( d , 2h , j = 7.3 hz ) , 8.00 ( d , 1h , j = 8.3 hz ) , 8.54 ( d , 2h , j = 15.6 hz ) , 9.8 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 112.6 , 115.9 , 119.9 , 122.9 , 124.2 , 125.0 , 125.2 , 128.1 , 128.5 , 128.9 , 129.2 , 131.2 , 132.2 , 134.7 , 136.2 , 139.6 , 149.5 , 150.5 , 154.4 , 158.0 ; hr - esi ( q - tof ) m / z : calcd for c26h25n4 [ m + h ] , 393.2079 ; found , 393.2088 . yield : 70 mg , 13% ; mp 245248 c ( dec ) ; ir max ( cm ) : 3029.9 , 1939.7 , 1599.1 , 1568.7 1525.3 , 1498.5 , 1446.4 , 1410.1 , 1385.1 , 1357.7 , 1300.1 , 1248.8 , 988.5 , 806.7 , 740.2 , 690.7 ; h nmr ( 500 mhz , dmso - d6 ) : 3.84 ( s , 3h ) , 6.77 ( d , 1h , j = 15.5 hz ) , 6.85 ( d , 1h , j = 15.5 hz ) , 6.95 ( d , 2h , j = 8.6 hz ) , 7.07 ( dd , 1h , j = 15.4 , 10.7 hz ) , 7.15 ( t , 1h , j = 7.3 hz ) , 7.29 ( dd , 1h , j = 15.5 , 10.7 hz ) , 7.42 ( t , 2h , j = 7.9 hz ) , 7.51 ( d , 2h , j = 8.5 hz ) , 7.73 ( d , 1h , j = 8.6 hz ) , 7.86 ( d , 2h , j = 7.7 hz ) , 8.04 ( d , 1h , j = 8.6 hz ) , 8.54 ( s , 1h ) , 8.59 ( s , 1h ) , 9.84 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 55.6 , 114.7 , 115.9 , 120.4 , 122.9 , 124.2 , 127.5 , 128.3 , 128.6 , 128.9 , 130.0 , 130.8 , 131.3 , 131.7 , 133.7 , 135.9 , 139.6 , 149.6 , 154.6 , 158.0 , 159.6 ; hr - esi ( q - tof ) m / z : calcd for c25h22n3o [ m + h ] , 380.1763 ; found , 380.1766 . yield : 70 mg , 14% ; mp 258260 c ( dec ) ; ir max ( cm ) : 3259.1 , 3055.1 , 1924.2 , 1604.2 , 1569.2 , 1528.3 , 1496.2 , 1440.3 , 1406.5 , 1388.2 , 1363.4 , 1329.5 , 1310.3 , 1253.8 , 978.0 , 901.4 , 750.3 , 686.5 ; h nmr ( 500 mhz , dmso - d6 ) : 6.81 ( d , 1h , j = 15.2 hz ) , 6.91 ( d , 1h , j = 15.2 hz ) , 7.11 ( t , 1h , j = 7.2 hz ) , 7.197.32 ( m , 3h ) , 7.38 ( d , 2h , j = 7.4 hz ) , 7.39 ( d , 2h , j = 7.4 hz ) , 7.56 ( d , 2h , j = 7.6 hz ) , 7.68 ( d , 1h , j = 8.5 hz ) , 7.80 ( d , 2h , j = 7.6 hz ) , 8.01 ( d , 1h , j = 8.5 hz ) , 8.47 ( s , 1h ) , 8.58 ( s , 1h ) , 9.84 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 116.1 , 121.1 , 123.0 , 123.8 , 126.9 , 128.2 , 128.4 , 128.8 , 129.2 , 129.8 , 131.0 , 132.3 , 133.7 , 135.3 , 137.4 , 140.7 , 149.9 , 154.9 , 158.1 ; hr - esi ( q - tof ) m / z : calcd for c24h20n3 [ m + h ] , 350.1657 ; found , 350.1673 . yield : 134 mg , 25% ; mp 242245 c ( dec ) ; ir max ( cm ) : 3365.8 , 3028.7 , 2224.5 , 1598.1 , 1568.6 , 1525.6 , 1496.8 , 1443.5 , 1402.4 , 1387.6 , 1361.7 , 1252.8 , 983.0 , 746.2 , 848.0 , 687.4 ; h nmr ( 500 mhz , dmso - d6 ) : 7.02 ( d , 1h , j = 15.2 hz ) , 7.16 ( d , 1h , j = 15.1 hz ) , 7.30 ( t , 1h , j = 7.4 hz ) , 7.467.60 ( m , 4h ) , 7.90 ( d , 3h , j = 8.5 hz ) , 7.96 ( d , 2h , j = 8.35 hz ) , 8.00 ( d , 2h , j = 7.65 hz ) , 8.22 ( dd , 1h , j = 8.7 , 1.0 hz ) , 8.69 ( s , 1h ) , 8.79 ( s , 1h ) , 9.98 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 109.9 , 115.8 , 119.4 , 121.4 , 122.9 , 124.2 , 127.5 , 128.7 , 128.9 , 130.6 , 131.3 , 131.8 , 133.0 , 133.5 , 134.4 , 135.2 , 139.5 , 142.1 , 150.1 , 154.9 , 158.1 ; hr - esi ( q - tof ) m / z : calcd for c25h19n4 [ m + h ] , 375.1609 ; found , 375.1610 . yield : 113 mg , 20% ; mp 273275 c ( dec ) ; ir max ( cm ) : 3421.3 , 3020.7 , 1927.9 , 1603.3 , 1585.6 , 1571.5 , 1557.7 , 1535.3 , 1494.4 , 1444.3 , 1331.5 , 1253.8 , 1109.3 , 987.1 , 743.6 , 804.3 ; h nmr ( 500 mhz , dmso - d6 ) : 6.92 ( d , 1h , j = 15.4 hz ) , 7.03 ( d , 1h , j = 15.3 hz ) , 7.13 ( t , 1h , j = 7.2 hz ) , 7.327.41 ( m , 3h ) , 7.48 ( dd , 1h , j = 15.4 , 10.7 hz ) , 7.73 ( d , 1h , j = 8.6 hz ) , 7.81 ( d , 2h , j = 8.8 hz ) , 7.83 ( d , 2h , j = 8.8 hz ) , 8.08 ( d , 1h , j = 8.6 hz ) , 8.20 ( d , 2h , j = 8.6 hz ) , 8.53 ( s , 1h ) , 8.64 ( s , 1h ) , 9.86 ( s , 1h ) ; c nmr ( 125 mhz , dmso - d6 ) : 115.8 , 121.5 , 123.0 , 124.3 , 124.4 , 127.6 , 128.7 , 128.9 , 130.6 , 131.4 , 132.0 , 134.5 , 135.1 , 135.2 , 139.5 , 144.2 , 146.6 , 150.1 , 155.0 , 158.1 ; hr - esi ( q - tof ) m / z : calcd for c24h19n4o2 [ m + h ] , 395.1508 ; found , 395.1503 .

fluorescent n - phenyl-4-aminoquinazoline probes targeting the atp - binding pocket of the erbb family of receptor tyrosine kinases are reported . extension of the aromatic quinazoline core with fluorophore arms through substitution at the 6- position of the quinazoline core with phenyl , styryl , and phenylbutadienyl moieties was predicted by means of td - dft calculations to produce probes with tunable photoexcitation energies and excited states possessing charge - transfer character . optical spectroscopy identified several synthesized probes that are nonemissive in aqueous solutions and exhibit emission enhancements in solvents of low polarity , suggesting good performance as turn - on fluorophores . ligand - induced erbb2 phosphorylation assays demonstrate that despite chemical modification to the quinazoline core these probes still function as erbb2 inhibitors in mcf7 cells . two probes were found to exhibit erbb2-induced fluorescence , demonstrating the utility of these probes as turn - on , fluoroescent kinase inhibitors .

Introduction Results and Discussion Conclusions Experimental Section

first , the structural and functional homology of kinases combined with the abundance of atp - binding folds throughout the cell make off - target binding events unavoidable . second , compensatory or alternate modes of pathway activation can lead to the development of resistance , requiring additional antineoplastic drugs or a different chemotherapy regimen . in an effort to develop molecular probes capable of interrogating kinase signaling pathways , we have introduced the concept of turn - on fluorescent ligands that target the atp - binding pocket of the egfr / erbb family of receptor tyrosine kinases . these molecular probes are built upon the 4-aminoquinazoline scaffold , which is an established egfr / erbb pharmacophore ; examples include gefitinib or erlotinib ( figure 1a , c ) , so - called type i inhibitors that preferentially binding the active kinase conformation , and lapatinib ( figure 1b , d ) , a type ii inhibitor targeting the inactive conformation . the preference for active and inactive conformations is addressed through substitution at the 4-amino position ( figure 1e ) . while the quinazoline core conveys binding to the nucleotide pocket , but with limited specificity for erbb - type receptors , the 4-amino aryl arm further increases specificity and contributes discrimination between activation states . this selectivity reflects increased access to the hydrophobic pocket adjacent to the nucleotide binding site in the inactive conformation . together , the quinazoline core and n - aryl arm constitute the pharmacophore . crystal structures of the kinase domain of egfr with either erlotinib ( 1m17 ) or lapatinib ( 1xkk ) ( figure 1a , b ) show that while the pharmacophore arm is oriented deep in the binding pocket , the 6-position is amenable to chemical modification without perturbing the key conserved contacts of the binding pocket . the structure of lapatinib demonstrates this point as the aromatic core is extended by the addition of a furan ring . thus , a potential strategy for synthesizing fluorescent kinase inhibitors is to modify the 4-aminoquinazoline core with fluorphore arms ( figure 1e ) at the 6-position . crystal structures of the egfr atp - binding pocket with ( a ) erlotinib ( pdb i d : 1m17 ) and ( b ) lapatinib ( pdb i d : 1xkk ) reveal the inhibitor binding modes . the arms at the 6-position of the quinazoline core ( in blue ; c , d ) may be replaced by fluorophore arms without disturbing the key binding contacts . herein , we investigate the effect of conjugation length and auxochrome substitution on the optical properties of a family of n - phenyl-4-aminoquinazoline probes . we find that extension of the framework effectively lowers the excitation and emission energies , yielding fluorescent probes that compare favorably with other fluorescent adenosine analogues . the introduction of strong electron donors or strong electron - withdrawing groups generates donor acceptor systems that are highly sensitive to solvent polarity . as a result , several probes exhibit high fluorescence on / off ratios , a feature that is key to their performance as self - reporting fluorescent kinase inhibitors . despite the modifications to the quinazoline core , we also found that several of these probes inhibit erbb2 phosphorylation in a live cell setting , demonstrating that binding to the atp pocket and cell permeability are preserved . overall , these fluorescent adenosine mimics compare favorably with other nucleobase analogues as they possess tunable optical properties and high fluorescence turn - on ratios and compete effectively with the native substrate to inhibit tyrosine phosphorylation . the optical properties of kinase - binding probes of general structure i can be addressed through the fluorophore arm depicted in figure 1e . we envisaged tuning two parameters : ( 1 ) the electron - donating ability of the substituent on the phenyl ring and ( 2 ) the conjugation length between the phenyl ring and the quinazoline core . td - dft calculations , vide infra , guided the selection of the electron - donating and electron - withdrawing substituents that span the range from the strongly donating dimethylamino group ( e.g. the 15-membered family of quinazolines , 1a3e ( figure 1 ) , was synthesized from a common intermediate , 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , by suzuki coupling of the appropriate arylboronic acid ( 1a1e , scheme 1 , route a ) or by heck coupling ( scheme1 , route b ) of the styryl ( 2a2e ) and phenyl butadiene ( 3a3e ) arms . two photophysical properties , the fluorescence on / off ratio and the optical band gap , of the kinase - binding probes can simultaneously be addressed by varying the donor or acceptor substitution and the conjugation length . to achieve this emission behavior , a probe should possess an excited state with a high degree of charge - transfer ( ct ) character and possibly access at twisted intramolecular charge - transfer ( tict ) excited state . td - dft calculations ( 6 - 31 g * , chcl3 , smd solvent model ) show that both strong electron donors and strong electron acceptors will lead to an s1 state with ct character . the presence of the strong electron - donating dimethylamino group more effectively lowers the homo lumo band gap by 0.50.3 ev when comparing 1a3a and 1a3c , whereas the strongly electron - withdrawing nitro group has the greatest effect , with differences of 0.8 to 0.5 ev when comparing 1a3c and 1a3e . in addition to manipulating the optical band gap , the presence of an electron - donating or electron - withdrawing substituent on the pendant phenyl arm influences the ct character of the excited state ; the polarization and spatial segregation of the homo and lumo is directly linked to the identity of the substituent . , 2a ; figure 2 ) , the homo is largely localized to the fluorophore arm , whereas the lumo is polarized toward the quinazoline core . in the case of 2e ( figure 2 ) , the homo is polarized toward the quinazoline core , whereas the lumo is largely localized to the styryl arm ; an excited state with high ct character still results from the promotion of an electron from the homo to the lumo . compounds 2b2d show a gradual redistribution of the homo and lumo densities between the two extreme cases , 2a and 2e . similar polarization of the frontier molecular orbitals is also observed in the 1a1e ( figure s16 ) and 3a3e series ( figure s17 ) . frontier molecular orbitals of 2a2e calculated at the cam - b3lyp/6 - 31 g * level : the polarization of the homo and lumo shifts across the series ( compare 2a and 2e ) ; strong ct character is expected for both 2a and 2e , although the localization of charge should be reversed . probes 1a3c were designed as turn - on fluorescent ligands and thus were expected to be nonemissive in solvents of high polarity ( e.g. this behavior can be qualitatively observed by eye as in chloroform ( et(30 ) = 39.1 kcal / mol ) solutions ; most of the probes appear bright , with emissions ranging from blue ( 2b and 3c ) to green ( 1a and 2a ) to yellow or orange ( 3a and 3e ) , figure 3 , whereas in aqueous solutions , weak or no emission was observed . chloroform solutions of selected probes ( 5 m ) under uv illumination ( 354 nm ) ; aqueous solutions showed weak or no emission ( vials not shown ) . despite the presence of ct character , the allowability of the s0 s1 transition is relatively high for most compounds , as is evident from the good to moderate molar absorptivities . increasing the conjugation length increases the extinction coefficient in a stepwise fashion : compounds 2a2e possess molar absorptivities roughly twice the values observed for 1a1e , whereas compounds 3a3c have molar absorptivities approximately three times greater than those of 1a1e this trend , which reflects the relative magnitudes of transition dipoles , can be directly linked to the spatial overlap of the contributing molecular orbitals , in this case the homo and lumo . in terms of optical compatibility , all of the synthesized probes are compatible with dapi , hoechst 33342 , or blue fluorescent protein filter sets for epifluorescence microscopy , whereas 2a , 2e , and 3a3e are optimally matched to the 405 nm diode laser . absorption ( solid lines ) and emission ( dashed lines ) spectra of ( a ) 1a1e , ( b ) 2a2e , and ( c ) 3a3e in chcl3 . the emission spectra of 1a3c in chloroform are shown in figure 4 ; most of the probes exhibit good to moderate quantum yields ( table 2 ) . emission intensities are enhanced in less polar solvents such as toluene and are reduced in more polar solvents such as acetonitrile , supporting the existence of excited states with significant ct character ( see supporting information , figure s18 ) . although many of the probes exhibit emission on the blue end of the visible spectrum , several probes show longer - wavelength emission owing to their longer conjugation length and/or the presence of strong electron - donating or -withdrawing groups . of the probes exhibiting strong emission , 3c has the bluest emission maximum ( 42 nm ) , whereas 3e has the reddest emission maximum ( 561 nm ) . indeed , 1a is the only member of the 4-phenylquinazoline series ( 1a1e ) of compounds that exhibits an appreciable degree of fluorescence ( em = 0.37 ) . the overall brightness ( em ) is one important parameter when considering the utility of the probes as optical reporters . an additional parameter , the turn - on ratio , should also be considered as a measure of the probes responsiveness to changes in their chemical microenvironment . compounds 1a , 2a , and 3e showed the largest on / off ratios with enhancements greater than 50-fold ; moderate ratios , between 20 and 40 were observed for 2d , 3a , 3b , and 3d . moderate enhancements were found for the remaining members of the 2 and 3 series , whereas the remaining 6-arylquinazolines ( series 1 ) showed essentially no emission enhancements . the extension of the aromatic system via the 6-position of the quinazoline core was not anticipated to affect the key binding contacts between the probes and the atp - binding fold of the erbb family . the crystallographically determined binding modes of gefitinib , erlotinib , and lapatinib show that the 6-position is amenable to substitution and , in some cases , decreases koff of inhibitors . to test this assumption , we evaluated selected probes as inhibitors of erbb2 phosphorylation in mcf7 cells initially using two probe concentrations , 10 m and 100 nm ( figure 6 ) . mcf7 cells are a well - established model system for the ligand - induced activation of erbb2erbb3 heterocomplexes by ligands of the neuregulin family ( nrg1 in this case ) . compounds 1a , 2a , 2d , 3d , and 3e were selected because they showed the highest turn - on ratios of their respective series . these results demonstrate two key features of these fluorescent quinazoline probes : first , despite chemical modification , the probes remain membrane permeable and are able to access the intracellular kinase binding domain of erbb2 . second , the pharmacophore remains an inhibitor of erbb2 phosphorylation despite the presence of bulky extensions of the aromatic system . ( a ) comparative inhibitory properties of selected probes : the inhibition of nrg1-induced erbb2 tyrosine phosphorylation in mcf7 cells was evaluated at 0.1 and 10 m concentrations of the indicated compounds . we next obtained the inhibition curves of 1a , which possesses the smallest modification at the 6-position , and 3d , which possesses one of the longest arms . the ki for these probes was similar with values of 10 m for 1a and 9 m for 3d . we are presently evaluating the binding kinetics of these probes and will report their binding modes separately . emission spectroscopy identified several probes ( 1a , 2a , and 3e ) that exhibit large turn - on ratios , and the phosphorylation inhibition studies demonstrate that the modified n - phenyl quinazolines are capable of accessing the atp - binding pocket of erbb2 . to determine if turn - on emission is observed upon binding to erbb2 , we obtained the fluorescence spectra of 1a , 2a , and 3e in pbs in the presence and absence of the soluble erbb2 kinase domain . in pbs alone , the emission of the probes is largely quenched , whereas the addition of the erbb2 kinase domain produced substantial emission enhancements for both of the dimethylamino - substituted probes . the lack of binding - induced emission enhancement may be a result of the longer conjugated arm projecting beyond the binding pocket and being exposed to the polar solvent environment . ( a ) 1a and ( b ) 2a exhibit turn - on emission upon binding to soluble erbb2 kinase domain . we have synthesized a family fluorescent of quinazoline probes targeting the atp - binding pocket of erbb2 and evaluated the influence of extended conjugation as well as substituent effects through the 6-position of the quinazoline core . our results demonstrate that the optical band gap can be manipulated by varying the conjugation length and auxochrome substitution . depending on the auxochrome identity , the quinazoline core can function either as an electron acceptor or an electron donor to achieve polar ct excited states . , dimethylamine , cyano , and nitro ) yield high on / off ratios , suggesting that they are the best candidates for designing future turn - on probes . importantly , the presence of a fluorophore arm at the 6-position of the quinazoline does not significantly attenuate the ability of the probes to function as inhibitors of erbb2 . two probes , 1a and 2a , were successfully demonstrated as turn - on probes that can report binding to the erbb2 kinase domain in solution . one limitation of the current family of fluorescent inhibitors is their relatively low solubility in aqueous solutions , although this is not unique to their modified structure , as lapatinib shows significant aggregation at physiologically relevant concentrations . our future work will focus on improving the solubility of the probes through chemical modification at either the 7-position of the core ( figure 8) or on the fluorophore arm . r1 , r2 , and r3 are possible sites for chemical modification of 1a and 2a to enhance aqueous solubility . ground - state geometries of the dyes were optimized by dft with the b3lyp/6 - 31g(d ) method using truhlar s smd solvation model . vertical transition energies were obtained by td - dft calculations with the b3lyp/6 - 31g(d ) with the smd model . erbb2 phosphorylation was evaluated for tyr1239 located close to the extreme cytoplasmic c - terminus of the receptor ( validated by pan - tyrp detection ( 4g10 ) ) . the signal obtained for ptyr1239 relative to the erbb2 receptor levels was determined as the relative receptor phosphorylation . three hundred forty seven milligrams ( 1.0 mmol ) of 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , 248 mg ( 1.5 mmol ) of boronic acid , 425 mg of csco3 ( 1.3 mmol ) , and 3 ml of dmf were placed in a 25 ml schlenk flask with a stirrer bar under argon purge . the reaction was heated at 80 c for 24 h , cooled , poured into 100 ml of h2o , and extracted with etoac ( 3 50 ml ) . five hundred milligrams ( 1.44 mmol ) of 6-iodo - n - phenyl-4-quinazolin - amine ( 4 ) , 289 mg ( 2.2 mmol ) of 4-vinylanisole , 0.38 ml of et3n ( 2.87 mmol ) , and 5 ml of dmf were placed in a 50 ml schlenk flask with a stirrer bar under argon purge . the reaction mixture was degassed for a further 20 min under a slow stream of argon , at which point 48 mg of pd(oac)2 and 56 mg of pph3 and the subsequent mixture was further degassed and heated at 8085 c for 24 h , cooled , poured into 100 ml of h2o , and extracted with etoac ( 3 50 ml ) .

the far eastern catfish silurus asotus is a member of the freshwater siluridae family . in korea , its annual production is about 8,000 metric tons ( kim et al . , 1988 ; although the demand for this species has increased in recent years , two factors limit the profitability of culturing far eastern catfish . first , the growth rate shows significant sexual dimorphism because females grow much faster than males ( kim et al . , 2001 ) . this hinders stock management and often results in cannibalism of young fish in farms . second , precocious maturation before the fish reach marketable sizes necessitates prolonged cultivation beyond sexual maturity . after reaching sexual maturity , catfish exhibit reduced growth and decreased feeding efficiency ( choi et al . , 1992 ) . cryopreservation of spermatozoa could be useful for artificial propagation , which may improve the genetic characteristics of this species . cyropreservation techniques have already been reported for spermatozoa from several fish species ( linhart et al . , 1993 ; conget et al . , 1996 ; linhart et al . , 2004 ) . cryopreserved spermatozoa may be used in research and in aquaculture by enabling the introduction of genes from wild or native populations into hatchery stocks if spawning times do not overlap , and increases the availability of spermatozoa for use in selective breeding programs , especially during seasonal shortages of males and/or females . furthermore , cryopreserved spermatozoa can be exchanged between countries located in both hemispheres , instead of exchanging fish , a particular advantage when the importation of live animals is prohibited ( conget et al . , 1996 ) . marian & krasznai ( 1987 ) were the first to successfully cryopreserve spermatozoa from european catfish , s. glanis . they used testicular spermatozoa for freezing , because artificially stripped spermatozoa were polluted with urine , which activated and destroyed the fertilization capacity within a few minutes ( linhart et al . , 1987 ) . this problem was overcome by storing spermatozoa in diluents , and the cryopreservation techniques were refined in later studies , in which the optimization of the diluents extended the viability and freezing rates of spermatozoa ( linhart et al . cryopreservation procedures have been successfully developed for five other catfish species : african catfish , clarias gariepinus ; channel catfish , ictalurus punctatus ; blue catfish , i. furcatus ; pseudoplatystoma corruscans ; and striped catfish , pangasius hypophthalmus , but only using testicular spermatozoa ( steyn et al . , 1985 ; christensen & tiersch , 1997 ; urbanyi et al . , 1999 ; horvath & urbanyi , 2000 ; rurangwa et al . , 2001 ; , 2003 ; kwantong & bart , 2003 ; lang et al . , 2003 ) . the aim of the present study was to optimize cryopreservation methods for ex situ cryopreservation of spermatozoa from far eastern catfish . the success of the cryopreservation procedure was evaluated in terms of the spermatozoa activity index ( sai ) and hatching rates of eggs fertilized with thawed cryopreserved sperm . far eastern catfish , silurus asotus were cultured according to the methods of kim et al . ( 2001 ) . mature females were induced to spawn by a single intraperitoneal ( ip ) injection of human chorionic gonadotropin ( hcg ; sigma , st . louis , mo , usa ) administered at 1,000 iu / kg body weight ( bw ) . spermatozoa were obtained by incision of testes from mature males that had also been given an ip injection of hcg at 500 iu / kg bw . the eggs were fertilized with spermatozoa diluted in saline using the wet method ( kim et al . , 2001 ) . the eggs were left to fertilize for 5 min and then rinsed to remove excess spermatozoa . the meanstandard deviation ( sd ) water temperature was 241.5 and the oxygen concentration was kept close to saturation ( meansd : 9.40.3 mg / l ) . the experimental fish were fed twice daily at 2% of the mean body weight throughout the experimental period ( 2 years ) . specimens were obtained from 2-year - old hatchlings at a meansd bw and standard length of 404.147.36 g and 42.95.11 cm , respectively . on 22 april 2010 , 50 fishes were randomly captured and anesthetized with 200 ppm lidocaine - hcl/1,000 ppm nahco3 to remove the testes . spermatozoa were obtained from 8 males by incision of the surgically removed testes after an ip injection of hcg at 1,000 iu / kg bw ( fig . 1 ) . spermatozoa were pooled from four fish with a spermatozoa motility index of > 80% , and were used for cryopreservation . the spermatozoa concentration was determined using a thoma hemocytometer ( depth : 0.0025 mm ; neubauer , germany ) under a microscope ( 1,000 ; ch 20 ; olympus , tokyo , japan ) and the value is expressed as the mean number of spermatozoa in 20 squares of the thoma cell . the diluents used in this study were alserver s solution ( 2.05 g glucose , 0.4 g sodium chloride , and 0.8 g sodium citrate/100 ml distilled water ) and 0.3 m glucose solution ( 54.3 g glucose/1,000 ml distilled water ) . a maximum of 8 ml of sperm was added to 40 ml of diluent to maintain a dilution rate of 1:5 ( sperm / diluent ) to prevent spontaneous initiation of spermatozoa activity ( linhart et al . , after collection , the containers were stored under aerobic conditions on ice at 0 for 1 hour . sperm activity was assessed ( table 1 ) and sai was assessed under a microscope and using video records ( n = 100 ) . a : testis in the inside of abdominal cavity ; b : sugically removed matured testis . sai : ( score % motile sperm)/100 ( after strssmann et al . , 1994 ) . we compared the effect of two cryoprotectants ( dimethyl sulfoxide [ dmso ] and ethylene glycol ) at different concentrations and in various combinations , as well as five different storage times . the final concentrations of dmso and ethylene glycol were 5% , 10% , 15% , and 20% . one milliliter of mixture was transferred into a 1.8 ml cryotube and immediately placed in a controlled rate cryofreezer ( planer kryo 10 series iii ; planer , sunbury - on - thames , uk ) that was set at + 4 and programmed to cool from + 4 to 9 at a rate of 4/min , and from 9 to 80 at a rate of 11/min . the tube was then transferred into liquid nitrogen and stored for 1 , 180 , 360 , 540 , or 720 days . after storage for the indicated time , frozen spermatozoa were thawed in a water bath at 15 , 25 , 35 , or 45 for 90 sec . for fertilization , 5 g of eggs ( 145 eggs per 1 g ) was placed in a 50 ml dish . then , 104,000 thawed or unfrozen spermatozoa per egg were dropped onto the eggs using a micropipette . after shaking the dish at 200 rpm with a 10 mm deflection , 5 ml of activation solution ( 17 mm nacl , 5 mm tris - hcl , ph 8) at 22 was added . after 3 min , about 100 eggs were placed into a special incubator cage containing 200 ml of ultraviolet - sterilized recirculated tap water at 23 ( 9 the dead eggs were counted and removed from each cage 1 day after fertilization and after hatching ( approximately 2.5 days after fertilization ) . the hatching rate ( hr ) was calculated for each cage using the following formula ( linhart et al . , 2005 ) : where , hl = number of hatched larvae and et = total number of eggs placed in the cage . to determine the proportion of immobile spermatozoa and for scanning electron microscopy ( sem ) analysis , 100 l of stored spermatozoa , diluted 1:100 in pbs , was placed in the center of a coverslip coated with 50 g / ml poly - l - lysine . cells were left to adhere for 30 min and fixed in 1% glutaraldehyde and 1% sucrose in pbs . they were then dehydrated in ethanol , subjected to critical point freezing , and sprayed with gold . we observed 100 spermatozoa per group at 720 days after storage , and counted the number of normal and abnormal spermatozoa in each group . one - way analysis of variance was used to test the statistical significance ( p<0.05 ) of differences among experimental groups . the spermatozoa from far eastern catfish , s. asotus showed good quality ( table 2 ) . the sai was greater for spermatozoa diluted in alserver s solution than for those diluted in glucose . the sai of spermatozoa diluted sperm in both solutions decreased with increasing duration of storage ; therefore , better results were obtained when spermatozoa were stored for 1 hour than when stored for 24 hours ( table 2 ) . sai : ( score % motile sperm)/100 ( after strssmann et al . , 1994 ) . values in same row having the different superscripts are significantly different ( n = 100 , p<0.05 ) . the sai values of far eastern catfish spermatozoa stored in two diluents and with different concentrations of the cryoprotectants are shown in table 3 . the sai gradually decreased with increasing storage duration in all of the experimental groups . the sai of spermatozoa diluted in alserver s solution and dmso gradually increased with increasing dmso concentration ( p<0.05 ) . the optimal concentration of dmso in alserver s solution was 20% for all storage durations . spermatozoa stored in 5% , 10% , or 15% dmso were immobilized after storage for 360 days , while those stored in 20% dmso were immobilized after 720 days . the sai of spermatozoa stored in alserver s solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the optimal concentration of ethylene glycol in alserver s solution was 15% for all storage durations . the spermatozoa were not immo bilized by storage in ethylene glycol for any storage duration . sai : ( score % motile sperm)/100 ( after strssmann et al . , 1994 ) . the temperature of storage is 80. meansd ( n=100 ) values are shown . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . experiments are triplicate . as shown in table 3 , the effects of dilution in glucose solution containing dmso or ethylene glycol were similar to those of alserver s solution . the sai of spermatozoa diluted in glucose solution and dmso gradually increased with increasing concentration of dmso ( p<0.05 ) . the sai was lower in spermatozoa diluted in glucose solution plus dmso than in spermatozoa diluted in alserver s solution plus dmso . the optimal concentration of dmso in glucose solution was 20% for all storage durations . spermatozoa stored in 5% , 10% , and 15% dmso were immobilized after 360 days , while those stored in 20% dmso were immobilized after 720 days . the sai of spermatozoa stored in glucose solution plus ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased when stored in 20% ethylene glycol ( p<0.05 ) . the sai of spermatozoa stored in glucose solution plus ethylene glycol was lower than that of spermatozoa stored in alserver s solution plus ethylene glycol . the optimal concentration of ethylene glycol in glucose solution was 15% for all storage durations . spermatozoa stored in glucose solution plus 5% or 10% ethylene glycol were immobilized after 540 days . by contrast , spermatozoa stored in 15% or 20% ethylene glycol were not immobilized at any storage duration . the trends in hatching rates were similar to those for sai and are shown in table 4 . the hatching rate in all of the experimental groups gradually decreased with increasing spermatozoa storage duration . the hatching rates of spermatozoa stored in alserver s solution and dmso gradually increased with increasing concentration of dmso ( p<0.05 ) . at all storage durations , the hatching rate was higher for spermatozoa stored in 20% dmso than for those stored in other concentrations of dmso . none of the eggs hatched after fertilization with spermatozoa stored in 5% , 10% , or 15% dmso for 360 days . the hatching rates for eggs fertilized with spermatozoa stored in alserver s solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the hatching rate was higher for eggs fertilized with spermatozoa stored in 15% ethylene glycol than in other concentrations of ethylene glycol . all of the eggs hatched after fertilization with spermatozoa stored in ethylene glycol for any duration . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . the hatching rate ( hr ) was calculated for each cage from the number of hatched larvae ( hl ) and from the total number of eggs placed in the cage ( et ) as follows : hr = ( hl / et ) 100 ( after linhart et al . , as shown in table 4 , the hatching rates of eggs fertilized with spermatozoa stored in glucose and dmso gradually increased with increasing concentration of dmso . at all storage durations , the hatching rate was highest for spermatozoa stored in 20% dmso than in other concentrations of dmso ( p<0.05 ) . none of the eggs hatched after fertilization with spermatozoa stored in 5% , 10% , or 15% dmso for 360 days , whereas all the eggs hatched after fertilization with spermatozoa stored in 20% dmso . the hatching rate of eggs fertilized with glucose solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the hatching rate was higher for eggs fertilized with spermatozoa stored in 15% ethylene glycol than in other concentrations of ethylene glycol . none of the eggs hatched after fertilization with spermatozoa stored in 5% or 10% ethylene glycol for 720 days . by contrast , the eggs hatched after fertilized with spermatozoa stored in 15% or 20% ethylene glycol for all of the storage durations . normal morphology was defined as the presence of a head and a flagellum ( fig . 2a , b ) , and the absence of a midpiece sleeve on sem images . the first type consisted of spermatozoa with a double - stranded flagellum ( type a ; fig . 2c ) , and these originate from abnormal spermatogenesis before cryopreservation . in the second type , the flagellum is cut by ice crystals or damaged during thawing ( type b ; fig . the proportion of immobile spermatozoa after storage for 720 days is shown in table 5 . the proportions of type a spermatozoa were not significantly different among the experimental groups . the proportion of type b spermatozoa stored in alserver s solution was lower at 5% dmso than at other concentrations of dmso . the proportions of type b spermatozoa were not significantly different among 10% , 15% , and 20% dmso . the proportion of type b spermatozoa stored in alserver s solution was lower at 5% ethylene glycol than at other concentrations . the trends in the proportions of type b spermatozoa were similar between ethylene glycol and dmso . a : fresh spermatozoa ; b : high power view of normal spermatozoa ; c : abnormal spermatozoa having a flagellum of two strands ; d : a flagellum of inability spermatozoa cut by ice crystal or thawing process after 720 days storage . bars are 1 m . the temperature of storage is 80. meansd ( n=100 ) values are shown . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . the proportions of type b spermatozoa were similar between those stored in glucose solution and alserver s solution when using dmso . the proportions of type b spermatozoa were also similar between the two diluents when using ethylene glycol . however , the proportions of type b spermatozoa stored in alserver s solution or glucose solution were significantly lower in ethylene glycol than in dmso . the proportion of immobile spermatozoa in the absence of a cryoprotectant was not significantly different between spermatozoa stored in alserver s solution or glucose solution . the proportion of immobile spermatozoa was significantly higher when stored without a diluent or a cryoprotectant compared with other groups . the proportion of immobile spermatozoa was lower for fresh sperm than for the other groups . the effects of storage duration and thawing temperature on the sai of spermatozoa are shown in fig . increased , sai gradually decreased as storage increased from 1 day to 360 days . however , sai after storage for 540 days and 720 days increased as the thawing temperature increased from 15 to 35 ( p<0.05 ) . the spermatozoa stored for 720 days were not immobilized by thawing at 45. the sai after storage for 540 days decreased as the thawing temperature increased from 35 to 45 ( p<0.05 ) . the mean sai after storage for 540 days and thawing at 35 and 45 was 0.30.07 and 0.1 0.07 , respectively . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . diluent and concentrations of cryoprotectant using this experiment were alsever s solution and 15% ethylene glycol . the effects of storage duration and thawing temperature on the hatching rate are shown in fig . 4 . the hatching rates gradually decreased with increasing thawing temperature for all storage durations ( p<0.05 ) . the hatching rates after storage for 1 or 180 days were not significantly affected by thawing temperatures of 15 - 35. similarly , the hatching rates at 360 , 540 , and 720 days were not significantly affected by thawing temperatures of 15 - 25. at all storage durations , the hatching rates were higher when fertilizing eggs with spermatozoa thawed at 15 or 25 compared with the other thawing temperatures . based on the sai and hatching rates , our results suggest that the optimal thawing temperature of far eastern catfish spermatozoa is 25. meansd ( n= 100 ) values are shown . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . diluent and concentrations of cryoprotectant using this experiment were alsever s solution and 15% ethylene glycol . in our study of far eastern catfish , 104,000 spermatozoa per egg were dropped onto unfertilized eggs using a micropipette . when optimizing the freezing protocol of cryopreserved spermatozoa , it is necessary to use a small or moderate number of spermatozoa per egg . for european catfish , silurus glanis , 80,00 - 80,000 fresh spermatozoa per egg are needed for artificial insemination , but hatcheries frequently use 100,000 - 200,000 spermatozoa per egg ( linhart et al . , 2004 ) . the use of a large number of spermatozoa per egg may mask the low viability or poor quality of thawed spermatozoa . moreover , it may not be practical to use large numbers of spermatozoa in artificial propagation because male european catfish generally produce a small volume of sperm . the dilution of sperm and eggs in water is another factor that must be adjusted . one part sperm to 25 parts water to 25 parts eggs was optimal for artificial propagation of european catfish in a previous study ( linhart et al . , 2004 ) . the freezing procedure applied in our study was based on cryopreservation studies of tiger puffer , takifugu rubripes , and european catfish , although the procedure was also successfully applied in far eastern catfish ( chang et al . , 1997 ; linhart et al . , 2005 ) . the cooling procedure was similar to that used for african catfish , clarias gariepinus , where a three - step program achieved better survival of thawed spermatozoa compared with a two - step program ( steyn et al . the first step of the freezing program involved slow cooling from + 4 to 40 at a rate of 2 - 10/min . while 5 min , the chamber temperature was maintained to 40 , and the samples were immediately plunged into liquid nitrogen ( from 40 to 200 ) . as mentioned steyn et al . ( 2001 ) , cooling rate of 5 - 11/min was considered to be optimal , and hatching rate was 81.75.0% in cooling rate of 5/min . in our study , cool rate was lower than that of previous study , however , hatching rate was similar to that of previous study . all previous studies have reported that the final temperature and its duration ( i.e. holding period ) just before plunging the frozen sperm into liquid nitrogen are very important . during the holding period , the temperature in the freezer remains constant , but the temperature of the freezing sperm continues to decrease until it reaches an equilibrium with the diluent . most of the freezing protocols for catfish cool the spermatozoa to a low temperature before transferring them into liquid nitrogen . for african catfish , 1985 ) used a holding temperature of 65 and horvath & urbanyi ( 2000 ) used a temperature of 80. for european catfish , linhart et al . ( 1993 ) used a holding temperature of 85. a holding temperature of 80 was used for striped catfish , pangasius hypophthalmus , by kwantong & bart ( 2003 ) and for channel catfish , ictalurus punctatus , by tiersch et al . frozen spermatozoa were thawed at 40 in most of those studies , while we used a temperature range of 25 - 40 , consistent with earlier studies . the use of ethylene glycol as the cryoprotectant resulted in significantly greater hatching rates and percentage of live spermatozoa compared with dmso . to our knowledge , no prior studies have optimized the cryoprotectant composition for cryopreservation of catfish spermatozoa . although many cryoprotectants have been tested for fish spermatozoa , those most commonly used for cryoprotection of catfish spermatozoa include dmso , glycerol , methanol , dimethyl acetamide , ethylene glycol , and propylene glycol ( christensen & tiersch , 1997 ; linhart et al . dmso was originally recommended for striped catfish , blue catfish ( i. furcatus ) , african catfish , and channel catfish ( guest et al . , 1976 ; bart et al . , 1998 ; horvath & urbanyi , 2000 ; kwantong & bart , 2003 ) . according to the present results , recent studies of african catfish and channel catfish showed greater sperm motility and fertility of thawed sperm using methanol as a cryoprotectant ( tiersch et al . , 1994 ; christensen & tiersch , 1997 ; horvath & urbanyi , 2000 ) . ( 2001 ) reported that osmotic shock derived from high concentrations of the cryoprotectant ( 15% vs. 10% ) increased the proportion of abnormal spermatozoa , as did cold shock during pre - freezing ( muchlisin & siti azizah , 2009 ) . osmotic shock injury of freeze - thawed spermatozoa is characterized by coiling and swelling of the distal end of the tail ( muchlisin & siti azizah , 2009 ) . ( 1992 ) reported that the morphological changes occurred before freezing and immediately after dilution with the cryosolution , and that the frequency of morphological changes increased with longer time in cryosolution . for freeze - thawed spermatozoa , osmotic shock refers to the cellular changes that occur after transferring spermatozoa from hypertonic conditions in a cryoprotectant , such as glycerol to isotonic conditions after thawing ( correa & zavos , 1996 ) . however , abnormalities were found in cryopreserved spermatozoa as well as fresh spermatozoa . in our study , 14.0% of fresh spermatozoa of far eastern catfish exhibited morphological abnormalities , which was comparable with that of baung , mystus nemurus , for which 13.96% of spermatozoa exhibited abnormalities ( muchlisin & siti azizah , 2009 ) . . the rate of thawing may be as important as the cooling rate in terms of the viability of cryopreserved spermatozoa ( christensen & tiersch , 2005 ) . in previous studies of blue catfish , the spermatozoa were thawed as 0.5 ml samples in french straws at 40 for 7 sec ( lang et al . , 2003 ) , a method that was originally developed for mammalian spermatozoa ( pickett & berndtson , 1974 ) . in the present study , it took 90 sec for the samples to completely thaw at 25 in a water bath . it may be more convenient and economical to use tap water rather than a water bath to thaw spermatozoa . we found that thawing at 15 or 25 did not affect spermatozoa mobility , but , if time is limited , 25 should be considered , especially when large numbers of samples need to be processed or if there are time constraints in the hatchery . cryopreservation of sperm is a useful and reliable technique for conservation of gene resources of far eastern catfish and for artificial propagation .

abstractthe aim of this study was to compare the efficacy of cryopreservation methods for ex situ conservation of spermatozoa from far eastern catfish , silurus asotus . the spermatozoa activity index ( sai ) and hatching rates were higher in spermatozoa stored in alserver s solution than those of spermatozoa stored in glucose solution . the sai and hatching rates in all experimental groups gradually decreased with increasing duration of storage . additionally , the sai and hatching rates gradually decreased with increasing thawing temperatures at all storage durations ( p<0.05 ) . based on the sai and hatching rates , our results suggest that the optimal cryopreservation conditions of catfish spermatozoa involve storage in alserver s solution with 15% ethylene glycol , and thawing at 25. cryopreservation of spermatozoa is a useful and reliable technique for conserving gene resources and for artificial propagation of far eastern catfish .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

the far eastern catfish silurus asotus is a member of the freshwater siluridae family . , 1988 ; although the demand for this species has increased in recent years , two factors limit the profitability of culturing far eastern catfish . first , the growth rate shows significant sexual dimorphism because females grow much faster than males ( kim et al . cryopreservation of spermatozoa could be useful for artificial propagation , which may improve the genetic characteristics of this species . cryopreserved spermatozoa may be used in research and in aquaculture by enabling the introduction of genes from wild or native populations into hatchery stocks if spawning times do not overlap , and increases the availability of spermatozoa for use in selective breeding programs , especially during seasonal shortages of males and/or females . marian & krasznai ( 1987 ) were the first to successfully cryopreserve spermatozoa from european catfish , s. glanis . this problem was overcome by storing spermatozoa in diluents , and the cryopreservation techniques were refined in later studies , in which the optimization of the diluents extended the viability and freezing rates of spermatozoa ( linhart et al . the aim of the present study was to optimize cryopreservation methods for ex situ cryopreservation of spermatozoa from far eastern catfish . the success of the cryopreservation procedure was evaluated in terms of the spermatozoa activity index ( sai ) and hatching rates of eggs fertilized with thawed cryopreserved sperm . far eastern catfish , silurus asotus were cultured according to the methods of kim et al . spermatozoa were pooled from four fish with a spermatozoa motility index of > 80% , and were used for cryopreservation . the spermatozoa concentration was determined using a thoma hemocytometer ( depth : 0.0025 mm ; neubauer , germany ) under a microscope ( 1,000 ; ch 20 ; olympus , tokyo , japan ) and the value is expressed as the mean number of spermatozoa in 20 squares of the thoma cell . the diluents used in this study were alserver s solution ( 2.05 g glucose , 0.4 g sodium chloride , and 0.8 g sodium citrate/100 ml distilled water ) and 0.3 m glucose solution ( 54.3 g glucose/1,000 ml distilled water ) . a maximum of 8 ml of sperm was added to 40 ml of diluent to maintain a dilution rate of 1:5 ( sperm / diluent ) to prevent spontaneous initiation of spermatozoa activity ( linhart et al . , after collection , the containers were stored under aerobic conditions on ice at 0 for 1 hour . sperm activity was assessed ( table 1 ) and sai was assessed under a microscope and using video records ( n = 100 ) . we compared the effect of two cryoprotectants ( dimethyl sulfoxide [ dmso ] and ethylene glycol ) at different concentrations and in various combinations , as well as five different storage times . the final concentrations of dmso and ethylene glycol were 5% , 10% , 15% , and 20% . they were then dehydrated in ethanol , subjected to critical point freezing , and sprayed with gold . one - way analysis of variance was used to test the statistical significance ( p<0.05 ) of differences among experimental groups . the spermatozoa from far eastern catfish , s. asotus showed good quality ( table 2 ) . the sai was greater for spermatozoa diluted in alserver s solution than for those diluted in glucose . the sai of spermatozoa diluted sperm in both solutions decreased with increasing duration of storage ; therefore , better results were obtained when spermatozoa were stored for 1 hour than when stored for 24 hours ( table 2 ) . values in same row having the different superscripts are significantly different ( n = 100 , p<0.05 ) . the sai values of far eastern catfish spermatozoa stored in two diluents and with different concentrations of the cryoprotectants are shown in table 3 . the sai gradually decreased with increasing storage duration in all of the experimental groups . the sai of spermatozoa diluted in alserver s solution and dmso gradually increased with increasing dmso concentration ( p<0.05 ) . the optimal concentration of dmso in alserver s solution was 20% for all storage durations . spermatozoa stored in 5% , 10% , or 15% dmso were immobilized after storage for 360 days , while those stored in 20% dmso were immobilized after 720 days . the sai of spermatozoa stored in alserver s solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the optimal concentration of ethylene glycol in alserver s solution was 15% for all storage durations . the spermatozoa were not immo bilized by storage in ethylene glycol for any storage duration . the temperature of storage is 80. meansd ( n=100 ) values are shown . as shown in table 3 , the effects of dilution in glucose solution containing dmso or ethylene glycol were similar to those of alserver s solution . the sai of spermatozoa diluted in glucose solution and dmso gradually increased with increasing concentration of dmso ( p<0.05 ) . the sai was lower in spermatozoa diluted in glucose solution plus dmso than in spermatozoa diluted in alserver s solution plus dmso . the optimal concentration of dmso in glucose solution was 20% for all storage durations . spermatozoa stored in 5% , 10% , and 15% dmso were immobilized after 360 days , while those stored in 20% dmso were immobilized after 720 days . the sai of spermatozoa stored in glucose solution plus ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased when stored in 20% ethylene glycol ( p<0.05 ) . the sai of spermatozoa stored in glucose solution plus ethylene glycol was lower than that of spermatozoa stored in alserver s solution plus ethylene glycol . the optimal concentration of ethylene glycol in glucose solution was 15% for all storage durations . spermatozoa stored in glucose solution plus 5% or 10% ethylene glycol were immobilized after 540 days . by contrast , spermatozoa stored in 15% or 20% ethylene glycol were not immobilized at any storage duration . the trends in hatching rates were similar to those for sai and are shown in table 4 . the hatching rate in all of the experimental groups gradually decreased with increasing spermatozoa storage duration . the hatching rates of spermatozoa stored in alserver s solution and dmso gradually increased with increasing concentration of dmso ( p<0.05 ) . at all storage durations , the hatching rate was higher for spermatozoa stored in 20% dmso than for those stored in other concentrations of dmso . none of the eggs hatched after fertilization with spermatozoa stored in 5% , 10% , or 15% dmso for 360 days . the hatching rates for eggs fertilized with spermatozoa stored in alserver s solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the hatching rate was higher for eggs fertilized with spermatozoa stored in 15% ethylene glycol than in other concentrations of ethylene glycol . all of the eggs hatched after fertilization with spermatozoa stored in ethylene glycol for any duration . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . the hatching rate ( hr ) was calculated for each cage from the number of hatched larvae ( hl ) and from the total number of eggs placed in the cage ( et ) as follows : hr = ( hl / et ) 100 ( after linhart et al . , as shown in table 4 , the hatching rates of eggs fertilized with spermatozoa stored in glucose and dmso gradually increased with increasing concentration of dmso . at all storage durations , the hatching rate was highest for spermatozoa stored in 20% dmso than in other concentrations of dmso ( p<0.05 ) . none of the eggs hatched after fertilization with spermatozoa stored in 5% , 10% , or 15% dmso for 360 days , whereas all the eggs hatched after fertilization with spermatozoa stored in 20% dmso . the hatching rate of eggs fertilized with glucose solution and ethylene glycol gradually increased as the ethylene glycol concentration increased from 5% to 15% , but decreased at 20% ethylene glycol ( p<0.05 ) . the hatching rate was higher for eggs fertilized with spermatozoa stored in 15% ethylene glycol than in other concentrations of ethylene glycol . none of the eggs hatched after fertilization with spermatozoa stored in 5% or 10% ethylene glycol for 720 days . by contrast , the eggs hatched after fertilized with spermatozoa stored in 15% or 20% ethylene glycol for all of the storage durations . 2a , b ) , and the absence of a midpiece sleeve on sem images . the first type consisted of spermatozoa with a double - stranded flagellum ( type a ; fig . 2c ) , and these originate from abnormal spermatogenesis before cryopreservation . in the second type , the flagellum is cut by ice crystals or damaged during thawing ( type b ; fig . the proportions of type a spermatozoa were not significantly different among the experimental groups . the proportion of type b spermatozoa stored in alserver s solution was lower at 5% dmso than at other concentrations of dmso . the proportions of type b spermatozoa were not significantly different among 10% , 15% , and 20% dmso . the proportion of type b spermatozoa stored in alserver s solution was lower at 5% ethylene glycol than at other concentrations . the trends in the proportions of type b spermatozoa were similar between ethylene glycol and dmso . the temperature of storage is 80. meansd ( n=100 ) values are shown . the proportions of type b spermatozoa were similar between those stored in glucose solution and alserver s solution when using dmso . the proportions of type b spermatozoa were also similar between the two diluents when using ethylene glycol . however , the proportions of type b spermatozoa stored in alserver s solution or glucose solution were significantly lower in ethylene glycol than in dmso . the proportion of immobile spermatozoa in the absence of a cryoprotectant was not significantly different between spermatozoa stored in alserver s solution or glucose solution . the effects of storage duration and thawing temperature on the sai of spermatozoa are shown in fig . however , sai after storage for 540 days and 720 days increased as the thawing temperature increased from 15 to 35 ( p<0.05 ) . the spermatozoa stored for 720 days were not immobilized by thawing at 45. the sai after storage for 540 days decreased as the thawing temperature increased from 35 to 45 ( p<0.05 ) . the mean sai after storage for 540 days and thawing at 35 and 45 was 0.30.07 and 0.1 0.07 , respectively . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . diluent and concentrations of cryoprotectant using this experiment were alsever s solution and 15% ethylene glycol . the effects of storage duration and thawing temperature on the hatching rate are shown in fig . the hatching rates gradually decreased with increasing thawing temperature for all storage durations ( p<0.05 ) . the hatching rates after storage for 1 or 180 days were not significantly affected by thawing temperatures of 15 - 35. similarly , the hatching rates at 360 , 540 , and 720 days were not significantly affected by thawing temperatures of 15 - 25. at all storage durations , the hatching rates were higher when fertilizing eggs with spermatozoa thawed at 15 or 25 compared with the other thawing temperatures . based on the sai and hatching rates , our results suggest that the optimal thawing temperature of far eastern catfish spermatozoa is 25. meansd ( n= 100 ) values are shown . means sharing the same letter superscript on given dates are not significantly different ( p>0.05 ) . diluent and concentrations of cryoprotectant using this experiment were alsever s solution and 15% ethylene glycol . in our study of far eastern catfish , 104,000 spermatozoa per egg were dropped onto unfertilized eggs using a micropipette . for european catfish , silurus glanis , 80,00 - 80,000 fresh spermatozoa per egg are needed for artificial insemination , but hatcheries frequently use 100,000 - 200,000 spermatozoa per egg ( linhart et al . moreover , it may not be practical to use large numbers of spermatozoa in artificial propagation because male european catfish generally produce a small volume of sperm . one part sperm to 25 parts water to 25 parts eggs was optimal for artificial propagation of european catfish in a previous study ( linhart et al . the freezing procedure applied in our study was based on cryopreservation studies of tiger puffer , takifugu rubripes , and european catfish , although the procedure was also successfully applied in far eastern catfish ( chang et al . while 5 min , the chamber temperature was maintained to 40 , and the samples were immediately plunged into liquid nitrogen ( from 40 to 200 ) . ( 2001 ) , cooling rate of 5 - 11/min was considered to be optimal , and hatching rate was 81.75.0% in cooling rate of 5/min . during the holding period , the temperature in the freezer remains constant , but the temperature of the freezing sperm continues to decrease until it reaches an equilibrium with the diluent . most of the freezing protocols for catfish cool the spermatozoa to a low temperature before transferring them into liquid nitrogen . ( 1993 ) used a holding temperature of 85. a holding temperature of 80 was used for striped catfish , pangasius hypophthalmus , by kwantong & bart ( 2003 ) and for channel catfish , ictalurus punctatus , by tiersch et al . the use of ethylene glycol as the cryoprotectant resulted in significantly greater hatching rates and percentage of live spermatozoa compared with dmso . to our knowledge , no prior studies have optimized the cryoprotectant composition for cryopreservation of catfish spermatozoa . although many cryoprotectants have been tested for fish spermatozoa , those most commonly used for cryoprotection of catfish spermatozoa include dmso , glycerol , methanol , dimethyl acetamide , ethylene glycol , and propylene glycol ( christensen & tiersch , 1997 ; linhart et al . dmso was originally recommended for striped catfish , blue catfish ( i. furcatus ) , african catfish , and channel catfish ( guest et al . osmotic shock injury of freeze - thawed spermatozoa is characterized by coiling and swelling of the distal end of the tail ( muchlisin & siti azizah , 2009 ) . ( 1992 ) reported that the morphological changes occurred before freezing and immediately after dilution with the cryosolution , and that the frequency of morphological changes increased with longer time in cryosolution . for freeze - thawed spermatozoa , osmotic shock refers to the cellular changes that occur after transferring spermatozoa from hypertonic conditions in a cryoprotectant , such as glycerol to isotonic conditions after thawing ( correa & zavos , 1996 ) . in our study , 14.0% of fresh spermatozoa of far eastern catfish exhibited morphological abnormalities , which was comparable with that of baung , mystus nemurus , for which 13.96% of spermatozoa exhibited abnormalities ( muchlisin & siti azizah , 2009 ) . in previous studies of blue catfish , the spermatozoa were thawed as 0.5 ml samples in french straws at 40 for 7 sec ( lang et al . we found that thawing at 15 or 25 did not affect spermatozoa mobility , but , if time is limited , 25 should be considered , especially when large numbers of samples need to be processed or if there are time constraints in the hatchery . cryopreservation of sperm is a useful and reliable technique for conservation of gene resources of far eastern catfish and for artificial propagation .

undifferentiated stem cells , which drive embryogenesis and development , are often responsible for adult tissue maintenance and regeneration in response to injury . stem cells are defined by their long - term ability to divide and self - renew and their ability to give rise to specialized cell types through differentiation . stem cells are classified by the range of their potential to differentiate into specialized cells . for example , pluripotent stem cells are able to give rise to any embryonic cell type whereas multipotent stem cells can give rise to smaller subsets of more closely related cell types . they are responsible for tissue homeostasis and regeneration after damage . while they are capable of self - renewal and differentiation , their stemness is reduced over time when grown in vitro [ 14 ] . unlike adult stem cells , pluripotent and embryonic stem cells can give rise to all tissues found in the human body and are virtually unlimited in their ability to proliferate in vitro , making them attractive for use in biomedical research and regenerative medicine . human pluripotent stem cells ( hpscs ) broadly refer to all pluripotent human stem cell types , including human embryonic stem cells ( hescs ) and induced pluripotent stem cells ( ipscs ) . hescs are most efficiently derived from the inner cell mass of human blastocytes generated during in vitro fertilization procedures . they are commonly grown in culture on either inactivated feeder layers of murine embryonic fibroblasts or under feeder - free conditions on tissue culture plates coated with a basement membrane protein substrate [ 57 ] . the development of conditions for feeder - free derivation and growth of hescs [ 6 , 811 ] has enabled standardized production of cells more amenable for use in therapeutic applications ( reviewed elsewhere ) . one major challenge when using hesc - derived cells for therapeutic purposes is generating tissue for transplantation that will not be rejected by the patient s immune system . another challenge for the use of hescs has been the ethical opposition to destroying human embryos during hesc isolation and accordingly , strict rules exist regarding derivation and use of new hesc lines in projects reliant on us federal funding . recently , methods to reprogram human or mouse adult - like somatic cells into embryonic - like stem cells was described by shinya yamanaka , a seminal discovery awarded the nobel prize in medicine in 2012 [ 13 , 14 ] . this method , called cellular reprogramming , was used to generate mouse and human ipscs from fibroblast cells through the forced expression of a specific set of transcription factors ( oct4 , sox2 , klf4 , c - myc ) [ 1518 ] . these reprogrammed cells resemble hescs in morphology and stem cell - defining characteristics , although detailed comparative gene expression studies suggest that there may be important differences between hescs and ipscs ( reviewed elsewhere ) . the original protocol for ipsc generation involved use of retroviral vectors which leave a genetic footprint in the reprogrammed cell , raising concerns for the safety of using ipscs for transplantation . in recent years , the methods have been further refined to eliminate the use of retroviruses , thus making it possible to generate patient - specific ipscs without changing the genetic composition of the cells . hpscs , embryonic and induced , can now be used to study and recapitulate many embryonic developmental stages in vitro . translational embryology is an emerging field in which researchers are able to use what we know about different stages of embryonic development to direct differentiation of pluripotent stem cells into specialized cells and tissues ( reviewed elsewhere [ 20 , 21 ] ) . directed differentiation is achieved by treating cells with recombinant proteins or small molecules that regulate important developmental signaling pathways , thereby mimicking key events during in vivo development . this approach has been used to generate a wide spectrum of cell types derived from all three primary germ layers ( ectoderm , mesoderm , and endoderm ) , holding great promise for studies and treatments of many diseases [ 21 , 22 ] . this review focuses on recent advances that used directed differentiation to generate intestinal tissue from hpscs . differentiation of hescs or ipscs into intestinal tissue requires a step - wise process mimicking major developmental events including definitive endoderm differentiation , gut specification and morphogenesis , and intestine development , growth , and homeostasis . the three primary germ layers , generated in the process of gastrulation during embryogenesis , all contribute to the development of the intestine . the enteric nervous system that innervates the gut arises from the ectoderm ; the smooth muscle , connective tissue , and vasculature of the gut arise from the mesoderm ; and intestinal epithelium arises from the endoderm [ 2326 ] . for the purpose of this review , we will focus on the development of the intestinal epithelium . variable levels of exposure to this growth factor control anterior posterior ( a p ) patterning of the endoderm . fibroblast growth factor ( fgf ) , wnt , bone morphogenetic protein ( bmp ) , and retinoic acid signaling are also involved in a p patterning and induction of tissue - specific transcription factors ( reviewed elsewhere [ 23 , 28 ] ) . simultaneous with formation of the gut tube , the endoderm is specified into future intestinal epithelium primarily through the induction of the transcription factor cdx2 [ 2932 ] . at this stage , the intestine is a single layer of cdx2 + cuboidal epithelial cells that transitions to become a pseudostratified epithelium . the subsequent rapid expansion and thickening of the pseudostratified epithelium is responsible for increasing the length and girth of the intestine [ 33 , 34 ] . the formation of the columnar epithelium is coincident with the emergence of villi and highly proliferative intervillus regions [ 3537 ] . the mechanistic details of villus morphogenesis and formation of the intervillus regions is not entirely known ; nevertheless , evidence suggests crosstalk between the epithelium and mesenchyme through the hedgehog , bmp , platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf)- , and wnt pathways are important for the process ( reviewed elsewhere [ 24 , 38 ] ) . transcription factors such as gata4 and gata6 are later involved in the specification of proximal versus distal intestine [ 3941 ] , although other less well understood factors are also likely involved . after villus morphogenesis is complete , epithelial proliferation becomes restricted to the intervillus regions of the embryonic intestine . it is generally speculated that the cells in the intervillus region may give rise to the adult stem cells residing in the crypts of lieberkhn , however , this has not been formally demonstrated . notably , many of the best characterized adult intestinal stem cell ( isc ) markers are not expressed in the proliferative embryonic intervillus domain [ 42 , 43 ] , suggesting that proliferating embryonic progenitor cells and adult stem cells in the crypt are not molecularly equivalent . five differentiated cell types appear during and after emergence of villi : enterocytes ( or colonocytes in the large intestine ) , goblet , tuft , enteroendocrine , and m - cells [ 44 , 45 ] . in the mouse , once the crypts emerge during intestinal maturation , wnt/-catenin and notch signaling are two critical pathways involved in the regulation of intestinal proliferation and cytodifferentiation ( reviewed elsewhere ) . since many molecular markers of isc are wnt target genes , it is likely that wnt signaling is essential for isc maintenance . wnt signaling also controls paneth cell differentiation , consistent with the spatial co - localization of iscs and paneth cells at the base of the crypts where wnt signals are high . the notch pathway is critical for isc self - renewal and differentiation [ 4951 ] ( reviewed in ) . during differentiation , notch / hes1 signaling in absorptive cells opposes expression of the secretory transcription factor atoh1/math1 [ 50 , 53 , 54 ] . further specification of secretory cell types occurs downstream of atoh1/math1 through neurogenin3 [ 55 , 56 ] for enteroendocrine cells , gfi1 , and spdef [ 5759 ] for goblet cells , and gfi1 , sox9 , and spdef for paneth cells [ 5862 ] . crosstalk between the wnt/-catenin and notch signaling pathways needs to be further elucidated given reports of wnt also playing a role in cell fate decisions [ 6366 ] . major breakthroughs in our understanding of isc regulation have been made over the last few years [ 36 , 49 , 6771 ] . these advances have , in turn , had several positive consequences including the identification of novel isc markers , an explosion in the development of new genetic tools to identify and study iscs in mice [ 7276 ] , opening new avenues to develop therapeutic drugs to treat intestinal cancer , and technological breakthroughs enabling researchers to grow robust primary epithelial cultures from mouse and human intestine [ 7880 ] . to date , many groups have published methodologies for growing primary intestinal epithelial cultures [ 7883 ] , which all include the core tenets that three - dimensional ( 3d ) structure and high levels of wnt signaling are critical to maintain long - term growth in vitro . due to their robustness , development of these 3d culture methods have yielded powerful , physiologically relevant systems that facilitate the study of intestinal homeostasis [ 48 , 51 ] , gene regulation and function [ 48 , 84 , 85 ] , and have additional promise for the performance of biochemical assays and drug screens . high wnt signaling and 3d intestinal culture conditions [ 78 , 79 ] enabled the expansion of human embryonic gut - like tissue derived from hpscs into more mature human intestinal organoids [ 31 , 86 ] ( see generating induced human intestinal organoids ( ihios ) section ) . given the diverse methods and tissue sources for primary intestinal cultures , there has been a recent initiative for the gastrointestinal research community to adopt a common nomenclature ( table 1 ) , although to date , a consensus has not been reached . this may be due , in part , to the fact that one of the original papers describing intestinal epithelial culture conditions referred to the resulting 3d structures as intestinal organoids . further , these epithelial - only cultures have been published and referred to as organoids in more than 30 publications since 2009 . stelzner et al . proposed that these epithelial cultures be re - coined enteroids , since they are solely derived from the enteric epithelium and that the term organoid be reserved for tissue that more closely resembles the organ proper , composed of multiple tissue types including the epithelial and mesenchymal components characteristic of native intestine . stelzner et al . also suggest referring to in vitro generation of intestinal tissue from non - intestinal sources as for the purposes of this review , we will refer to intestine derived from human embryonic or ipscs as induced human intestinal organoids we caution readers not to confuse ihios with epithelial - only cultures derived from isolated intestinal epithelium , crypts or single iscs ( table 1).table 1description of terms and abbreviationsabbreviationsfull namedescriptionheschuman embryonic stem cellpluripotent cells derived from human embryoipscinduced pluripotent stem cellspluripotent cells derived from reprogrammed somatic cellsspheroidintestinal spheroid or mid / hindgut spheroid3d structure generated from human endoderm , resembling early embryonic gut tissue . spheroids expand and give rise to ihiosorganoidorganoid3d organ - like structure grown in vitro that resembles a complex organ in vivo , including multiple cell and tissue typesihioinduced human intestinal organoid3d intestinal tissue generated from pluripotent stem cells , comprised of intestinal epithelial and mesenchymal tissueenteroidenteroidprimary intestinal epithelium grown in culture , can be generated from mouse or human intestinal epithelium description of terms and abbreviations as already highlighted , human embryonic and ipscs have been highly touted for their potential to treat or cure disease through cell - based transplantation therapies . hpscs also hold amazing potential to accurately mimic human development , homeostasis , and disease in vitro . work over the past decade has focused on understanding regulation of pluripotency and differentiation in hpscs . more recently , directed differentiation has emerged as the most efficient approach to achieving in vitro generation of a cell or tissue of interest [ 48 , 82 , 83 , 88102 ] . many breakthroughs in pluripotent stem cell differentiation have occurred using two - dimensional ( 2d ) culture conditions directed at generating a single cell type of interest . in contrast , generation of 3d organ units ( organoids ) or more complex tissue from hpscs has only recently been recognized as a viable approach for differentiation [ 31 , 103 , 104 ] . such 3d models will offer complex multi - lineage , multi - cellular systems that can more closely recapitulate both normal physiology as well as pathological conditions in vitro , forming the basis for new in vitro human models designed to help understand normal homeostasis , complex multigenic diseases , perform drug screens , and validate the efficacy of new drugs prior to clinical trials . using directed differentiation , we were able to successfully generate 3d intestinal tissue by recapitulating embryonic development of the intestine in vitro [ 31 , 86 ] . we employed a step - wise differentiation protocol that included endoderm induction , a p patterning , intestinal lineage commitment , and intestinal growth and differentiation ( fig . 1 ) . in the embryo , as cells migrate through the primitive streak , they are exposed to nodal , a tgf family member . depending on the concentration and time of exposure to nodal signaling , cells adopt either a mesodermal or endodermal fate en route to forming their proper germ layer [ 106117 ] . in hpscs , robust and efficient endoderm induction is achieved by mimicking nodal signaling using activin a [ 20 , 88 , 118 ] . we are routinely able to generate human endoderm cultures with > 85 % efficiency , determined by co - staining of the transcription factors sox17 and foxa2 using a well - established 3-day differentiation protocol [ 31 , 88 ] . following induction , we consider the resulting human foxa2+/sox17 + endodermal tissue to be naive , capable of giving rise to all endodermal lineages including pancreatic ( pdx1 + ) , hepatic ( alb+ ) , biliary ( sox17 + ) , and intestinal ( cdx2 + ) [ 31 , 119].fig . 1schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . a small proportion of cells ( ~25 % ) also differentiate into brachyury ( t)-positive mesoderm . induction of the intestinal epithelial transcription factor cdx2 is achieved by activating fgf and wnt signaling for 4 days . the mesenchymal population expands and expresses the intestine mesenchyme transcription factor foxf1 . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . spheroids are then cultured in a 3d matrix ( matrigel ) in high wnt conditions ( wnt3a and/or rspo1 ) along with egf and noggin ( nog ) . during the first month in culture , spheroids expand drastically in size , giving rise to ihios . ihios can be split and re - cultured , and maintained for many months in vitro schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . a small proportion of cells ( ~25 % ) also differentiate into brachyury ( t)-positive mesoderm . induction of the intestinal epithelial transcription factor cdx2 is achieved by activating fgf and wnt signaling for 4 days . the mesenchymal population expands and expresses the intestine mesenchyme transcription factor foxf1 . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish spheroids are then cultured in a 3d matrix ( matrigel ) in high wnt conditions ( wnt3a and/or rspo1 ) along with egf and noggin ( nog ) . during the first month in culture , spheroids expand drastically in size , giving rise to ihios . ihios can be split and re - cultured , and maintained for many months in vitro during endoderm induction , the embryo simultaneously undergoes complex morphogenetic movements and patterning events to give rise to the early gut tube , which is patterned into different domains along the a p and dorsal ventral ( d v ) axes with the different domains giving rise to different subsets of endodermal organs [ 23 , 109 , 120124 ] . of note , work done in a host of vertebrate organisms has shown that an increasing anterior - to - posterior gradient of fgf , wnt and bmp signaling acts to posteriorize the endoderm [ 28 , 125129 ] ; wnt and/or fgf signaling is able to induce human endoderm towards cdx2 + intestinal lineages [ 31 , 32 ] . in fgf4 + wnt3a treated induced human endoderm , we observed robust and stable induction of cdx2 in ~95 % of cells after 96 h of treatment . remarkably , we also observed dramatic morphogenetic movements in the tissue culture dish , which gave rise to gut - like spheroids , which were small 3d clusters of cells that budded from the underlying monolayer . although the mechanisms downstream of fgf and/or wnt signaling that govern these complex in vitro morphogenetic tissue movements are unclear , this system will likely be an excellent tool to study how complex tissue movements and tube formation occurs . following spheroid formation , we took advantage of pro - intestinal conditions established by sato et al . and continued to culture spheroids embedded in matrigel and medium supplemented with recombinant human growth factors that promoted high levels of wnt signaling ( wnt3a and/or rspo1 ) . spheroid size and complexity increased remarkably over 1 month , giving rise to an epithelial and mesenchymal layer . the epithelium expressed molecular markers typical of many small intestinal cell types , including enteroendocrine cells ( chromogranin a ) , goblet cells ( mucin2 ) , paneth cells ( lysozyme ) , and enterocytes ( dipeptidyl peptidase ( dpp)4 , villin ) . we also observed that when ihios were cultured for > 2 months , expression of isc markers such as achaete - scute complex homolog 2 ( ascl2 ) and leucine - rich repeat - containing g - protein coupled receptor 5 ( lgr5 ) were observed . using ihios , we have published a limited number of experiments to show that the epithelium appears to be functional and behaves in a normal physiological manner . for example , the epithelium in ihios is turned over every 67 days , similar to intestinal cell turnover in vivo [ 84 , 130132 ] . we have also demonstrated that ihios have a functional enterocyte peptide transport system by visualizing transport of a fluorescently labeled dipeptide [ 31 , 133 ] . major breakthroughs in our understanding of isc regulation have been made over the last few years [ 36 , 49 , 6771 ] . these advances have , in turn , had several positive consequences including the identification of novel isc markers , an explosion in the development of new genetic tools to identify and study iscs in mice [ 7276 ] , opening new avenues to develop therapeutic drugs to treat intestinal cancer , and technological breakthroughs enabling researchers to grow robust primary epithelial cultures from mouse and human intestine [ 7880 ] . to date , many groups have published methodologies for growing primary intestinal epithelial cultures [ 7883 ] , which all include the core tenets that three - dimensional ( 3d ) structure and high levels of wnt signaling are critical to maintain long - term growth in vitro . due to their robustness , development of these 3d culture methods have yielded powerful , physiologically relevant systems that facilitate the study of intestinal homeostasis [ 48 , 51 ] , gene regulation and function [ 48 , 84 , 85 ] , and have additional promise for the performance of biochemical assays and drug screens . high wnt signaling and 3d intestinal culture conditions [ 78 , 79 ] enabled the expansion of human embryonic gut - like tissue derived from hpscs into more mature human intestinal organoids [ 31 , 86 ] ( see generating induced human intestinal organoids ( ihios ) section ) . given the diverse methods and tissue sources for primary intestinal cultures , there has been a recent initiative for the gastrointestinal research community to adopt a common nomenclature ( table 1 ) , although to date , a consensus has not been reached . this may be due , in part , to the fact that one of the original papers describing intestinal epithelial culture conditions referred to the resulting 3d structures as intestinal organoids . further , these epithelial - only cultures have been published and referred to as organoids in more than 30 publications since 2009 . stelzner et al . proposed that these epithelial cultures be re - coined enteroids , since they are solely derived from the enteric epithelium and that the term organoid be reserved for tissue that more closely resembles the organ proper , composed of multiple tissue types including the epithelial and mesenchymal components characteristic of native intestine . stelzner et al . also suggest referring to in vitro generation of intestinal tissue from non - intestinal sources as for the purposes of this review , we will refer to intestine derived from human embryonic or ipscs as induced human intestinal organoids we caution readers not to confuse ihios with epithelial - only cultures derived from isolated intestinal epithelium , crypts or single iscs ( table 1).table 1description of terms and abbreviationsabbreviationsfull namedescriptionheschuman embryonic stem cellpluripotent cells derived from human embryoipscinduced pluripotent stem cellspluripotent cells derived from reprogrammed somatic cellsspheroidintestinal spheroid or mid / hindgut spheroid3d structure generated from human endoderm , resembling early embryonic gut tissue . spheroids expand and give rise to ihiosorganoidorganoid3d organ - like structure grown in vitro that resembles a complex organ in vivo , including multiple cell and tissue typesihioinduced human intestinal organoid3d intestinal tissue generated from pluripotent stem cells , comprised of intestinal epithelial and mesenchymal tissueenteroidenteroidprimary intestinal epithelium grown in culture , can be generated from mouse or human intestinal epithelium description of terms and abbreviations as already highlighted , human embryonic and ipscs have been highly touted for their potential to treat or cure disease through cell - based transplantation therapies . hpscs also hold amazing potential to accurately mimic human development , homeostasis , and disease in vitro . work over the past decade has focused on understanding regulation of pluripotency and differentiation in hpscs . more recently , directed differentiation has emerged as the most efficient approach to achieving in vitro generation of a cell or tissue of interest [ 48 , 82 , 83 , 88102 ] . many breakthroughs in pluripotent stem cell differentiation have occurred using two - dimensional ( 2d ) culture conditions directed at generating a single cell type of interest . in contrast , generation of 3d organ units ( organoids ) or more complex tissue from hpscs has only recently been recognized as a viable approach for differentiation [ 31 , 103 , 104 ] . such 3d models will offer complex multi - lineage , multi - cellular systems that can more closely recapitulate both normal physiology as well as pathological conditions in vitro , forming the basis for new in vitro human models designed to help understand normal homeostasis , complex multigenic diseases , perform drug screens , and validate the efficacy of new drugs prior to clinical trials . using directed differentiation , we were able to successfully generate 3d intestinal tissue by recapitulating embryonic development of the intestine in vitro [ 31 , 86 ] . we employed a step - wise differentiation protocol that included endoderm induction , a p patterning , intestinal lineage commitment , and intestinal growth and differentiation ( fig . 1 ) . in the embryo , as cells migrate through the primitive streak , they are exposed to nodal , a tgf family member . depending on the concentration and time of exposure to nodal signaling , cells adopt either a mesodermal or endodermal fate en route to forming their proper germ layer [ 106117 ] . in hpscs , robust and efficient endoderm induction is achieved by mimicking nodal signaling using activin a [ 20 , 88 , 118 ] . we are routinely able to generate human endoderm cultures with > 85 % efficiency , determined by co - staining of the transcription factors sox17 and foxa2 using a well - established 3-day differentiation protocol [ 31 , 88 ] . following induction , we consider the resulting human foxa2+/sox17 + endodermal tissue to be naive , capable of giving rise to all endodermal lineages including pancreatic ( pdx1 + ) , hepatic ( alb+ ) , biliary ( sox17 + ) , and intestinal ( cdx2 + ) [ 31 , 119].fig . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . a small proportion of cells ( ~25 % ) also differentiate into brachyury ( t)-positive mesoderm . induction of the intestinal epithelial transcription factor cdx2 is achieved by activating fgf and wnt signaling for 4 days . the mesenchymal population expands and expresses the intestine mesenchyme transcription factor foxf1 . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . spheroids are then cultured in a 3d matrix ( matrigel ) in high wnt conditions ( wnt3a and/or rspo1 ) along with egf and noggin ( nog ) . during the first month in culture , spheroids expand drastically in size , giving rise to ihios . ihios can be split and re - cultured , and maintained for many months in vitro schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . a small proportion of cells ( ~25 % ) induction of the intestinal epithelial transcription factor cdx2 is achieved by activating fgf and wnt signaling for 4 days . the mesenchymal population expands and expresses the intestine mesenchyme transcription factor foxf1 . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . spheroids are then cultured in a 3d matrix ( matrigel ) in high wnt conditions ( wnt3a and/or rspo1 ) along with egf and noggin ( nog ) . during the first month in culture , spheroids expand drastically in size , giving rise to ihios . ihios can be split and re - cultured , and maintained for many months in vitro during endoderm induction , the embryo simultaneously undergoes complex morphogenetic movements and patterning events to give rise to the early gut tube , which is patterned into different domains along the a p and dorsal ventral ( d v ) axes with the different domains giving rise to different subsets of endodermal organs [ 23 , 109 , 120124 ] . of note , work done in a host of vertebrate organisms has shown that an increasing anterior - to - posterior gradient of fgf , wnt and bmp signaling acts to posteriorize the endoderm [ 28 , 125129 ] ; wnt and/or fgf signaling is able to induce human endoderm towards cdx2 + intestinal lineages [ 31 , 32 ] . in fgf4 + wnt3a treated induced human endoderm , we observed robust and stable induction of cdx2 in ~95 % of cells after 96 h of treatment . remarkably , we also observed dramatic morphogenetic movements in the tissue culture dish , which gave rise to gut - like spheroids , which were small 3d clusters of cells that budded from the underlying monolayer . although the mechanisms downstream of fgf and/or wnt signaling that govern these complex in vitro morphogenetic tissue movements are unclear , this system will likely be an excellent tool to study how complex tissue movements and tube formation occurs . following spheroid formation , we took advantage of pro - intestinal conditions established by sato et al . and continued to culture spheroids embedded in matrigel and medium supplemented with recombinant human growth factors that promoted high levels of wnt signaling ( wnt3a and/or rspo1 ) . spheroid size and complexity increased remarkably over 1 month , giving rise to an epithelial and mesenchymal layer . the epithelium expressed molecular markers typical of many small intestinal cell types , including enteroendocrine cells ( chromogranin a ) , goblet cells ( mucin2 ) , paneth cells ( lysozyme ) , and enterocytes ( dipeptidyl peptidase ( dpp)4 , villin ) . we also observed that when ihios were cultured for > 2 months , expression of isc markers such as achaete - scute complex homolog 2 ( ascl2 ) and leucine - rich repeat - containing g - protein coupled receptor 5 ( lgr5 ) were observed . using ihios , we have published a limited number of experiments to show that the epithelium appears to be functional and behaves in a normal physiological manner . for example , the epithelium in ihios is turned over every 67 days , similar to intestinal cell turnover in vivo [ 84 , 130132 ] . we have also demonstrated that ihios have a functional enterocyte peptide transport system by visualizing transport of a fluorescently labeled dipeptide [ 31 , 133 ] . epithelial - only enteroids generated from adult mouse or human intestinal epithelium have a proven track record as a highly useful tool for studying physiologically relevant events , such as isc regulation and differentiation . in this light , it is important to highlight some of the advantages that ihios have to offer , as well as to point out potential disadvantages compared to enteroid cultures . one of the most significant advantages ihios offer is the ability to use this model to study human embryonic events in vitro . we have demonstrated that , much like the developing intestine in vivo , ihios transition from an early hindgut - like stage into a pseudostratified epithelium , which then undergoes tissue rearrangements that give rise to a columnar epithelium that has villus - like structures and proliferating intervillus - like domains [ 31 , 38 ] . further , development and differentiation of specialized cell types in the normal intestine are reflected in ihios . for example , we demonstrated that knockdown or overexpression of neurog3 led to a respective decrease or increase of enteroendocrine cells in ihios , consistent with the described role of neurog3 in humans and mice [ 31 , 55 , 134136 ] . recently , it has also been demonstrated that the transcription factor arx is also critical for proper enteroendocrine development in the developing mouse intestine and in ihios . that is , ihios have an endodermally derived epithelial layer , and a mesodermally derived mesenchymal layer . the presence of both germ layers is a byproduct of the activina endoderm differentiation protocol used in the first step in ihio generation . while differentiation is very efficient , and routinely generates ~90 % endoderm , a small population of cells differentiate into brachyury ( t)-positive mesodermal progenitors that give rise to foxf1 + intestinal mesenchymal progenitors . the ihio mesenchymal layer , while disorganized , is comprised of multiple cell types , including smooth muscle ( smooth muscle actin + ) , intestinal subepithelial myofibroblasts ( isemf ) , and fibroblasts . since ihios possess this mesenchymal layer , they can be used to study intestinal mesenchymal biology in addition to epithelial - mesenchymal interactions . a third advantage of ihios is the ability to generate patient - specific intestinal tissue for the study of human disease . with the recent advances in generating induced pluripotent stem cell lines [ 15 , 16 ] , although likely possible , it is currently unclear whether enteroids ( epithelium only ) can be generated from diseased or damaged epithelium . coupling these two in vitro systems ( ihios and enteroids ) to study intestinal disease will be ideal given their distinct advantages . for example , ihios will enable the study of healthy intestinal tissue from an individual whereas enteroids from the same individual will have been exposed to the disease environment in vivo . for example , ihios generated from individuals with inflammatory bowel disease ( ibd ) will have healthy , non - inflamed intestinal tissue , with no exposure to the immune system whereas enteroids from the same patient will have had exposure to inflammation and the immune system . therefore , intestinal tissue that has been in a disease - state ( enteroids ) and a non - disease state ( ihios ) from this patient can be examined , compared and experimentally manipulated . in this light , ihios will be a very powerful tool to elucidate the pathogenesis of complex multigenic human diseases . emerging evidence suggests that ihios will be useful for a wide variety of studies , such as infectious diseases . as ihios are more widely adopted as a model to study human intestinal biology and pathobiology , their true utility and limitations will become more apparent . the significance of having complex , multi - lineage ( mesenchyme , epithelium ) 3d human intestinal tissue as a model experimental system will likely be unparalleled . in this light , it will be exciting to see how the scientific community puts this novel system to use .

many significant advances in our understanding of intestine development , intestinal stem cell homeostasis and differentiation have been made in recent years . these advances include novel techniques to culture primary human and mouse intestinal epithelium in three - dimensional matrices , and de novo generation of human intestinal tissue from embryonic and induced pluripotent stem cells . this short review will focus on the directed differentiation of human pluripotent stem cells into intestinal tissue , highlight novel uses of this tissue , and compare and contrast this system to primary intestinal epithelial cultures .

Introduction Growing the Intestine In Vivo Growing Intestinal Tissue In Vitro Primary Intestinal Epithelial Culture Methods and Nomenclature Generating Induced Human Intestinal Organoids (iHIOs) Experimental Utility of iHIOs and Enteroids Perspectives

undifferentiated stem cells , which drive embryogenesis and development , are often responsible for adult tissue maintenance and regeneration in response to injury . stem cells are defined by their long - term ability to divide and self - renew and their ability to give rise to specialized cell types through differentiation . stem cells are classified by the range of their potential to differentiate into specialized cells . for example , pluripotent stem cells are able to give rise to any embryonic cell type whereas multipotent stem cells can give rise to smaller subsets of more closely related cell types . human pluripotent stem cells ( hpscs ) broadly refer to all pluripotent human stem cell types , including human embryonic stem cells ( hescs ) and induced pluripotent stem cells ( ipscs ) . hescs are most efficiently derived from the inner cell mass of human blastocytes generated during in vitro fertilization procedures . recently , methods to reprogram human or mouse adult - like somatic cells into embryonic - like stem cells was described by shinya yamanaka , a seminal discovery awarded the nobel prize in medicine in 2012 [ 13 , 14 ] . in recent years , the methods have been further refined to eliminate the use of retroviruses , thus making it possible to generate patient - specific ipscs without changing the genetic composition of the cells . hpscs , embryonic and induced , can now be used to study and recapitulate many embryonic developmental stages in vitro . translational embryology is an emerging field in which researchers are able to use what we know about different stages of embryonic development to direct differentiation of pluripotent stem cells into specialized cells and tissues ( reviewed elsewhere [ 20 , 21 ] ) . directed differentiation is achieved by treating cells with recombinant proteins or small molecules that regulate important developmental signaling pathways , thereby mimicking key events during in vivo development . this approach has been used to generate a wide spectrum of cell types derived from all three primary germ layers ( ectoderm , mesoderm , and endoderm ) , holding great promise for studies and treatments of many diseases [ 21 , 22 ] . this review focuses on recent advances that used directed differentiation to generate intestinal tissue from hpscs . differentiation of hescs or ipscs into intestinal tissue requires a step - wise process mimicking major developmental events including definitive endoderm differentiation , gut specification and morphogenesis , and intestine development , growth , and homeostasis . the enteric nervous system that innervates the gut arises from the ectoderm ; the smooth muscle , connective tissue , and vasculature of the gut arise from the mesoderm ; and intestinal epithelium arises from the endoderm [ 2326 ] . for the purpose of this review , we will focus on the development of the intestinal epithelium . fibroblast growth factor ( fgf ) , wnt , bone morphogenetic protein ( bmp ) , and retinoic acid signaling are also involved in a p patterning and induction of tissue - specific transcription factors ( reviewed elsewhere [ 23 , 28 ] ) . it is generally speculated that the cells in the intervillus region may give rise to the adult stem cells residing in the crypts of lieberkhn , however , this has not been formally demonstrated . notably , many of the best characterized adult intestinal stem cell ( isc ) markers are not expressed in the proliferative embryonic intervillus domain [ 42 , 43 ] , suggesting that proliferating embryonic progenitor cells and adult stem cells in the crypt are not molecularly equivalent . five differentiated cell types appear during and after emergence of villi : enterocytes ( or colonocytes in the large intestine ) , goblet , tuft , enteroendocrine , and m - cells [ 44 , 45 ] . the notch pathway is critical for isc self - renewal and differentiation [ 4951 ] ( reviewed in ) . further specification of secretory cell types occurs downstream of atoh1/math1 through neurogenin3 [ 55 , 56 ] for enteroendocrine cells , gfi1 , and spdef [ 5759 ] for goblet cells , and gfi1 , sox9 , and spdef for paneth cells [ 5862 ] . major breakthroughs in our understanding of isc regulation have been made over the last few years [ 36 , 49 , 6771 ] . these advances have , in turn , had several positive consequences including the identification of novel isc markers , an explosion in the development of new genetic tools to identify and study iscs in mice [ 7276 ] , opening new avenues to develop therapeutic drugs to treat intestinal cancer , and technological breakthroughs enabling researchers to grow robust primary epithelial cultures from mouse and human intestine [ 7880 ] . to date , many groups have published methodologies for growing primary intestinal epithelial cultures [ 7883 ] , which all include the core tenets that three - dimensional ( 3d ) structure and high levels of wnt signaling are critical to maintain long - term growth in vitro . due to their robustness , development of these 3d culture methods have yielded powerful , physiologically relevant systems that facilitate the study of intestinal homeostasis [ 48 , 51 ] , gene regulation and function [ 48 , 84 , 85 ] , and have additional promise for the performance of biochemical assays and drug screens . high wnt signaling and 3d intestinal culture conditions [ 78 , 79 ] enabled the expansion of human embryonic gut - like tissue derived from hpscs into more mature human intestinal organoids [ 31 , 86 ] ( see generating induced human intestinal organoids ( ihios ) section ) . this may be due , in part , to the fact that one of the original papers describing intestinal epithelial culture conditions referred to the resulting 3d structures as intestinal organoids . further , these epithelial - only cultures have been published and referred to as organoids in more than 30 publications since 2009 . also suggest referring to in vitro generation of intestinal tissue from non - intestinal sources as for the purposes of this review , we will refer to intestine derived from human embryonic or ipscs as induced human intestinal organoids we caution readers not to confuse ihios with epithelial - only cultures derived from isolated intestinal epithelium , crypts or single iscs ( table 1).table 1description of terms and abbreviationsabbreviationsfull namedescriptionheschuman embryonic stem cellpluripotent cells derived from human embryoipscinduced pluripotent stem cellspluripotent cells derived from reprogrammed somatic cellsspheroidintestinal spheroid or mid / hindgut spheroid3d structure generated from human endoderm , resembling early embryonic gut tissue . spheroids expand and give rise to ihiosorganoidorganoid3d organ - like structure grown in vitro that resembles a complex organ in vivo , including multiple cell and tissue typesihioinduced human intestinal organoid3d intestinal tissue generated from pluripotent stem cells , comprised of intestinal epithelial and mesenchymal tissueenteroidenteroidprimary intestinal epithelium grown in culture , can be generated from mouse or human intestinal epithelium description of terms and abbreviations as already highlighted , human embryonic and ipscs have been highly touted for their potential to treat or cure disease through cell - based transplantation therapies . hpscs also hold amazing potential to accurately mimic human development , homeostasis , and disease in vitro . work over the past decade has focused on understanding regulation of pluripotency and differentiation in hpscs . more recently , directed differentiation has emerged as the most efficient approach to achieving in vitro generation of a cell or tissue of interest [ 48 , 82 , 83 , 88102 ] . many breakthroughs in pluripotent stem cell differentiation have occurred using two - dimensional ( 2d ) culture conditions directed at generating a single cell type of interest . in contrast , generation of 3d organ units ( organoids ) or more complex tissue from hpscs has only recently been recognized as a viable approach for differentiation [ 31 , 103 , 104 ] . such 3d models will offer complex multi - lineage , multi - cellular systems that can more closely recapitulate both normal physiology as well as pathological conditions in vitro , forming the basis for new in vitro human models designed to help understand normal homeostasis , complex multigenic diseases , perform drug screens , and validate the efficacy of new drugs prior to clinical trials . using directed differentiation , we were able to successfully generate 3d intestinal tissue by recapitulating embryonic development of the intestine in vitro [ 31 , 86 ] . we employed a step - wise differentiation protocol that included endoderm induction , a p patterning , intestinal lineage commitment , and intestinal growth and differentiation ( fig . depending on the concentration and time of exposure to nodal signaling , cells adopt either a mesodermal or endodermal fate en route to forming their proper germ layer [ 106117 ] . following induction , we consider the resulting human foxa2+/sox17 + endodermal tissue to be naive , capable of giving rise to all endodermal lineages including pancreatic ( pdx1 + ) , hepatic ( alb+ ) , biliary ( sox17 + ) , and intestinal ( cdx2 + ) [ 31 , 119].fig . 1schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . ihios can be split and re - cultured , and maintained for many months in vitro schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish spheroids are then cultured in a 3d matrix ( matrigel ) in high wnt conditions ( wnt3a and/or rspo1 ) along with egf and noggin ( nog ) . ihios can be split and re - cultured , and maintained for many months in vitro during endoderm induction , the embryo simultaneously undergoes complex morphogenetic movements and patterning events to give rise to the early gut tube , which is patterned into different domains along the a p and dorsal ventral ( d v ) axes with the different domains giving rise to different subsets of endodermal organs [ 23 , 109 , 120124 ] . and continued to culture spheroids embedded in matrigel and medium supplemented with recombinant human growth factors that promoted high levels of wnt signaling ( wnt3a and/or rspo1 ) . the epithelium expressed molecular markers typical of many small intestinal cell types , including enteroendocrine cells ( chromogranin a ) , goblet cells ( mucin2 ) , paneth cells ( lysozyme ) , and enterocytes ( dipeptidyl peptidase ( dpp)4 , villin ) . major breakthroughs in our understanding of isc regulation have been made over the last few years [ 36 , 49 , 6771 ] . these advances have , in turn , had several positive consequences including the identification of novel isc markers , an explosion in the development of new genetic tools to identify and study iscs in mice [ 7276 ] , opening new avenues to develop therapeutic drugs to treat intestinal cancer , and technological breakthroughs enabling researchers to grow robust primary epithelial cultures from mouse and human intestine [ 7880 ] . to date , many groups have published methodologies for growing primary intestinal epithelial cultures [ 7883 ] , which all include the core tenets that three - dimensional ( 3d ) structure and high levels of wnt signaling are critical to maintain long - term growth in vitro . due to their robustness , development of these 3d culture methods have yielded powerful , physiologically relevant systems that facilitate the study of intestinal homeostasis [ 48 , 51 ] , gene regulation and function [ 48 , 84 , 85 ] , and have additional promise for the performance of biochemical assays and drug screens . high wnt signaling and 3d intestinal culture conditions [ 78 , 79 ] enabled the expansion of human embryonic gut - like tissue derived from hpscs into more mature human intestinal organoids [ 31 , 86 ] ( see generating induced human intestinal organoids ( ihios ) section ) . given the diverse methods and tissue sources for primary intestinal cultures , there has been a recent initiative for the gastrointestinal research community to adopt a common nomenclature ( table 1 ) , although to date , a consensus has not been reached . also suggest referring to in vitro generation of intestinal tissue from non - intestinal sources as for the purposes of this review , we will refer to intestine derived from human embryonic or ipscs as induced human intestinal organoids we caution readers not to confuse ihios with epithelial - only cultures derived from isolated intestinal epithelium , crypts or single iscs ( table 1).table 1description of terms and abbreviationsabbreviationsfull namedescriptionheschuman embryonic stem cellpluripotent cells derived from human embryoipscinduced pluripotent stem cellspluripotent cells derived from reprogrammed somatic cellsspheroidintestinal spheroid or mid / hindgut spheroid3d structure generated from human endoderm , resembling early embryonic gut tissue . spheroids expand and give rise to ihiosorganoidorganoid3d organ - like structure grown in vitro that resembles a complex organ in vivo , including multiple cell and tissue typesihioinduced human intestinal organoid3d intestinal tissue generated from pluripotent stem cells , comprised of intestinal epithelial and mesenchymal tissueenteroidenteroidprimary intestinal epithelium grown in culture , can be generated from mouse or human intestinal epithelium description of terms and abbreviations as already highlighted , human embryonic and ipscs have been highly touted for their potential to treat or cure disease through cell - based transplantation therapies . hpscs also hold amazing potential to accurately mimic human development , homeostasis , and disease in vitro . more recently , directed differentiation has emerged as the most efficient approach to achieving in vitro generation of a cell or tissue of interest [ 48 , 82 , 83 , 88102 ] . many breakthroughs in pluripotent stem cell differentiation have occurred using two - dimensional ( 2d ) culture conditions directed at generating a single cell type of interest . in contrast , generation of 3d organ units ( organoids ) or more complex tissue from hpscs has only recently been recognized as a viable approach for differentiation [ 31 , 103 , 104 ] . such 3d models will offer complex multi - lineage , multi - cellular systems that can more closely recapitulate both normal physiology as well as pathological conditions in vitro , forming the basis for new in vitro human models designed to help understand normal homeostasis , complex multigenic diseases , perform drug screens , and validate the efficacy of new drugs prior to clinical trials . using directed differentiation , we were able to successfully generate 3d intestinal tissue by recapitulating embryonic development of the intestine in vitro [ 31 , 86 ] . we employed a step - wise differentiation protocol that included endoderm induction , a p patterning , intestinal lineage commitment , and intestinal growth and differentiation ( fig . depending on the concentration and time of exposure to nodal signaling , cells adopt either a mesodermal or endodermal fate en route to forming their proper germ layer [ 106117 ] . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . ihios can be split and re - cultured , and maintained for many months in vitro schematic of human pluripotent stem cell - derived intestinal organoids . human - induced pluripotent ( ipsc ) or embryonic ( hesc ) stem cells are differentiated into foxa2/sox17 positive endoderm with > 85 % efficiency . a small proportion of cells ( ~25 % ) induction of the intestinal epithelial transcription factor cdx2 is achieved by activating fgf and wnt signaling for 4 days . during this 4-day induction , 3d spheroids comprised of cdx2 + epithelial and foxf1 + mesenchymal layers form , and delaminate from the tissue culture dish . and continued to culture spheroids embedded in matrigel and medium supplemented with recombinant human growth factors that promoted high levels of wnt signaling ( wnt3a and/or rspo1 ) . the epithelium expressed molecular markers typical of many small intestinal cell types , including enteroendocrine cells ( chromogranin a ) , goblet cells ( mucin2 ) , paneth cells ( lysozyme ) , and enterocytes ( dipeptidyl peptidase ( dpp)4 , villin ) . epithelial - only enteroids generated from adult mouse or human intestinal epithelium have a proven track record as a highly useful tool for studying physiologically relevant events , such as isc regulation and differentiation . further , development and differentiation of specialized cell types in the normal intestine are reflected in ihios . that is , ihios have an endodermally derived epithelial layer , and a mesodermally derived mesenchymal layer . while differentiation is very efficient , and routinely generates ~90 % endoderm , a small population of cells differentiate into brachyury ( t)-positive mesodermal progenitors that give rise to foxf1 + intestinal mesenchymal progenitors . the ihio mesenchymal layer , while disorganized , is comprised of multiple cell types , including smooth muscle ( smooth muscle actin + ) , intestinal subepithelial myofibroblasts ( isemf ) , and fibroblasts . a third advantage of ihios is the ability to generate patient - specific intestinal tissue for the study of human disease . with the recent advances in generating induced pluripotent stem cell lines [ 15 , 16 ] , although likely possible , it is currently unclear whether enteroids ( epithelium only ) can be generated from diseased or damaged epithelium . for example , ihios will enable the study of healthy intestinal tissue from an individual whereas enteroids from the same individual will have been exposed to the disease environment in vivo . for example , ihios generated from individuals with inflammatory bowel disease ( ibd ) will have healthy , non - inflamed intestinal tissue , with no exposure to the immune system whereas enteroids from the same patient will have had exposure to inflammation and the immune system . therefore , intestinal tissue that has been in a disease - state ( enteroids ) and a non - disease state ( ihios ) from this patient can be examined , compared and experimentally manipulated . as ihios are more widely adopted as a model to study human intestinal biology and pathobiology , their true utility and limitations will become more apparent . the significance of having complex , multi - lineage ( mesenchyme , epithelium ) 3d human intestinal tissue as a model experimental system will likely be unparalleled . in this light , it will be exciting to see how the scientific community puts this novel system to use .

nitric oxide ( no ) , free radical produced by the inducible no synthase ( inos ) isoform , is an essential component of the host innate immune and inflammatory response to a variety of pathogens . it has diverse physiological roles and may also contribute towards pathological processes . when no is synthesized in large quantities by activated inflammatory cells , it has cytotoxic properties and may be involved in the pathogenesis of acute and chronic inflammatory conditions . in particular , no is claimed to contribute to damage of the joint cartilage in rheumatoid arthritis , to mucosal injury in inflammatory bowel disease and to degeneration of neurons in neurodegenerative diseases such as multiple scleroses , parkinson 's disease , and alzheimer 's disease . in most neurodegenerative disorders , a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response , where activated astrocytes and microglia and their cytotoxic agents play a crucial pathological role . glial cells consisting of astrocytes and microglia can produce cytokines , reactive oxygen radicals , and no in response to ischaemic , traumatic , and infectious insults , leading to exaggeration of the disease processes . coinduction or coregulation of cyclooxygenase-2 ( cox-2 ) and inos have been demonstrated in a number of cell culture studies and animal inflammatory model [ 6 , 7 ] . both cox-2 and inos are inducible form of enzymes up - regulated in response to inflammation challenge . in inflammatory process , cox-2 is expressed in many cells including fibroblast and macrophages , and produces prostaglandins that contribute to the pain and swelling of inflammation . expression of these inflammatory genes such as inos and cox-2 can be regulated by the activation of the nuclear factor-b ( nf-b ) . research of the literature reveals that there are one nf-b consensus dna sequence within cox-2 promoter , and two nf-b dna consensus sequences within inos promoter that are responsible for lps - induced nf-b dna binding activity . the most common active form of the nf-b family is the p50/p65 or p52/p65 heterodimer . in most cell types , inactive nf-b complexes are sequestered in the cytoplasm via their noncovalent interaction with inhibitory proteins known as ibs . in response to multiple stimuli , including cytokines , virus , and stress - inducing agents , the latent cytoplasmic nf-b / ib complex is activated by phosphorylation on conserved serine residues in the n - terminal portion of ib . after that , activated nf-b translocates to the nucleus and binds to its cognate dna binding site in the promoter or enhancer regions of specific genes . nf-b is a major transcription factor that plays an essential role in several aspects of human health including the development of inflammation and immunity . the dysregulation of nf-b is associated with many disease states such as atherosclerosis , arthritis , cancer . therefore , appropriate regulation and control of nf-b activity would provide a potential approach for the management of nf-b - related human diseases . isodon excisus , named oh ri bang pul in korea , belongs to the genus isodon and is distributed in korea and japan . the extracts have been used in fork medicine in korea for treating a bruise , inflammation and pain . the genus isodon ( also called rabdosia ) is a rich source of diterpenes , especially the highly oxidized kaurene diterpenes . ent - kaurene is the main diterpene intermediate involved in the biosynthesis of gibberellins , a widespread family of plant hormones with isoprenoid structure that control various physiological plant functions such as growth , germination , and flowering . some kaurene ditrerpene compounds have been demonstrated to exhibit not only cytotoxic activity against various cancer cell lines but also inhibitory activity of the nf-b pathway in macrophages [ 1416 ] . for example , linearol , a kaurene diterpene that impaired the inflammatory signaling by inhibiting nf-b inducing kinase in lps - induced j774 macrophages . kamebakaurin , another kaurane diterpene also inhibited tnf--induced nf-b activation by direct covalent modification of cysteine 62 in the p50 in mcf-7 cells . therefore , much interest has recently been shown in the biological effects of kaurene diterpenes . in the present study , we investigated antiinflammatory activity of inflexinol and its possible mechanisms in cultured raw 264.7 cells and astrocytes . inflexinol inhibited lps - induced no production as well as lps - induced expression of inos and cox-2 in raw 264.7 cells and astrocytes . using gel shift assay and nf-b luciferase assay , we showed that inflexinol inhibited activation of the transcriptional factor nf-b , a central regulator of inos and inflammatory response of body . these our data provide evidence that inflexinol has inhibitory effect on no production through inhibition of nf-b activation . inflexinol ( figure 1 ) was isolated from isodon excisus ( labiatae ) . the dried aerial parts of i. excisus ( 1.6 kg ) were pulverized and extracted with meoh ( 3 1.5 l ) at room temperature ( 24 hours ) . the extract was filtered and concentrated , in vacau and suitably diluted with water , then partitioned with n - hexane ( 3 1.5 l ) and ch2cl2 ( 3 1.5 l ) , respectively . the ch2cl2 extract ( 13.7 g ) was subjected to column chromatography on silica gel ( 9 25 cm , 70230 mesh ) eluting with n - hexane - acetone ( 5 : 1 , 3 : 1 , 3 : 2 , acetone ) affording five fractions ( iec-1 iec-5 ) . fraction iec-3 was subjected to flash column chromatography on rp-18 ( 2 30 cm , 4063 m ) eluting with ch3cn : h2o ( 30 : 70 ) and semiprepatative hplc ( column : ymc - odc , 20 150 mm ) eluting with ch3cn : h2o ( 23 : 77 ) at the flow speed of 6.5 ml / min . the structure of this compound was determined as ent-1,3,6,11-tetrahydroxykaur-16-ene-15-one-3,11-diacetate ( inflexinol ) , by comparison of its physicochemical and spectral data with those of literature . lps was obtained from sigma aldrich ( st louis , mo , usa ) dulbecco 's modified eagle 's medium ( dmem ) , fetal bovine serum , penicillin , and streptomycin were purchased from invitrogen ( carlsbad , calif , usa ) . raw 264.7 cells were obtained from the american type culture collection ( rockville , md , usa ) . these cells were maintained at subconfluence in a 95% air , 5% co2 humidified atmosphere at 37c . the medium used for routine subcultivation was dulbecco 's modified eagle 's medium ( dmem , invitrogen , carlsbad , calif , usa ) , supplemented with 10% fetal bovine serum ( fbs ) , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) . cells were counted with a hemocytometer and the number of viable cells was determined through trypan blue dye exclusion . the sprague - dawley rats were maintained in accodance with the policy of the national institute of toxicological research , which is in accord with the korea food and drug administration 's guideline for the care and use of laboratory animals . sprague - dawley rats weighing 200300 g were housed under 12-hour light / dark cycles , at 23c , and 60 5% humidity . all animals had free access to food ( samyang foods , seoul , south korea ) and water . cerebral cortical cells were isolated from neonatal rat brain ( day 1 ) in pbs ( 0.1 mol ) . after washing with dulbecco 's modified eagle 's medium ( dmem ) , the isolated cells were incubated for 15 minutes in dmem containing 0.2% trypsin . cells were dissociated by trituration and plated into polyethyleneimine - coated plastic ( 5 10 cells/60 mm dish ) containing minimum essential medium with eagle 's salts supplemented with 10% heat - inactivated fetal bovine serum , 2 mm l - glutamine , 1 mm pyruvate , 20 mm kcl , 10 mm sodium bicarbonate , and 1 mm hepes ( ph 7.2 ) . after 3 days in culture , the culture medium was replaced with dmem containing 10% fetal bovine serum , and medium was changed every 3 days of culture . cells were cultured for designated time . the cultured cells contained < 10% neuronal cells . the cytotoxicity of inflexinol was evaluated using the wst-8 assay ( dojindo laboratories , tokyo , japan ) . wst-8 [ 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2h - tetrazolium , monosodium salt ] is reduced by dehydrogenases in cells to give a yellow - colored product ( formazan ) , which is soluble in the culture medium . the amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells . in brief , 1 10 cells per well were plated into 96-well plates , incubated at 37c for 24 hours , and given a fresh change of medium . cells were then incubated with or without lps ( 1 g / ml ) in the absence or presence of various concentrations of inflexinol at 37c for an additional 24 hours . at that point , 10 l of the wst-8 solution were added to the wells and incubation was continued for another 1 hour . the resulting color was assayed at 450 nm using a microplate absorbance reader ( sunrise , tecan , switzerland ) . cells were grown in 96-well plates and then incubated with or without lps ( 1 g / ml ) in the absence or presence of various concentrations of inflexinol for 24 hours . each 50 l of culture supernatant was mixed with an equal volume of griess reagent [ 0.1% n-(1-naphthyl)-ethylenediamine , 1% sulfanilamide in 5% phosphoric acid ] and incubated at room temperature for 10 minutes . the absorbance at 540 nm was measured in a microplate absorbance reader , and a series of known concentrations of sodium nitrite was used as a standard . cells were homogenized with protein extraction solution ( pro - prep , intron biotechnology , south korea ) , and lysed by 40-minute incubation on ice . equal amount of proteins ( 40 g ) were separated on an sds/10%-polyacrylamide gel , and then transferred to a polyvinylidene difluoride ( pvdf ) membrane ( ge water & process technologies , trevose , pa , usa ) . blots were blocked for 2 hours at room temperature with 5% ( w / v ) nonfat dried milk in tris - buffered saline tween-20 [ tbst : 10 mm tris ( ph 8.0 ) and 150 mm nacl solution containing 0.05% tween-20 ] . after a short wash in tbst , rabbit polyclonal antibodies against inos and cox-2 ( 1 : 1000 ) ( cayman chemical , ann arbor , mich , usa ) , and rabbit polyclonal antibodies against p65 and ib ( 1 : 500 ) , and mouse monoclonal antibody against p50 ( 1 : 500 ) ( santa cruz biotechnology inc . santa cruz , calif , usa ) were used in study . the blot was then incubated with the corresponding conjugated antirabbit or mouse immunoglobulin g - horseradish peroxidase ( santa cruz biotechnology inc . gel shift assays were performed according to the manufacturer 's recommendations ( promega , madison , wis , usa ) . briefly , 5 10 cells was washed twice with 1 pbs , followed by the addition of 1 ml of pbs , and the cells were scraped into a cold eppendorf tube . cells were spun down at 13 000 rpm for 5 minutes , and the resulting supernatant was removed . cells were suspended in 400 l of solution a containing 10 mm hepes , ph 7.9 , 1.5 mm mgcl2 , 10 mm kcl , 0.5 mm dithiothreitol , 0.2 mm phenylmethylsulfonyl fluoride ; vigorously vortexed ; allowed to incubate on ice for 10 minutes ; and centrifuged at 12 000 rpm for 6 minutes . the pelleted nuclei were resuspended in solution c ( solution a + 420 mm nacl , 20% glycerol ) and allowed to incubate on ice for 20 minutes . the cells were centrifuged at 15 000 rpm for 15 minutes , and the resulting nuclear extract supernatant was collected in a chilled eppendorf tube . consensus oligonucleotides were end - labeled using t4 polynucleotide kinase and [ -p ] atp for 10 minutes at 37c . gel shift reactions were assembled and allowed to incubate at room temperature for 10 minutes followed by the addition of 1 l ( 50 000200 000 cpm ) of p end - labeled oligonucleotide and another 20 minutes of incubation at room temperature . subsequently 1 l of gel loading buffer was added to each reaction and loaded onto a 6% nondenaturing gel and electrophoresis until the dye was four - fifths of the way down the gel . the gel was dried at 80c for 1 hour and exposed to film overnight at 70c . raw 264.7 cells and astrocytes were plated at a density of 1 10 cells per 24-well plate . after 24 hours of growth to 90% confluence , the cells were transfected with pnf-b - luc plasmid ( 5 nf-b ; stratagene , calif , usa ) using a mixture of plasmid and lipofectamine plus in opti - men according to manufacture 's specification ( invitrogen , carlsbad , calif , usa ) . luciferase activity was measured by using the luciferase assay kit ( promega , madison , wis , usa ) according to the manufacturer 's instructions ( winglow , bad wildbad , germany ) . the data represent the mean ( se ) of three independent experiments performed in triplicate . statistical analysis was performed by one - way anova , followed by a dunnett test as post hoc comparison . after raw 264.7 cells were incubated with inflexinol in the absence of lps , inflexinol increased slightly cell viability at lower concentrations ( 1 , 5 m ) and showed mild reduction ( < 20% ) of cell viability at highest concentration ( 10 m ) used ( figure 2(a ) ) . when raw 264.7 cells were incubated with inflexinol in the presence of lps , lps remarkably increased the cell viability . although mild reduction of cell viability ( < 20% ) was showed by 10 m of inflexinol like previous case , it is considered that inhibitory effect of inflammatory mediators by infleixnol is not related with cytotoxic effect ( figure 2(b ) ) . moreover , inflexinol ( with or without lps ) did not decrease the cell viability at the various concentrations ( 1 , 5 , 10 m ) used in astrocytes whether inflexinol was treated with or without lps ( figures 2(c ) and 2(d ) ) . we have examined the inhibitory effect of inflexinol on no production of raw 264.7 cells and astrocytes induced by lps ( 1 g / ml ) . to evaluate the effect of inflexinol on no production in lps - induced raw 264.7 cells and astrocytes , after co - treatment with lps and inflexinol ( 1 , 5 , 10 m ) for 24 hours , lps - induced nitrite concentrations in the medium were decreased remarkably in a concentration - dependent manner . in raw 264.7 cells ( figure 3(a ) ) and astrocytes ( figure 3(b ) ) , the ic50 values of inflexinol on inhibiting lps - induced no production were 3.43 m and 2.66 m , respectively . to investigate whether inflexinol inhibits the no production via inhibition of corresponding gene expression , we determined inos expression by western blot analysis . we also determined cox-2 expression since inos can be modulated by cox-2 . as shown in figures 3(c ) and 3(d ) , the cells expressed extremely low levels of inos and cox-2 protein in an unstimulated condition . however , inos and cox-2 protein expression was markedly increased in response to lps ( 1 g / ml ) after 24 hours . treatment with inflexinol ( 1 , 5 , 10 m ) caused concentration - dependent decreases in lps - induced inos expression in raw 264.7 cells ( figure 3(c ) ) and astrocytes ( figure 3(d ) ) . this result is consistent with the profile of the inhibitory effect of inflexinol on no production . a similar inhibitory effect of inflexinol on the lps - induced cox-2 expression was found ( figures 3(c ) and 3(d ) ) . nf-b controls the expression of enzymes including inos and cox-2 whose products contribute to the pathogenesis of the inflammatory process . to investigate whether inflexinol is able to attenuate lps - induced nf-b - mediated promoter activity , we used a luciferase reporter gene expressed under the control of five b cis - acting elements . raw 264.7 cells and astrocytes were transiently transfected with the nf-b - dependent luciferase reporter construct according to manufacture 's specification ( invitrogen ) , and then cell treated with lps ( 1 g / ml ) or cotreated with lps and inflexinol for 8 hours . treatment of raw 264.7 cells ( figure 4(a ) ) and astrocytes ( figure 4(b ) ) with inflexinol resulted in a dose - dependent suppression of luciferase activity induced by lps . in raw 264.7 cells and astrocytes , the ic50 values of inflexinol on inhibiting lps - induced luciferase activity were 2.77 m and 3.88 m , respectively . these doses inhibiting nf-b luciferase activity were similar to the doses inhibiting no production . because activation of nf-b is critical for induction of both cox-2 and inos by lps or other inflammatory cytokines , we determined whether inflexinol might suppress nf-b activation in lps - activated raw 264.7 cells and astrocytes . to investigate whether inflexinol can also inhibit nf-b activation , raw 264.7 cells and astrocytes were cotreated with lps and inflexinol for 60 minutes and 90 minutes , respectively , which it is the time to activate nf-b maximally of its lps treatment ( data are not shown ) . nuclear extracts from cotreated cells were prepared and assayed nf-b dna binding by emsa . in raw 264.7 cells ( figure 4(c ) ) and astrocytes ( figure 4(d ) ) , lps induced a strong nf-b binding activity , which was markedly inhibited by cotreatment with inflexinol in a dose - dependent manner . it has been demonstrated that lps activates nf-b transcription factor that leads to the induction of the expression of many immediate early genes . to clarify the inhibitory mechanism of action of inflexinol for lps - induced nf-b , translocation of p50 and p65 as well as ib degradation were examined . treatment with lps increased nuclear translocation of p50 and p65 . in the presence of inflexinol , nuclear translocation of p50 and p65 was inhibited in a dose - dependent manner in raw 264.7 cells and astrocytes . moreover , inflexinol inhibited the lps - induced degradation of ib ( figures 5(a ) and 5(b ) ) . these results indicate that inflexinol may inhibit the lps - induced activation of nf-b via an inhibition of ib degradation as well as a translocation of p50 and p65 into the nuclear , and this effect may result in the inhibition of the lps - induced no production as well as inos and cox-2 expression . macrophage overproduction of inflammatory mediators such as cytokine and no has been implicated in inflammatory diseases such as rheumatoid arthritis , septic shock , cerebral malaria , and autoimmune diabetes . astrocytes play a key role in regulating aspects of inflammation in the central nervous system . several enzymes , such as the inos or cox-2 , along with different inflammatory mediators such as the free radical no or proinflammatory cytokines , have been proposed to be involved in the cell damage associated with neuroinflammation . in this study , we investigated the inhibitory effects of inflexinol on lps - induced no production and expression of inos , cox-2 in raw 264.7 cells and astrocytes . inflexinol ( 1 , 5 , 10 m ) that significantly inhibited lps - induced no production in a dose - dependent manner . inflexinol strongly inhibited lps - induced no production in raw 264.7 cells and astrocytes with ic50 values of 3.43 m and 2.66 m , respectively . these inhibitory effects may not be related with their cytotoxic effects since no effects on cell viability were observed at the concentration up to 10 m in raw 264.7 cells and astrocytes . comparison with ic50 value of indomethacin ( 53.8 m ) and lornoxicam ( 65 m ) being known as nonsteroidal antiinflammatory drugs in lps - stimulated raw 264.7 cells indicates that inflexinol has superior effects on inhibition of no production [ 24 , 25 ] . this inhibitory effect of no production could be related with gene expression of inos since inflexinol inhibited inos protein in raw 264.7 cells and astrocytes . these results showed that inflexinol could interfere lps - induced signaling involving the production of proinflammatory molecules . however , our data showed that the expression of cox-2 was found to be less sensitive than that of inos to the inflexinol . this could account for the higher sensitivity of inos gene transcription toward the inflexinol compared with that of cox-2 . in fact , structurally different diterpenoids displayed differential inhibition of inos and cox-2 expression even though the dna binding activity of nf-b is similar . since lps - induced inos and cox-2 expression is primarily regulated by nf-b , we examined the effect of inflexinol on lps - induced activation of nf-b using an nf-b reporter system as well as dna binding activity using emsa . consistent with the inhibitory effect on inos and cox-2 expression , inflexinol decreased nf-b transcriptional activity in raw 264.7 cells and astrocyte with ic50 values of 2.77 m and 3.88 m , respctively . inflexinol also inhibited nf-b - specific dna binding activity dose dependently . at the gene level , the expression of inos the promoter of the inos gene contains two major discrete regions synergistically functioning for binding of transcription factors : one for nf-b , which is mainly activated by lps . therefore , inhibition of nf-b activation could contribute to the inhibitory effect of inflexinol on inos and cox-2 expression . several studies have shown that antiinflammatory agents inhibit activation of nf-b via prevention of ib degradation . ib specifically binds and masks the nuclear translocation signals of p50 and p65 , thereby preventing the nuclear translocation of the nf-b heterodimer . hehner et al . have showed that sesquiterpene lactones prevented the induced degradation of ib and ib by diverse stimuli and therefore interfered with a common step in the signaling cascade leading to the activation of nf-b . have also demonstrated that prevention of ib degradation by 2-hydroxycinnamaldehyde contributed to inactivation of nf-b ( p50 ) in antiinflammatory reaction in raw 264.7 cells as well as tnf--treated colon cancer cell death . however , it is noteworthy that inflexinol contains an -methylenecyclopentanone moiety as a commom functional group which is known to react with nucleophiles , especially cystein sulfhydryl groups in protein , by a michael - type addition . a c-20-nonoxygenated - ent - kaurane diterpenoid ( kamebakaurin , ka ) , isolated from isodon japonicus , was suggested to interact with cysteine of dna binding domain of the p50 subunit of nf-b . recently , ka was found to be able to interact with both p50 and p65 subunits of nf-b . hehner et al . also demonstrated that sesquiterpene lactones interfered with the activation of nf-b by preventing a degradation of ib and ib but lacking either the lactone or the exomethylene group in the -position to the lactone function displayed no inhibitory effect on pathway leading to the activation of nf-b . similarly , the exomethylene group of inflexinol may be essential for the covalent modification via interaction with cysteine as above compounds . moreover , data presented herein show that inflexinol significantly inhibits nf-b activation by reducing the degradation of ib . kwok et al . have reported that exocylic methylene of sesquiterpene lactone parthenolide is required for in vivo and in vitro antiinflammatory activity and modification of cysteine 179 of ikk has been proposed to mediate the pathological effects of arsenite and parthenolide . therefore , our data suggest a possibility that inflexinol inhibit the upstream proteins of ib such as ikk or 26s proteasome . on the basis of the current results and those of other reports , we propose that inflxinol inhibit the expression of inos , cox-2 , and no production by inhibition of nf-b dna binding activity and transcriptional activation through prevention of ib degradation , and suggest that inflexinol may be useful as an antiinflammatory agent .

inflexinol , an ent - kaurane diterpenoid , was isolated from the leaves of isodon excisus . many diterpenoids isolated from the genus isodon ( labiatae ) have antitumor and antiinflammatory activities . we investigated the antiinflammatory effect of inflexinol in raw 264.7 cells and astrocytes . as a result , we found that inflexinol ( 1 , 5 , 10 m ) suppressed the expression of inducible nitric oxide synthase ( inos ) and cyclooxygenase-2 ( cox-2 ) as well as the production of nitric oxide ( no ) in lps - stimulated raw 264.7 cells and astrocytes . consistent with the inhibitory effect on inos and cox-2 expression , inflexinol also inhibited transcriptional and dna binding activity of nf-b via inhibition of ib degradation as well as p50 and p65 translocation into nucleus . these results suggest that inflexinol inhibits inos and cox-2 expression through inhibition of nf-b activation , thereby inhibits generation of inflammatory mediators in raw 264.7 cells and astrocytes , and may be useful for treatment of inflammatory diseases .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION

nitric oxide ( no ) , free radical produced by the inducible no synthase ( inos ) isoform , is an essential component of the host innate immune and inflammatory response to a variety of pathogens . when no is synthesized in large quantities by activated inflammatory cells , it has cytotoxic properties and may be involved in the pathogenesis of acute and chronic inflammatory conditions . coinduction or coregulation of cyclooxygenase-2 ( cox-2 ) and inos have been demonstrated in a number of cell culture studies and animal inflammatory model [ 6 , 7 ] . expression of these inflammatory genes such as inos and cox-2 can be regulated by the activation of the nuclear factor-b ( nf-b ) . research of the literature reveals that there are one nf-b consensus dna sequence within cox-2 promoter , and two nf-b dna consensus sequences within inos promoter that are responsible for lps - induced nf-b dna binding activity . in response to multiple stimuli , including cytokines , virus , and stress - inducing agents , the latent cytoplasmic nf-b / ib complex is activated by phosphorylation on conserved serine residues in the n - terminal portion of ib . after that , activated nf-b translocates to the nucleus and binds to its cognate dna binding site in the promoter or enhancer regions of specific genes . therefore , appropriate regulation and control of nf-b activity would provide a potential approach for the management of nf-b - related human diseases . isodon excisus , named oh ri bang pul in korea , belongs to the genus isodon and is distributed in korea and japan . the genus isodon ( also called rabdosia ) is a rich source of diterpenes , especially the highly oxidized kaurene diterpenes . ent - kaurene is the main diterpene intermediate involved in the biosynthesis of gibberellins , a widespread family of plant hormones with isoprenoid structure that control various physiological plant functions such as growth , germination , and flowering . for example , linearol , a kaurene diterpene that impaired the inflammatory signaling by inhibiting nf-b inducing kinase in lps - induced j774 macrophages . kamebakaurin , another kaurane diterpene also inhibited tnf--induced nf-b activation by direct covalent modification of cysteine 62 in the p50 in mcf-7 cells . in the present study , we investigated antiinflammatory activity of inflexinol and its possible mechanisms in cultured raw 264.7 cells and astrocytes . inflexinol inhibited lps - induced no production as well as lps - induced expression of inos and cox-2 in raw 264.7 cells and astrocytes . using gel shift assay and nf-b luciferase assay , we showed that inflexinol inhibited activation of the transcriptional factor nf-b , a central regulator of inos and inflammatory response of body . these our data provide evidence that inflexinol has inhibitory effect on no production through inhibition of nf-b activation . inflexinol ( figure 1 ) was isolated from isodon excisus ( labiatae ) . the dried aerial parts of i. excisus ( 1.6 kg ) were pulverized and extracted with meoh ( 3 1.5 l ) at room temperature ( 24 hours ) . fraction iec-3 was subjected to flash column chromatography on rp-18 ( 2 30 cm , 4063 m ) eluting with ch3cn : h2o ( 30 : 70 ) and semiprepatative hplc ( column : ymc - odc , 20 150 mm ) eluting with ch3cn : h2o ( 23 : 77 ) at the flow speed of 6.5 ml / min . raw 264.7 cells were obtained from the american type culture collection ( rockville , md , usa ) . cerebral cortical cells were isolated from neonatal rat brain ( day 1 ) in pbs ( 0.1 mol ) . cells were dissociated by trituration and plated into polyethyleneimine - coated plastic ( 5 10 cells/60 mm dish ) containing minimum essential medium with eagle 's salts supplemented with 10% heat - inactivated fetal bovine serum , 2 mm l - glutamine , 1 mm pyruvate , 20 mm kcl , 10 mm sodium bicarbonate , and 1 mm hepes ( ph 7.2 ) . in brief , 1 10 cells per well were plated into 96-well plates , incubated at 37c for 24 hours , and given a fresh change of medium . cells were then incubated with or without lps ( 1 g / ml ) in the absence or presence of various concentrations of inflexinol at 37c for an additional 24 hours . cells were grown in 96-well plates and then incubated with or without lps ( 1 g / ml ) in the absence or presence of various concentrations of inflexinol for 24 hours . the absorbance at 540 nm was measured in a microplate absorbance reader , and a series of known concentrations of sodium nitrite was used as a standard . after a short wash in tbst , rabbit polyclonal antibodies against inos and cox-2 ( 1 : 1000 ) ( cayman chemical , ann arbor , mich , usa ) , and rabbit polyclonal antibodies against p65 and ib ( 1 : 500 ) , and mouse monoclonal antibody against p50 ( 1 : 500 ) ( santa cruz biotechnology inc . the blot was then incubated with the corresponding conjugated antirabbit or mouse immunoglobulin g - horseradish peroxidase ( santa cruz biotechnology inc . briefly , 5 10 cells was washed twice with 1 pbs , followed by the addition of 1 ml of pbs , and the cells were scraped into a cold eppendorf tube . raw 264.7 cells and astrocytes were plated at a density of 1 10 cells per 24-well plate . after raw 264.7 cells were incubated with inflexinol in the absence of lps , inflexinol increased slightly cell viability at lower concentrations ( 1 , 5 m ) and showed mild reduction ( < 20% ) of cell viability at highest concentration ( 10 m ) used ( figure 2(a ) ) . when raw 264.7 cells were incubated with inflexinol in the presence of lps , lps remarkably increased the cell viability . although mild reduction of cell viability ( < 20% ) was showed by 10 m of inflexinol like previous case , it is considered that inhibitory effect of inflammatory mediators by infleixnol is not related with cytotoxic effect ( figure 2(b ) ) . moreover , inflexinol ( with or without lps ) did not decrease the cell viability at the various concentrations ( 1 , 5 , 10 m ) used in astrocytes whether inflexinol was treated with or without lps ( figures 2(c ) and 2(d ) ) . we have examined the inhibitory effect of inflexinol on no production of raw 264.7 cells and astrocytes induced by lps ( 1 g / ml ) . to evaluate the effect of inflexinol on no production in lps - induced raw 264.7 cells and astrocytes , after co - treatment with lps and inflexinol ( 1 , 5 , 10 m ) for 24 hours , lps - induced nitrite concentrations in the medium were decreased remarkably in a concentration - dependent manner . in raw 264.7 cells ( figure 3(a ) ) and astrocytes ( figure 3(b ) ) , the ic50 values of inflexinol on inhibiting lps - induced no production were 3.43 m and 2.66 m , respectively . to investigate whether inflexinol inhibits the no production via inhibition of corresponding gene expression , we determined inos expression by western blot analysis . as shown in figures 3(c ) and 3(d ) , the cells expressed extremely low levels of inos and cox-2 protein in an unstimulated condition . however , inos and cox-2 protein expression was markedly increased in response to lps ( 1 g / ml ) after 24 hours . treatment with inflexinol ( 1 , 5 , 10 m ) caused concentration - dependent decreases in lps - induced inos expression in raw 264.7 cells ( figure 3(c ) ) and astrocytes ( figure 3(d ) ) . this result is consistent with the profile of the inhibitory effect of inflexinol on no production . a similar inhibitory effect of inflexinol on the lps - induced cox-2 expression was found ( figures 3(c ) and 3(d ) ) . nf-b controls the expression of enzymes including inos and cox-2 whose products contribute to the pathogenesis of the inflammatory process . to investigate whether inflexinol is able to attenuate lps - induced nf-b - mediated promoter activity , we used a luciferase reporter gene expressed under the control of five b cis - acting elements . raw 264.7 cells and astrocytes were transiently transfected with the nf-b - dependent luciferase reporter construct according to manufacture 's specification ( invitrogen ) , and then cell treated with lps ( 1 g / ml ) or cotreated with lps and inflexinol for 8 hours . treatment of raw 264.7 cells ( figure 4(a ) ) and astrocytes ( figure 4(b ) ) with inflexinol resulted in a dose - dependent suppression of luciferase activity induced by lps . in raw 264.7 cells and astrocytes , the ic50 values of inflexinol on inhibiting lps - induced luciferase activity were 2.77 m and 3.88 m , respectively . because activation of nf-b is critical for induction of both cox-2 and inos by lps or other inflammatory cytokines , we determined whether inflexinol might suppress nf-b activation in lps - activated raw 264.7 cells and astrocytes . to investigate whether inflexinol can also inhibit nf-b activation , raw 264.7 cells and astrocytes were cotreated with lps and inflexinol for 60 minutes and 90 minutes , respectively , which it is the time to activate nf-b maximally of its lps treatment ( data are not shown ) . in raw 264.7 cells ( figure 4(c ) ) and astrocytes ( figure 4(d ) ) , lps induced a strong nf-b binding activity , which was markedly inhibited by cotreatment with inflexinol in a dose - dependent manner . it has been demonstrated that lps activates nf-b transcription factor that leads to the induction of the expression of many immediate early genes . to clarify the inhibitory mechanism of action of inflexinol for lps - induced nf-b , translocation of p50 and p65 as well as ib degradation were examined . treatment with lps increased nuclear translocation of p50 and p65 . in the presence of inflexinol , nuclear translocation of p50 and p65 was inhibited in a dose - dependent manner in raw 264.7 cells and astrocytes . moreover , inflexinol inhibited the lps - induced degradation of ib ( figures 5(a ) and 5(b ) ) . these results indicate that inflexinol may inhibit the lps - induced activation of nf-b via an inhibition of ib degradation as well as a translocation of p50 and p65 into the nuclear , and this effect may result in the inhibition of the lps - induced no production as well as inos and cox-2 expression . macrophage overproduction of inflammatory mediators such as cytokine and no has been implicated in inflammatory diseases such as rheumatoid arthritis , septic shock , cerebral malaria , and autoimmune diabetes . several enzymes , such as the inos or cox-2 , along with different inflammatory mediators such as the free radical no or proinflammatory cytokines , have been proposed to be involved in the cell damage associated with neuroinflammation . in this study , we investigated the inhibitory effects of inflexinol on lps - induced no production and expression of inos , cox-2 in raw 264.7 cells and astrocytes . inflexinol ( 1 , 5 , 10 m ) that significantly inhibited lps - induced no production in a dose - dependent manner . inflexinol strongly inhibited lps - induced no production in raw 264.7 cells and astrocytes with ic50 values of 3.43 m and 2.66 m , respectively . these inhibitory effects may not be related with their cytotoxic effects since no effects on cell viability were observed at the concentration up to 10 m in raw 264.7 cells and astrocytes . comparison with ic50 value of indomethacin ( 53.8 m ) and lornoxicam ( 65 m ) being known as nonsteroidal antiinflammatory drugs in lps - stimulated raw 264.7 cells indicates that inflexinol has superior effects on inhibition of no production [ 24 , 25 ] . this inhibitory effect of no production could be related with gene expression of inos since inflexinol inhibited inos protein in raw 264.7 cells and astrocytes . these results showed that inflexinol could interfere lps - induced signaling involving the production of proinflammatory molecules . however , our data showed that the expression of cox-2 was found to be less sensitive than that of inos to the inflexinol . in fact , structurally different diterpenoids displayed differential inhibition of inos and cox-2 expression even though the dna binding activity of nf-b is similar . since lps - induced inos and cox-2 expression is primarily regulated by nf-b , we examined the effect of inflexinol on lps - induced activation of nf-b using an nf-b reporter system as well as dna binding activity using emsa . consistent with the inhibitory effect on inos and cox-2 expression , inflexinol decreased nf-b transcriptional activity in raw 264.7 cells and astrocyte with ic50 values of 2.77 m and 3.88 m , respctively . inflexinol also inhibited nf-b - specific dna binding activity dose dependently . at the gene level , the expression of inos the promoter of the inos gene contains two major discrete regions synergistically functioning for binding of transcription factors : one for nf-b , which is mainly activated by lps . therefore , inhibition of nf-b activation could contribute to the inhibitory effect of inflexinol on inos and cox-2 expression . several studies have shown that antiinflammatory agents inhibit activation of nf-b via prevention of ib degradation . ib specifically binds and masks the nuclear translocation signals of p50 and p65 , thereby preventing the nuclear translocation of the nf-b heterodimer . have showed that sesquiterpene lactones prevented the induced degradation of ib and ib by diverse stimuli and therefore interfered with a common step in the signaling cascade leading to the activation of nf-b . have also demonstrated that prevention of ib degradation by 2-hydroxycinnamaldehyde contributed to inactivation of nf-b ( p50 ) in antiinflammatory reaction in raw 264.7 cells as well as tnf--treated colon cancer cell death . however , it is noteworthy that inflexinol contains an -methylenecyclopentanone moiety as a commom functional group which is known to react with nucleophiles , especially cystein sulfhydryl groups in protein , by a michael - type addition . a c-20-nonoxygenated - ent - kaurane diterpenoid ( kamebakaurin , ka ) , isolated from isodon japonicus , was suggested to interact with cysteine of dna binding domain of the p50 subunit of nf-b . recently , ka was found to be able to interact with both p50 and p65 subunits of nf-b . also demonstrated that sesquiterpene lactones interfered with the activation of nf-b by preventing a degradation of ib and ib but lacking either the lactone or the exomethylene group in the -position to the lactone function displayed no inhibitory effect on pathway leading to the activation of nf-b . similarly , the exomethylene group of inflexinol may be essential for the covalent modification via interaction with cysteine as above compounds . moreover , data presented herein show that inflexinol significantly inhibits nf-b activation by reducing the degradation of ib . therefore , our data suggest a possibility that inflexinol inhibit the upstream proteins of ib such as ikk or 26s proteasome . on the basis of the current results and those of other reports , we propose that inflxinol inhibit the expression of inos , cox-2 , and no production by inhibition of nf-b dna binding activity and transcriptional activation through prevention of ib degradation , and suggest that inflexinol may be useful as an antiinflammatory agent .

the epidemiologic evidence of the prevalence and consequences of pediatric obesity is well documented . currently , there is considerable emphasis on establishing effective prevention and treatment protocols for pediatric obesity [ 2 , 3 ] . in 2007 , an expert committee put forth recommendations for the assessment , prevention , and treatment of pediatric obesity . in this report , a staged approach to the prevention and treatment under the chronic care model was outlined . in brief , four treatment stages consisting of increasing intensity were recommended . patients generally begin at the least - intensive stage and advance depending on responses to treatment , age , severity of obesity , health risks , and motivation . stage 1 is referred to as prevention plus and generally takes place in the primary care office setting . key lifestyle behaviors are promoted and if , after 3 to 6 months , the child has not made appropriate improvement the provider can offer the next level of obesity care . stage 2 entails structured weight management and often includes a multidisciplinary team of health care providers . this level of obesity treatment is distinguished from prevention plus less by differences in the targeted health behaviors and more by the support and structure that is provided to the child in achieving health behaviors . stages 3 and 4 occur within specialty clinics and include a multidisciplinary team where the intensity of behavior changes and frequency of visits increases . in stage 4 obesity care , medications , very low calorie diets , or bariatric surgery this paper describes the methodology of fitkids360 , a stage 2 pediatric weight management program , and provides outcomes in patients who have completed one of 33 fitkids360 classes conducted between 2010 and 2013 . fitkids360 is a multicomponent , family - based , low - cost healthy lifestyle program targeting overweight and obese children and adolescents of 516 years of age and their families . fitkids360 was conceived and designed by a multidisciplinary team of medical professionals ( pediatricians , nurses , dieticians , social workers , exercise specialists , or leaders ) . the overall goal of fitkids360 is to provide an evidence - based approach to the treatment of pediatric overweight and obesity by achieving the following primary aims : ( 1 ) improving the patients ' physical activity , screen time , and dietary behaviors and ( 2 ) improving the family 's obesogenic risk score . the 360 portion of the program name was suggested by the marketing agency that designed the fitkids360 material . it stems from the logo being 360 degrees and is intended to depict people coming full circle , being well rounded , and the program being part of everything in their life . in addition , it was to represent that the program was of full service to encompass all aspects of obesity , not just the physical component , and to support our belief that a 360-degree perspective is required to address the complex issue of pediatric obesity . the involvement of the family and assisting participants by creating a supportive environment for healthy behaviors is a major component of fitkids360 . indeed , the family component is essential in the development of an environment that supports children 's efforts to implement changes in nutrition , physical activity , and screen time [ 4 , 5 ] . furthermore , parents and other family members provide the primary social learning environment in which attitudes and behaviors regarding eating , physical activity , and the use of screen media are formed . the strong influence of the relationship between the parent / caregiver and child , including modeling of health behaviors , creating an environment conducive to active lifestyles , choosing and preparing food , and encouraging and reinforcing eating and physical activity patterns , suggests that parents and caregivers must be involved in interventions designed to increase healthy eating and physical activity in childhood . epstein and wing cited 3 reasons for parental and familial involvement in obesity interventions . ( 1 ) because obesity runs in families , it may be unrealistic to intervene with one family member while other family members are modeling and supporting behaviors that may counteract the intervention 's effectiveness ; ( 2 ) specific parental behaviors that facilitate overeating and inactivity are important in the development of unhealthy behaviors ; ( 3 ) to achieve maximal behavior change in children , use of specific behavior - change strategies ( such as positive reinforcement ) by parents may be warranted . for background purposes , it is important to note that this program was developed based on a collaborative design ; there was no grant funding or hired staff . beginning in 2009 , key players were actively recruited , including researchers , marketing experts , nonprofits , insurance company representatives , fitness centers , and primary care physicians . as the concept was designed , volunteers filled the needs in the constructing of the curriculum , program development , brand design , and so forth . the original objectives of the program were to be ( 1 ) evidence - based , ( 2 ) free of charge , and ( 3 ) widely available at multiple sites and times within the community ( up to 20 or more sessions per year ) . patients must be 5 to 16 years of age and have a body mass index ( bmi ) 85th percentile ( i.e. , overweight or obese ) according to the centers for disease control growth chart . in addition , since fitkids360 utilizes a family - based approach , a parent or guardian must accompany the patient to each class , including the orientation session , and is expected to be involved and assist in making positive changes and offer vital encouragement throughout the duration of the program . each session is limited to 2025 children and their parents in order to give individual attention to each family and build relationships with them . to date , we have targeted low - income , underserved youth and their families with approximately 85% of participants enrolled in medicaid . all patients must be referred by a physician or health care provider to the fitkids360 program coordinator . in addition , patients and their parents or support partners are screened for readiness to change . the program coordinator enters the patient 's information into a wait list and , upon announcement of a new class , places a phone call to the patient ( descending order on the referral list ) . following three attempts to contact the patient , the program coordinator will refer the patient back to the referring physician . patients who are contacted and ready to initiate the program are mailed a welcome letter and also receive a reminder phone call the day before the class is scheduled to start . fitkids360 is conducted by a multidisciplinary team of experts in their respective discipline and in pediatric obesity . table 1 provides a description of the role and responsibilities for each staff member involved in fitkids360 . the intervention begins with a two - hour orientation and assessment period followed by six weekly sessions . each weekly session is two hours in duration and consists of physical activity and nutrition education and behavioral counseling for the patients and their social support person(s ) . in addition , physical activity is performed intermittently throughout the session for a total of 3060 minutes . between weekly sessions , the orientation consists of an introduction of the fitkids360 staff , a description of the purpose of the program , program expectations , class structure , and a review of the weekly log sheets and other paperworks . besides general introductions of all participants , we also conduct getting to know you / icebreaker activitiesto create a connection to the group . for example , if your name were given as the description for any one word in the dictionary , what word would that be ? and why ? we also try to provide a positive perception of pediatric obesity treatment by starting the discussion with the following questions.what do you think this group will be about and why do you think you were asked to participate?what are your perceptions of the group ? what things do you think you have in common?what needs to happen for your family to be successful ? what do you think this group will be about and why do you think you were asked to participate ? what things do you think you have in common ? what needs to happen for your family to be successful ? the curriculum was developed based on an evidence - based approach to key features related to pediatric obesity . healthy counts ( 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 ) are the cornerstone of the fitkids360 curriculum and more broadly our primary care and public health education campaign:8 to 11 hours of sleep every night;7 breakfasts every week;6 home - cooked meals around the table every week;5 servings of fruit and vegetables every day;4 positive self - messages per day;3 servings of low - fat dairy per day;2 hours or less of screen time per day;1 hour or more of physical activity per day;0 sugar - sweetened beverages per day . 8 to 11 hours of sleep every night ; 7 breakfasts every week ; 6 home - cooked meals around the table every week ; 5 servings of fruit and vegetables every day ; 4 positive self - messages per day ; 3 servings of low - fat dairy per day ; 2 hours or less of screen time per day ; 1 hour or more of physical activity per day ; 0 sugar - sweetened beverages per day . more specifically , the cornerstone lifestyle behaviors of physical activity / sedentary behavior and nutrition were coupled with behavioral strategies and issues . the fitkids360 development team relied upon the expert committee recommendations to develop the curriculum , which covered various behavioral , nutritional , and physical activity topics across the duration of the program . families monitor their progress towards their goals via tracking logs for diet ( grains , vegetables , fruits , dairy , meat , and beans ) , physical activity , and screen time . patients and their families set weekly goals with support from the clinical staff . besides monitoring goals , the tracking logs are also part of the incentive point system ( table 3 ) . weekly prizes ( e.g. , hacky sacks , inflatable beach balls , water bottles , jump ropes , hula hoops , and other fun , active prizes ) are offered to incentivize attending each class and participating in all activities . during the last class session , grand prizes are awarded based on incentive points earned . to date , 258 of 418 ( 62% , 38% attrition rate ) patients who started the program have completed it . in comparison , a recent review reported attrition rates ranging from 27% to 73% ( with most on higher end ) . aside from individual patient and family motivation , first , the program or site coordinator makes weekly phone calls 2448 hours before class to remind parents about the class and confirm attendance . second , transportation via taxi is provided free of charge to patients if they are not able to get to class on their own . parents are encouraged to bring siblings over the age of 5 years to the class . while the weekly curriculum provides patients and their families with the foundation for a healthy lifestyle , the buddy program presents the opportunity for enhanced and extended support especially since compliance and adherence are major barriers in the treatment of childhood obesity . the buddy program serves as a transition program that helps children and their families translate knowledge from weekly sessions into lasting behavior changes . the mentoring approach used here is similar to that used in public schools for a variety of goals ( e.g. , educational , life skills , and health habits ) [ 10 , 11 ] . a recent school - based intervention has also incorporated mentoring by trained college allied health and medical students using adapted facets of goal setting methodology previously used with adults [ 13 , 14 ] , with insights and guidance from the limited published research on individualized goal setting for health behavior change with youth [ 15 , 16 ] . the first phase of the buddy program , which occurs concurrently with the fitkids360 weekly sessions , matches participating families with student mentors ( buddies ) for accountability , goal setting , motivation , and weekly planning . the buddies are first and second year students at the michigan state university college of human medicine . each medical student is assigned one or two families during the first week of class . the minimum requirements for each contact include ( 1 ) asking about progress towards individual and family goals , ( 2 ) remembering to complete physical activity and screen time logs , and ( 3 ) addressing any remaining concerns . the second phase of the buddy program occurs during the 6 months following completion of the fitkids360 weekly sessions . this phase builds on the relationships formed during the fitkids360 class session and includes continuation of phone calls or email checkups to facilitate structured goal setting and monthly family events to review themes from the fitkids360 classes and demonstrate healthy lifestyle choices ( e.g. , healthy cooking demonstrations ) . this opportunity for medical students to engage in a pediatric obesity treatment program is unique , since the medical school curriculum does not typically cover details of nutrition , physical activity , healthy lifestyle , or obesity prevention and treatment . this is important since residency and physician training after residency graduation consist of individuals choosing courses on their own to meet continuing medical education requirements without mandatory update training in any specific areas of medicine . furthermore , training of future physicians in obesity prevention and treatment is critical regardless of their future career paths . whether they choose primary care or subspecialty paths , obesity does not spare any particular underlying disease process and its presence will complicate the disease processes of children with special health care needs . the assessment team has consisted of site staff , nurses , medical students , and undergraduate and graduate health science students . the assessment battery includes lifestyle behaviors ( physical activity , screen time , and diet ) as well as physical measures ( height , weight , waist circumference , and percent body fat ) . physical activity , screen time ( viewing television , playing video games , and online computer use ) , and diet are determined by self - report . the self - report question for physical activity is the same question used in the youth risk behavior survey , asking how many days per week children are engaged in moderate - to - vigorous physical activity ( mvpa ) . total screen time is determined from self - reported television viewing , computer use , and video games on weekdays and weekends . dietary consumption of 100% fruit juices , fruit , vegetables , whole grains , sugar - sweetened beverages , sweets / desserts , and dairy was reported as times consumed per day or week . since the family environment is instrumental to the physical activity , screen time , and nutritional behaviors of the patient , the impact of the program on the family obesogenic environment the fnpa screening tool was developed by ihmels and colleagues through comprehensive evidence analyses supported by the american dietetic association and designed to determine the strength of evidence linking physical activity and diet behaviors with overweight / obesity in children . the evidence analyses identified ten primary factors ( breakfast and family meals , nutrition modeling , nutrient dense foods , high calorie beverages , restriction and reward , parent modeling physical activity , child 's physical activity , screen time , tv in the bedroom , sleep , and routine schedule ) that were positively associated with becoming overweight and obese . the score ranges from 20 to 80 with lower scores indicating an adverse , obesogenic family environment . two papers have shown its predictive validity of assessing the prevalence of overweight at baseline and 1-year change in bmi after accounting for initial bmi , parent bmi , and other demographic variables . stature is measured without shoes to the nearest 0.1 cm using a shorrboard stadiometer ( shorrproduction , olney , md ) . body mass is measured to the nearest 0.1 kg and body fatness ( % bf ) is estimated using a foot - to - foot bioelectric impedance scale ( tanita bc-534 , tokyo , japan , and us service center arlington heights , il ) . body mass index ( weight in kg / height in m ) is then calculated from measured stature and body mass . age- and sex - specific percentiles for height , weight , and bmi are determined using the cdc sas growth software ( http://www.cdc.gov/nccdphp/dnpao/growthcharts/resources/sas.htm ) . waist circumference is measured as a proxy for abdominal or visceral adiposity using a gullick tape to the nearest 0.1 cm at the superior border of the iliac crest . upon the completion of a session , the site coordinator is issued a report on the composite results , that is , attrition rate and mean values from pre- and postassessments and so forth . patients are mailed a copy of a report card and cover letter that includes baseline and follow - up values for sleep , screen time , physical activity , fruit and vegetable consumption , low - fat dairy consumption , and the fnpa . in addition , the recommendation for each item is also indicated along with a brief statement about the behavior ( e.g. , children should have less than 2 hours of screen time per day ) . a separate report is sent to the referring physician and also includes educational insight into the key lifestyle behaviors and obesity assessment . in addition , the physician is reminded of the recommended visit schedule for stage 2 obesity treatment ( monthly ) . the program is of relatively low cost about $ 196 per child at a participation rate of 20 children per seven - week session . however , given the interest in pediatric obesity within some communities and by some individuals , costs may be lower as some institutions will allow free facility rental or donate items ( snacks , prizes , etc . ) . in addition , this does not include the cost ( salary and benefits ) for a program coordinator if multiple classes are run in a single community . to increase engagement amongst those interested in fitkids360 , quarterly workgroup meetings are held and attended by key personnel , staff from existing fitkids360 sites , and any other interested persons or parties . these 1 - 2 hour meetings include presentations and discussion on topics such as class updates , recent results , funding opportunities , procedural changes , brainstorming sessions , and alerts to related opportunities in the community ( i.e. , 5 k walks , summer camps , etc . ) . as needed , subcommittees are formed and meet periodically . the entire program is overseen by a steering committee comprised of six individuals including two pediatricians , an exercise physiologist , a registered dietician , a researcher , and the program coordinator . all teams interested in conducting a fitkids360 class are required to participate in our 1-day training program . training sessions are held on a quarterly basis , depending on interest , and are provided by the program coordinator and several members of the fitkids360 steering committee , including the dietician , exercise physiologist , and researcher . the training session begins with an overview and introduction of the fitkids360 program ( 60 minutes ) . given the ethnic and socioeconomic variation in our patients and their perceptions and beliefs about a healthy lifestyle , nutrition , food preparation , and so forth , we provide training on cultural competency and sensitivity ( 60 minutes ) led by a community health educator with experience on this topic . goal setting and motivational interviewing are taught and time is allowed to practice motivational interviewing techniques ( 50 minutes ) . following the lunch break , an overview of assessments and measurements is provided ( 60 minutes ) , and then breakout groups are formed based on area of expertise ( site coordinators and physicians , registered dieticians , behavioral health specialists , and exercise specialists ) . this portion of the training lasts about 2 hours to allow an in - depth training on each week of the curriculum . while lesson plans have been developed to engage the entire family , there are cognitive and developmental differences inherent in the wide age range of our patients . therefore , these breakout sessions include training for adapting the curriculum to different age groups and provide examples of accommodations for younger and older participants . ( described previously ) also plays a major role in providing individualized support to both older and younger students . over the past 4 years a total of 33 fitkids360 classes have been administered at over dozen sites throughout the state of michigan . specifically , baseline characteristics were calculated as means standard deviations ( sd ) , and changes in primary and secondary outcomes over the 7-week program were evaluated using within - subjects repeated measures analysis of variance . before analyses , data were screened for outliers as identified by values > 3 sd from the mean . all analyses were performed using pc - sas ( version 9.3 ) and alpha was set at p < 0.05 . a total of 418 overweight or obese ( bmi 85th centile ) patients aged 516 years old were enrolled in one of 33 fitkids360 classes . 258 patients completed the program and the follow - up evaluation ( 62% retention ) . upon enrollment , 365 patients ( 87.3% ) were obese ( bmi 95th centile ) and 173 ( 41.4% ) of the total sample were severely obese ( bmi 99th centile ) . 41% of patients reported meeting current physical activity recommendations ( mvpa 60 min / d ) . overall , patients increased mpva by 14 min / d after completing fitkids360 ( p = 0.019 ) . among those not meeting recommended activity levels at baseline , mvpa increased by 31 min / d and 56% reported mvpa levels sufficient to meet the 60 min / d guideline by follow - up . on average , patients reduced screen time by 44 min / d ( 0.7 hr / d ) ( p < 0.001 ) due to significant decreases in tv viewing ( p < 0.001 ) and video game playing ( p = 0.027 ) . among the 70% reporting > 2 hr / d of tv viewing at baseline , 42% reduced to < 2 hr / d after completing fitkids360 . significant changes in reported nutrition behaviors included a reduction in the frequency of consumed sweets / desserts ( p < 0.001 ) and sugar - sweetened soft drinks ( p = 0.016 ) and an increase in the frequency of consumed whole grains ( p < 0.001 ) and fruits and vegetables ( p = 0.017 ) ( table 4 ) . in addition , fnpa scores increased by 5.4 points ( 9% ) in the overall sample ( p < 0.001 ) , and scores improved by 8.5 points ( 16% ) among families identified as having high - risk family environments and behaviors according to baseline fnpa levels . on average , patients increased both height ( p < 0.001 ) and weight ( p = 0.030 ) over the 7-week program resulting in a small but significant reduction in bmi ( p = 0.011 ) . similarly , a significant decrease was measured in age- and sex - adjusted bmi z - scores ( p < 0.001 ) . based on the results , it appears that fitkids360 may be an effective program for initiating positive health behavior changes and supporting weight management efforts , at least in the short term . patients who completed fitkids360 made meaningful improvements to their physical activity and sedentary behaviors and reported significant progress in their nutrition habits , including consuming whole grains , fruits , and vegetables more frequently and sweets / desserts and sugar - sweetened soft drinks less frequently . perhaps most significantly , participants ( and their families ) improved overall health behaviors ( i.e. , household rules and family structure regarding diet , physical activity , sedentary time , and sleep ) as indicated by the fnpa survey . have shown that youth in the lowest tertile for fnpa scores had 1.7 times higher odds of being overweight or obese ( bmi 85th percentile ) when compared to youth in the highest tertile . in the current study , 50% of patients had baseline fnpa scores within this lowest tertile range , but 69% of these high - risk patients improved fnpa scores sufficiently to move them into the middle or upper tertiles after completing fitkids360 . due to the short duration of the fitkids360 curriculum , substantial changes in body composition patients who completed the program did experience small , but significant improvements in both bmi and bmi z - scores over the 7-week program . this is a notable result since typical bmi trajectories for growing children in this age range are positive . future research is needed to assess the long - term sustainability and health impact of the health behavior and anthropometric changes that took place during fitkids360 classes . one limitation of this study is the lack of a control / comparison group , which introduces several potential threats to internal validity , including confounding , regression toward the mean , and mortality . in addition , several of the lifestyle behaviors evaluated in this study were self- or parent - reported , which introduces potential social desirability bias . however , the self - report tools used in fitkids360 consist of surveys and questions which have been previously validated . in addition , the results presented here are fairly robust , as they include a relatively large sample from over 30 different fitkids360 interventions . moreover , the fitkids360 program was designed as an evidence - based , yet feasible and practical pediatric weight management program rather than a randomized clinical trial . to this end , real - world indication of short - term outcomes after participating in the fitkids360 program . the fitkids360 team is currently working to develop a standard follow - up procedure to allow long - term tracking of patients . we have initiated the process by sending a report to the patients ' physicians asking them to continue long - term follow - up . based on the expert recommendations , monthly office visits for 36 months are probably most appropriate at this level . in summary , we have described the program design and intervention and measurement components of a low - cost stage 2 pediatric obesity program called fitkids360 , which has been implemented in several locations throughout michigan and has now expanded outside of the state as well . in addition , we have reported outcomes on over 250 youth who have completed the fitkids360 program , including improvements in physical activity , nutrition , and sedentary behaviors , as well as bmi . we hope that this report will be useful to researchers , public health professionals , and medical professionals seeking to develop similar stage 2 pediatric obesity programs .

this paper describes fitkids360 , a stage 2 pediatric weight management program . fitkids360 is a physician - referred , multicomponent , low - cost healthy lifestyle program for overweight and obese youth 516 years of age and their families . fitkids360 provides an evidence - based approach to the treatment of pediatric overweight by targeting patients ' physical activity , screen time , and dietary behaviors using a family - centered approach . the intervention begins with a two - hour orientation and assessment period followed by six weekly sessions . assessments include lifestyle behaviors , anthropometry , and the family nutrition and physical activity ( fnpa ) survey , which screens for obesogenic risk factors in the home environment . outcomes are presented from 258 patients who completed one of 33 fitkids360 classes . after completing fitkids360 , patients increased moderate to vigorous physical activity by 14 minutes ( p = 0.019 ) , reduced screen time by 44 minutes ( p < 0.001 ) , and improved key dietary behaviors . overall , fnpa scores increased by 9% ( p < 0.001 ) and 69% of patients with high risk fnpa scores at baseline dropped below the high risk range by followup . patients also lowered bmis ( p = 0.011 ) and age- and sex - adjusted bmi z - scores ( p < 0.001 ) after completing the 7-week program . we hope this report will be useful to medical and public health professionals seeking to develop stage 2 pediatric obesity programs .

1. Introduction 2. What Is FitKids360? 3. Assessments 4. Costs 5. Meetings and Training Program 6. Outcomes 7. Discussion 8. Summary

the epidemiologic evidence of the prevalence and consequences of pediatric obesity is well documented . currently , there is considerable emphasis on establishing effective prevention and treatment protocols for pediatric obesity [ 2 , 3 ] . in 2007 , an expert committee put forth recommendations for the assessment , prevention , and treatment of pediatric obesity . in this report , a staged approach to the prevention and treatment under the chronic care model was outlined . stage 1 is referred to as prevention plus and generally takes place in the primary care office setting . key lifestyle behaviors are promoted and if , after 3 to 6 months , the child has not made appropriate improvement the provider can offer the next level of obesity care . stage 2 entails structured weight management and often includes a multidisciplinary team of health care providers . this level of obesity treatment is distinguished from prevention plus less by differences in the targeted health behaviors and more by the support and structure that is provided to the child in achieving health behaviors . in stage 4 obesity care , medications , very low calorie diets , or bariatric surgery this paper describes the methodology of fitkids360 , a stage 2 pediatric weight management program , and provides outcomes in patients who have completed one of 33 fitkids360 classes conducted between 2010 and 2013 . fitkids360 is a multicomponent , family - based , low - cost healthy lifestyle program targeting overweight and obese children and adolescents of 516 years of age and their families . the overall goal of fitkids360 is to provide an evidence - based approach to the treatment of pediatric overweight and obesity by achieving the following primary aims : ( 1 ) improving the patients ' physical activity , screen time , and dietary behaviors and ( 2 ) improving the family 's obesogenic risk score . it stems from the logo being 360 degrees and is intended to depict people coming full circle , being well rounded , and the program being part of everything in their life . in addition , it was to represent that the program was of full service to encompass all aspects of obesity , not just the physical component , and to support our belief that a 360-degree perspective is required to address the complex issue of pediatric obesity . the involvement of the family and assisting participants by creating a supportive environment for healthy behaviors is a major component of fitkids360 . indeed , the family component is essential in the development of an environment that supports children 's efforts to implement changes in nutrition , physical activity , and screen time [ 4 , 5 ] . furthermore , parents and other family members provide the primary social learning environment in which attitudes and behaviors regarding eating , physical activity , and the use of screen media are formed . the strong influence of the relationship between the parent / caregiver and child , including modeling of health behaviors , creating an environment conducive to active lifestyles , choosing and preparing food , and encouraging and reinforcing eating and physical activity patterns , suggests that parents and caregivers must be involved in interventions designed to increase healthy eating and physical activity in childhood . ( 1 ) because obesity runs in families , it may be unrealistic to intervene with one family member while other family members are modeling and supporting behaviors that may counteract the intervention 's effectiveness ; ( 2 ) specific parental behaviors that facilitate overeating and inactivity are important in the development of unhealthy behaviors ; ( 3 ) to achieve maximal behavior change in children , use of specific behavior - change strategies ( such as positive reinforcement ) by parents may be warranted . beginning in 2009 , key players were actively recruited , including researchers , marketing experts , nonprofits , insurance company representatives , fitness centers , and primary care physicians . as the concept was designed , volunteers filled the needs in the constructing of the curriculum , program development , brand design , and so forth . the original objectives of the program were to be ( 1 ) evidence - based , ( 2 ) free of charge , and ( 3 ) widely available at multiple sites and times within the community ( up to 20 or more sessions per year ) . patients must be 5 to 16 years of age and have a body mass index ( bmi ) 85th percentile ( i.e. , overweight or obese ) according to the centers for disease control growth chart . in addition , since fitkids360 utilizes a family - based approach , a parent or guardian must accompany the patient to each class , including the orientation session , and is expected to be involved and assist in making positive changes and offer vital encouragement throughout the duration of the program . each session is limited to 2025 children and their parents in order to give individual attention to each family and build relationships with them . to date , we have targeted low - income , underserved youth and their families with approximately 85% of participants enrolled in medicaid . all patients must be referred by a physician or health care provider to the fitkids360 program coordinator . in addition , patients and their parents or support partners are screened for readiness to change . following three attempts to contact the patient , the program coordinator will refer the patient back to the referring physician . patients who are contacted and ready to initiate the program are mailed a welcome letter and also receive a reminder phone call the day before the class is scheduled to start . fitkids360 is conducted by a multidisciplinary team of experts in their respective discipline and in pediatric obesity . the intervention begins with a two - hour orientation and assessment period followed by six weekly sessions . each weekly session is two hours in duration and consists of physical activity and nutrition education and behavioral counseling for the patients and their social support person(s ) . in addition , physical activity is performed intermittently throughout the session for a total of 3060 minutes . between weekly sessions , the orientation consists of an introduction of the fitkids360 staff , a description of the purpose of the program , program expectations , class structure , and a review of the weekly log sheets and other paperworks . besides general introductions of all participants , we also conduct getting to know you / icebreaker activitiesto create a connection to the group . for example , if your name were given as the description for any one word in the dictionary , what word would that be ? we also try to provide a positive perception of pediatric obesity treatment by starting the discussion with the following questions.what do you think this group will be about and why do you think you were asked to participate?what are your perceptions of the group ? what do you think this group will be about and why do you think you were asked to participate ? the curriculum was developed based on an evidence - based approach to key features related to pediatric obesity . healthy counts ( 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 ) are the cornerstone of the fitkids360 curriculum and more broadly our primary care and public health education campaign:8 to 11 hours of sleep every night;7 breakfasts every week;6 home - cooked meals around the table every week;5 servings of fruit and vegetables every day;4 positive self - messages per day;3 servings of low - fat dairy per day;2 hours or less of screen time per day;1 hour or more of physical activity per day;0 sugar - sweetened beverages per day . 8 to 11 hours of sleep every night ; 7 breakfasts every week ; 6 home - cooked meals around the table every week ; 5 servings of fruit and vegetables every day ; 4 positive self - messages per day ; 3 servings of low - fat dairy per day ; 2 hours or less of screen time per day ; 1 hour or more of physical activity per day ; 0 sugar - sweetened beverages per day . more specifically , the cornerstone lifestyle behaviors of physical activity / sedentary behavior and nutrition were coupled with behavioral strategies and issues . the fitkids360 development team relied upon the expert committee recommendations to develop the curriculum , which covered various behavioral , nutritional , and physical activity topics across the duration of the program . families monitor their progress towards their goals via tracking logs for diet ( grains , vegetables , fruits , dairy , meat , and beans ) , physical activity , and screen time . patients and their families set weekly goals with support from the clinical staff . in comparison , a recent review reported attrition rates ranging from 27% to 73% ( with most on higher end ) . parents are encouraged to bring siblings over the age of 5 years to the class . while the weekly curriculum provides patients and their families with the foundation for a healthy lifestyle , the buddy program presents the opportunity for enhanced and extended support especially since compliance and adherence are major barriers in the treatment of childhood obesity . the buddy program serves as a transition program that helps children and their families translate knowledge from weekly sessions into lasting behavior changes . a recent school - based intervention has also incorporated mentoring by trained college allied health and medical students using adapted facets of goal setting methodology previously used with adults [ 13 , 14 ] , with insights and guidance from the limited published research on individualized goal setting for health behavior change with youth [ 15 , 16 ] . the first phase of the buddy program , which occurs concurrently with the fitkids360 weekly sessions , matches participating families with student mentors ( buddies ) for accountability , goal setting , motivation , and weekly planning . the minimum requirements for each contact include ( 1 ) asking about progress towards individual and family goals , ( 2 ) remembering to complete physical activity and screen time logs , and ( 3 ) addressing any remaining concerns . the second phase of the buddy program occurs during the 6 months following completion of the fitkids360 weekly sessions . this phase builds on the relationships formed during the fitkids360 class session and includes continuation of phone calls or email checkups to facilitate structured goal setting and monthly family events to review themes from the fitkids360 classes and demonstrate healthy lifestyle choices ( e.g. this opportunity for medical students to engage in a pediatric obesity treatment program is unique , since the medical school curriculum does not typically cover details of nutrition , physical activity , healthy lifestyle , or obesity prevention and treatment . the assessment battery includes lifestyle behaviors ( physical activity , screen time , and diet ) as well as physical measures ( height , weight , waist circumference , and percent body fat ) . physical activity , screen time ( viewing television , playing video games , and online computer use ) , and diet are determined by self - report . the self - report question for physical activity is the same question used in the youth risk behavior survey , asking how many days per week children are engaged in moderate - to - vigorous physical activity ( mvpa ) . total screen time is determined from self - reported television viewing , computer use , and video games on weekdays and weekends . dietary consumption of 100% fruit juices , fruit , vegetables , whole grains , sugar - sweetened beverages , sweets / desserts , and dairy was reported as times consumed per day or week . since the family environment is instrumental to the physical activity , screen time , and nutritional behaviors of the patient , the impact of the program on the family obesogenic environment the fnpa screening tool was developed by ihmels and colleagues through comprehensive evidence analyses supported by the american dietetic association and designed to determine the strength of evidence linking physical activity and diet behaviors with overweight / obesity in children . the evidence analyses identified ten primary factors ( breakfast and family meals , nutrition modeling , nutrient dense foods , high calorie beverages , restriction and reward , parent modeling physical activity , child 's physical activity , screen time , tv in the bedroom , sleep , and routine schedule ) that were positively associated with becoming overweight and obese . two papers have shown its predictive validity of assessing the prevalence of overweight at baseline and 1-year change in bmi after accounting for initial bmi , parent bmi , and other demographic variables . stature is measured without shoes to the nearest 0.1 cm using a shorrboard stadiometer ( shorrproduction , olney , md ) . body mass is measured to the nearest 0.1 kg and body fatness ( % bf ) is estimated using a foot - to - foot bioelectric impedance scale ( tanita bc-534 , tokyo , japan , and us service center arlington heights , il ) . age- and sex - specific percentiles for height , weight , and bmi are determined using the cdc sas growth software ( http://www.cdc.gov/nccdphp/dnpao/growthcharts/resources/sas.htm ) . waist circumference is measured as a proxy for abdominal or visceral adiposity using a gullick tape to the nearest 0.1 cm at the superior border of the iliac crest . patients are mailed a copy of a report card and cover letter that includes baseline and follow - up values for sleep , screen time , physical activity , fruit and vegetable consumption , low - fat dairy consumption , and the fnpa . in addition , the recommendation for each item is also indicated along with a brief statement about the behavior ( e.g. , children should have less than 2 hours of screen time per day ) . a separate report is sent to the referring physician and also includes educational insight into the key lifestyle behaviors and obesity assessment . in addition , the physician is reminded of the recommended visit schedule for stage 2 obesity treatment ( monthly ) . to increase engagement amongst those interested in fitkids360 , quarterly workgroup meetings are held and attended by key personnel , staff from existing fitkids360 sites , and any other interested persons or parties . these 1 - 2 hour meetings include presentations and discussion on topics such as class updates , recent results , funding opportunities , procedural changes , brainstorming sessions , and alerts to related opportunities in the community ( i.e. the entire program is overseen by a steering committee comprised of six individuals including two pediatricians , an exercise physiologist , a registered dietician , a researcher , and the program coordinator . training sessions are held on a quarterly basis , depending on interest , and are provided by the program coordinator and several members of the fitkids360 steering committee , including the dietician , exercise physiologist , and researcher . the training session begins with an overview and introduction of the fitkids360 program ( 60 minutes ) . given the ethnic and socioeconomic variation in our patients and their perceptions and beliefs about a healthy lifestyle , nutrition , food preparation , and so forth , we provide training on cultural competency and sensitivity ( 60 minutes ) led by a community health educator with experience on this topic . following the lunch break , an overview of assessments and measurements is provided ( 60 minutes ) , and then breakout groups are formed based on area of expertise ( site coordinators and physicians , registered dieticians , behavioral health specialists , and exercise specialists ) . over the past 4 years a total of 33 fitkids360 classes have been administered at over dozen sites throughout the state of michigan . specifically , baseline characteristics were calculated as means standard deviations ( sd ) , and changes in primary and secondary outcomes over the 7-week program were evaluated using within - subjects repeated measures analysis of variance . all analyses were performed using pc - sas ( version 9.3 ) and alpha was set at p < 0.05 . a total of 418 overweight or obese ( bmi 85th centile ) patients aged 516 years old were enrolled in one of 33 fitkids360 classes . 258 patients completed the program and the follow - up evaluation ( 62% retention ) . upon enrollment , 365 patients ( 87.3% ) were obese ( bmi 95th centile ) and 173 ( 41.4% ) of the total sample were severely obese ( bmi 99th centile ) . 41% of patients reported meeting current physical activity recommendations ( mvpa 60 min / d ) . overall , patients increased mpva by 14 min / d after completing fitkids360 ( p = 0.019 ) . among those not meeting recommended activity levels at baseline , mvpa increased by 31 min / d and 56% reported mvpa levels sufficient to meet the 60 min / d guideline by follow - up . on average , patients reduced screen time by 44 min / d ( 0.7 hr / d ) ( p < 0.001 ) due to significant decreases in tv viewing ( p < 0.001 ) and video game playing ( p = 0.027 ) . among the 70% reporting > 2 hr / d of tv viewing at baseline , 42% reduced to < 2 hr / d after completing fitkids360 . significant changes in reported nutrition behaviors included a reduction in the frequency of consumed sweets / desserts ( p < 0.001 ) and sugar - sweetened soft drinks ( p = 0.016 ) and an increase in the frequency of consumed whole grains ( p < 0.001 ) and fruits and vegetables ( p = 0.017 ) ( table 4 ) . in addition , fnpa scores increased by 5.4 points ( 9% ) in the overall sample ( p < 0.001 ) , and scores improved by 8.5 points ( 16% ) among families identified as having high - risk family environments and behaviors according to baseline fnpa levels . on average , patients increased both height ( p < 0.001 ) and weight ( p = 0.030 ) over the 7-week program resulting in a small but significant reduction in bmi ( p = 0.011 ) . similarly , a significant decrease was measured in age- and sex - adjusted bmi z - scores ( p < 0.001 ) . based on the results , it appears that fitkids360 may be an effective program for initiating positive health behavior changes and supporting weight management efforts , at least in the short term . patients who completed fitkids360 made meaningful improvements to their physical activity and sedentary behaviors and reported significant progress in their nutrition habits , including consuming whole grains , fruits , and vegetables more frequently and sweets / desserts and sugar - sweetened soft drinks less frequently . perhaps most significantly , participants ( and their families ) improved overall health behaviors ( i.e. , household rules and family structure regarding diet , physical activity , sedentary time , and sleep ) as indicated by the fnpa survey . have shown that youth in the lowest tertile for fnpa scores had 1.7 times higher odds of being overweight or obese ( bmi 85th percentile ) when compared to youth in the highest tertile . in the current study , 50% of patients had baseline fnpa scores within this lowest tertile range , but 69% of these high - risk patients improved fnpa scores sufficiently to move them into the middle or upper tertiles after completing fitkids360 . due to the short duration of the fitkids360 curriculum , substantial changes in body composition patients who completed the program did experience small , but significant improvements in both bmi and bmi z - scores over the 7-week program . future research is needed to assess the long - term sustainability and health impact of the health behavior and anthropometric changes that took place during fitkids360 classes . one limitation of this study is the lack of a control / comparison group , which introduces several potential threats to internal validity , including confounding , regression toward the mean , and mortality . in addition , several of the lifestyle behaviors evaluated in this study were self- or parent - reported , which introduces potential social desirability bias . moreover , the fitkids360 program was designed as an evidence - based , yet feasible and practical pediatric weight management program rather than a randomized clinical trial . to this end , real - world indication of short - term outcomes after participating in the fitkids360 program . the fitkids360 team is currently working to develop a standard follow - up procedure to allow long - term tracking of patients . we have initiated the process by sending a report to the patients ' physicians asking them to continue long - term follow - up . in summary , we have described the program design and intervention and measurement components of a low - cost stage 2 pediatric obesity program called fitkids360 , which has been implemented in several locations throughout michigan and has now expanded outside of the state as well . in addition , we have reported outcomes on over 250 youth who have completed the fitkids360 program , including improvements in physical activity , nutrition , and sedentary behaviors , as well as bmi . we hope that this report will be useful to researchers , public health professionals , and medical professionals seeking to develop similar stage 2 pediatric obesity programs .

all protocols were carried out in accordance with the principles of the declaration of helsinki . participants were composed of 128 patients who underwent initial therapy at the study hospital between november 2004 and december 2010 and had been pathologically given a diagnosis of eoc . informed consent was obtained from all patients for the use of surgical specimens for research purposes . the clinical stage of each specimen was decided in accordance with the international federation of gynecology and obstetrics 2014 classifications . although 181 patients were treated during the study period , 53 patients who underwent nac were excluded considering the impact of anticancer drug exposure on immunostaining in tumor cells . the clinical stages of these 53 patients were stage ii in 2 patients , stage iii in 33 patients , and stage iv in 18 patients . the histological types were serous carcinoma in 45 patients , ccc in 5 patients , mucinous carcinoma in 2 patients , and undifferentiated carcinoma in 1 patient . twelve patients in whom nac was not expected to lead to the total elimination of cancer in the initial surgery , 23 patients in whom surgery needed to be cut short because of complications including vte or a poor general condition , and 18 patients in the nac group who took part in a phase iii trial of upfront debulking surgery versus nac for stage iii / iv ovarian , tubal , and peritoneal cancers9 were ultimately chosen for treatment , bringing the total number of patients included in this study to 128 . immunostaining was performed on surgical specimens obtained from 126 patients who had undergone radical surgery for eoc and biopsy tissues obtained from 2 patients who had undergone exploratory laparotomy . we performed immunostaining on tissue specimens from all patients in this study , using the same technique reported in our 2007 investigation.8 in brief , we prepared 3-mm - thick sections from 3 sites of paraffin block specimens from each patient . the anti - tf antibody ( cedarlane laboratories , burlington , nc ) used as the primary antibody was diluted 50-fold and biotinylated by the avidin - biotin - peroxidase complex method ( vector abc elite kit ; vector laboratories , burlingame , ca ) , followed by color development with diaminobenzidine tetrahydrochloride . as positive controls , we used sections of the umbilical cord , which is known to stain brightly for tf,10 whereas negative controls were sections that had been incubated in normal mouse serum . the intensity of tf expression ( itfe ) was graded into the following 4 levels based on the proportion of all cell populations that stained positively for tf : negative , no apparent positive tumor cells ; weakly positive , less than 50% positive tumor cells ; moderately positive , greater than or equal to 50% positive tumor cells with weak intensity ; and strongly positive , greater than or equal to 50% positive tumor cells with strong intensity ( fig . percentages are based on the proportion of the entire tumor cell population positive for tf . all evaluations of the immunohistological results were performed by 2 independent observers blinded to the results of hematological examinations and histological diagnoses . examination for vte was performed using the same method we have reported elsewhere.4,8 leg vein ultrasonography was performed on all patients using an atl hdi5000 system ( philips medical systems , bothell , wa ) with a 3- to 7.5-mhz transducer , to detect deep vein thrombosis ( dvt ) . output , pulse repetition frequency , and wall thump filter settings were adjusted for venous vascular studies . bilateral iliac , femoral , great saphenous , popliteal , peroneal , posterior tibial , and soleal veins were assessed for the presence / absence of dvt . the iliac and femoral veins were examined with the patient supine , whereas all other veins were assessed in the upright position . all vessels were imaged in both short - axis cross - section ( transverse image ) and long - axis cross - section ( longitudinal image ) . manual compression using a transducer and color doppler imaging were performed to observe the lumina of veins and search for blood clots . however , the absence of a response to the valsalva maneuver would raise the suspicion of a blood flow disorder in the proximal vein , so thrombi in the pelvic veins were diagnosed based on the results of contrast - enhanced computed tomography ( ct ) . all patients underwent extensive imaging using ct and magnetic resonance imaging to detect the spread of pelvic tumors and blood clots in the iliac veins and inferior vena cava . all patients in whom dvt was detected on leg vein ultrasonography or pelvic contrast - enhanced ct were further examined for the presence or absence of pulmonary thromboembolism on contrast - enhanced ct of the chest or pulmonary blood flow scintigraphy using tc-99 . categorical data are summarized as frequency and percentage , whereas continuous data are summarized as mean and standard deviation . continuous data were tested using student t test for differences between 2 groups and by an analysis of variance for comparisons of 3 or more groups . the development of vte was investigated using univariate and multivariate analyses with a logistic model . age , body mass index ( bmi ) , stage , pretreatment dimerized plasmin fragment d ( d - dimer or dd ) level , histological type , and itfe were used as variables in the univariate analysis to identify potential risk factors for vte development . variables showing significance were then subjected to multivariate analysis . variables showing strong confounding suggestive of multicollinearity as well as a low p value were selected and included in the multivariate analysis model . to evaluate the contribution of itfe and histology in each stage , we used additional multivariate models that included itfe , stage , and dd level ; the interaction between itfe and stage ; the interaction among histological type , stage , and dd level ; and the interaction between histological type and stage . all analyses were performed using sas version 9.4 software ( sas institute , cary , nc ) , and values of p < 0.05 were considered statistically significant . all protocols were carried out in accordance with the principles of the declaration of helsinki . participants were composed of 128 patients who underwent initial therapy at the study hospital between november 2004 and december 2010 and had been pathologically given a diagnosis of eoc . informed consent was obtained from all patients for the use of surgical specimens for research purposes . the clinical stage of each specimen was decided in accordance with the international federation of gynecology and obstetrics 2014 classifications . although 181 patients were treated during the study period , 53 patients who underwent nac were excluded considering the impact of anticancer drug exposure on immunostaining in tumor cells . the clinical stages of these 53 patients were stage ii in 2 patients , stage iii in 33 patients , and stage iv in 18 patients . the histological types were serous carcinoma in 45 patients , ccc in 5 patients , mucinous carcinoma in 2 patients , and undifferentiated carcinoma in 1 patient . twelve patients in whom nac was not expected to lead to the total elimination of cancer in the initial surgery , 23 patients in whom surgery needed to be cut short because of complications including vte or a poor general condition , and 18 patients in the nac group who took part in a phase iii trial of upfront debulking surgery versus nac for stage iii / iv ovarian , tubal , and peritoneal cancers9 were ultimately chosen for treatment , bringing the total number of patients included in this study to 128 . immunostaining was performed on surgical specimens obtained from 126 patients who had undergone radical surgery for eoc and biopsy tissues obtained from 2 patients who had undergone exploratory laparotomy . we performed immunostaining on tissue specimens from all patients in this study , using the same technique reported in our 2007 investigation.8 in brief , we prepared 3-mm - thick sections from 3 sites of paraffin block specimens from each patient . the anti - tf antibody ( cedarlane laboratories , burlington , nc ) used as the primary antibody was diluted 50-fold and biotinylated by the avidin - biotin - peroxidase complex method ( vector abc elite kit ; vector laboratories , burlingame , ca ) , followed by color development with diaminobenzidine tetrahydrochloride . as positive controls , we used sections of the umbilical cord , which is known to stain brightly for tf,10 whereas negative controls were sections that had been incubated in normal mouse serum . the intensity of tf expression ( itfe ) was graded into the following 4 levels based on the proportion of all cell populations that stained positively for tf : negative , no apparent positive tumor cells ; weakly positive , less than 50% positive tumor cells ; moderately positive , greater than or equal to 50% positive tumor cells with weak intensity ; and strongly positive , greater than or equal to 50% positive tumor cells with strong intensity ( fig . percentages are based on the proportion of the entire tumor cell population positive for tf . all evaluations of the immunohistological results were performed by 2 independent observers blinded to the results of hematological examinations and histological diagnoses . examination for vte was performed using the same method we have reported elsewhere.4,8 leg vein ultrasonography was performed on all patients using an atl hdi5000 system ( philips medical systems , bothell , wa ) with a 3- to 7.5-mhz transducer , to detect deep vein thrombosis ( dvt ) . output , pulse repetition frequency , and wall thump filter settings were adjusted for venous vascular studies . bilateral iliac , femoral , great saphenous , popliteal , peroneal , posterior tibial , and soleal veins were assessed for the presence / absence of dvt . the iliac and femoral veins were examined with the patient supine , whereas all other veins were assessed in the upright position . all vessels were imaged in both short - axis cross - section ( transverse image ) and long - axis cross - section ( longitudinal image ) . manual compression using a transducer and color doppler imaging were performed to observe the lumina of veins and search for blood clots . however , the absence of a response to the valsalva maneuver would raise the suspicion of a blood flow disorder in the proximal vein , so thrombi in the pelvic veins were diagnosed based on the results of contrast - enhanced computed tomography ( ct ) . all patients underwent extensive imaging using ct and magnetic resonance imaging to detect the spread of pelvic tumors and blood clots in the iliac veins and inferior vena cava . all patients in whom dvt was detected on leg vein ultrasonography or pelvic contrast - enhanced ct were further examined for the presence or absence of pulmonary thromboembolism on contrast - enhanced ct of the chest or pulmonary blood flow scintigraphy using tc-99 . categorical data are summarized as frequency and percentage , whereas continuous data are summarized as mean and standard deviation . continuous data were tested using student t test for differences between 2 groups and by an analysis of variance for comparisons of 3 or more groups . the development of vte was investigated using univariate and multivariate analyses with a logistic model . age , body mass index ( bmi ) , stage , pretreatment dimerized plasmin fragment d ( d - dimer or dd ) level , histological type , and itfe were used as variables in the univariate analysis to identify potential risk factors for vte development . variables showing strong confounding suggestive of multicollinearity as well as a low p value were selected and included in the multivariate analysis model . to evaluate the contribution of itfe and histology in each stage , we used additional multivariate models that included itfe , stage , and dd level ; the interaction between itfe and stage ; the interaction among histological type , stage , and dd level ; and the interaction between histological type and stage . all analyses were performed using sas version 9.4 software ( sas institute , cary , nc ) , and values of p < 0.05 were considered statistically significant . at the time of initial treatment , mean age was 56.6 years ( range , 3188 years ) , and mean bmi was 22.4 kg / m ( range , 13.335.4 kg / m ) . mean pretreatment dd level was 4.2 g / ml ( range , 0.120.0 g / ml ) . for the 128 patients , stage was i in 55 patients , ii in 22 patients , iii in 37 patients , and iv in 14 patients , whereas the histological type was serous in 42 patients , mucinous in 12 patients , endometrioid in 15 patients , ccc in 53 patients , and undifferentiated in 6 patients ( table 1 ) . patient characteristics ( n = 128 ) tissue factor was expressed in 71 patients ( weakly positive , n = 23 ; moderately positive , n = 32 ; strongly positive , n = 16 ) , representing 55.5% of the 128 patients . the itfe did not correlate with patient age , bmi , or pretreatment dd level . according to stage , itfe was 81.8% for stage i and 45.4% for stage ii but was significantly lower for stages iii and iv , at 29.7% and 35.7% , respectively ( p < 0.001 ) . incidences according to histological type were 94.3% ( 50/53 ) for ccc , 23.8% ( 10/42 ) for serous carcinoma , 26.7% ( 4/15 ) for endometrioid carcinoma , 58.3% ( 7/12 ) for mucinous carcinoma , and 0% ( 0/6 ) for undifferentiated carcinoma . the itfe was significantly higher in ccc than in non - ccc ( p < 0.001 ) and was also stronger in ccc ( table 2 ) . as itfe increased from negative to mild , moderate , and strong , the incidence of vte increased significantly ( ptrend = 0.014 ) to 16% , 17% , 38% , and 38% , respectively ( table 3 ) . patient characteristics according to itfe in cancer tissues incidence of vte for each itfe venous thromboembolism developed before starting treatment in 31 patients ( 24.2% ) ( dvt alone , n = 20 ; dvt with pe , n = 8 ; pe alone , n = 3 ) , and the rate of subclinical disease was 96.8% . screening was performed according to a dd level with a negative predictive value of 96.1%.12 nonetheless , patients with silent pe alone were likely overlooked among the patients who did not undergo ct because no dvt was identified we consider that the rate is extremely low . mean ( sd ) dd level was 7.4 ( 6.4 ) g / ml in patients with vte , which is significantly higher than in patients without vte ( 3.2 [ 5.0 ] g / ml ; p < 0.001 ) . venous thromboembolism developed in 34.0% ( 18/53 ) of the patients with ccc , which is significantly higher than the incidence of 17.3% ( 13/75 ) among patients with non - ccc ( p = 0.03 ) . tissue factor positive patients developed vte at an incidence of 31.0% ( 22/71 ) , which is significantly higher than the incidence of 15.8% ( 9/57 ) in tf - negative patients ( p = 0.0496 ) . comparison of the negative / weakly positive patient group with the moderately / strongly positive patient group showed a significant difference in vte incidences , at 16.3% ( 13/80 ) and 37.5% ( 18/48 ; p = 0.01 ) , respectively . the itfe was therefore entered into the multivariate analysis using those 2 groups ( negative / weakly positive vs moderately / strongly positive ) . a strong correlation was found between ccc and itfe , and because multicollinearity was seen ( spearman correlation coefficient , 0.66 ) , simultaneous incorporation of ccc and itfe in the multivariate analysis model was considered statistically inappropriate . accordingly , when itfe was selected as the factor with the lower p value and multivariate analysis was performed , the results showed that pretreatment dd level and itfe represented significant independent risk factors for the development of vte before the start of treatment ( table 4 ) . we also examined the impact of histology and tf on vte development before the start of treatment by early- and advanced - stage diseases . although no significant difference was seen between ccc and non - ccc in either early- or advanced - stage diseases , a tendency was seen for vte development to increase in ccc . when tf was moderately / strongly positive , no significant difference was seen in early - stage diseases , but a tendency toward an increased risk of vte development was seen . in advanced - stage diseases , the risk of vte development was significantly increased ( p = 0.021 ; table 5 ) . logistic regression analysis for vte effect of tf and histological type on the risk of vte in early- and advanced - stage diseases at the time of initial treatment , mean age was 56.6 years ( range , 3188 years ) , and mean bmi was 22.4 kg / m ( range , 13.335.4 kg / m ) . mean pretreatment dd level was 4.2 g / ml ( range , 0.120.0 g / ml ) . for the 128 patients , stage was i in 55 patients , ii in 22 patients , iii in 37 patients , and iv in 14 patients , whereas the histological type was serous in 42 patients , mucinous in 12 patients , endometrioid in 15 patients , ccc in 53 patients , and undifferentiated in 6 patients ( table 1 ) . tissue factor was expressed in 71 patients ( weakly positive , n = 23 ; moderately positive , n = 32 ; strongly positive , n = 16 ) , representing 55.5% of the 128 patients . the itfe did not correlate with patient age , bmi , or pretreatment dd level . according to stage , itfe was 81.8% for stage i and 45.4% for stage ii but was significantly lower for stages iii and iv , at 29.7% and 35.7% , respectively ( p < 0.001 ) . incidences according to histological type were 94.3% ( 50/53 ) for ccc , 23.8% ( 10/42 ) for serous carcinoma , 26.7% ( 4/15 ) for endometrioid carcinoma , 58.3% ( 7/12 ) for mucinous carcinoma , and 0% ( 0/6 ) for undifferentiated carcinoma . the itfe was significantly higher in ccc than in non - ccc ( p < 0.001 ) and was also stronger in ccc ( table 2 ) . as itfe increased from negative to mild , moderate , and strong , the incidence of vte increased significantly ( ptrend = 0.014 ) to 16% , 17% , 38% , and 38% , respectively ( table 3 ) . venous thromboembolism developed before starting treatment in 31 patients ( 24.2% ) ( dvt alone , n = 20 ; dvt with pe , n = 8 ; pe alone , n = 3 ) , and the rate of subclinical disease was 96.8% . screening was performed according to a dd level with a negative predictive value of 96.1%.12 nonetheless , patients with silent pe alone were likely overlooked among the patients who did not undergo ct because no dvt was identified we consider that the rate is extremely low . mean ( sd ) dd level was 7.4 ( 6.4 ) g / ml in patients with vte , which is significantly higher than in patients without vte ( 3.2 [ 5.0 ] g / ml ; p < 0.001 ) . venous thromboembolism developed in 34.0% ( 18/53 ) of the patients with ccc , which is significantly higher than the incidence of 17.3% ( 13/75 ) among patients with non - ccc ( p = 0.03 ) . tissue factor positive patients developed vte at an incidence of 31.0% ( 22/71 ) , which is significantly higher than the incidence of 15.8% ( 9/57 ) in tf - negative patients ( p = 0.0496 ) . comparison of the negative / weakly positive patient group with the moderately / strongly positive patient group showed a significant difference in vte incidences , at 16.3% ( 13/80 ) and 37.5% ( 18/48 ; p = 0.01 ) , respectively . the itfe was therefore entered into the multivariate analysis using those 2 groups ( negative / weakly positive vs moderately / strongly positive ) . a strong correlation was found between ccc and itfe , and because multicollinearity was seen ( spearman correlation coefficient , 0.66 ) , simultaneous incorporation of ccc and itfe in the multivariate analysis model was considered statistically inappropriate . accordingly , when itfe was selected as the factor with the lower p value and multivariate analysis was performed , the results showed that pretreatment dd level and itfe represented significant independent risk factors for the development of vte before the start of treatment ( table 4 ) . we also examined the impact of histology and tf on vte development before the start of treatment by early- and advanced - stage diseases . although no significant difference was seen between ccc and non - ccc in either early- or advanced - stage diseases , a tendency was seen for vte development to increase in ccc . when tf was moderately / strongly positive , no significant difference was seen in early - stage diseases , but a tendency toward an increased risk of vte development was seen . in advanced - stage diseases , the risk of vte development was significantly increased ( p = 0.021 ; table 5 ) . logistic regression analysis for vte effect of tf and histological type on the risk of vte in early- and advanced - stage diseases this study was able to confirm that tf is involved in the pretreatment development of vte in eoc , particularly in ccc . uno et al8 studied 32 patients with ovarian cancer and revealed a relationship between tf expression in tumor tissues and the development of vte before the start of treatment . however , that result was only shown in univariate analysis because of the small size of the study cohort . this study focused on eoc as the histological type and enrolled 4 times as many patients , enabling multivariate analysis but generating the same results and confirming the earlier findings . we were also able to verify that the incidence of vte development before the start of treatment increased significantly as itfe increased . tissue factor binds to blood coagulation factor vii , which is released on damage to the vascular endothelial cells and others and promotes extrinsic coagulation.13 we surmised that the mechanism involves tf present in tumors being released into the blood vessels and leading to an increased incidence of vte development . uno et al8 reported pretreatment vte development in 45.5% ( 5/11 ) of patients with ccc , which is significantly higher than the frequency in patients with non - ccc . some subsequent studies found development rates of 15% to 42%,1416 which are higher than those in patients with non - ccc . likewise , this study found pretreatment vte development in 33.9% ( 18/53 ) of patients with ccc and confirmed that the rate was significantly elevated . the possibility was suggested that the histological type of ovarian cancer represents a confounding factor that skews tf expression and vte development . for that reason , this study applied multivariate analysis using 2 factors : itfe with a lower p value and pretreatment dd level . that analysis confirmed both factors as significant risk factors ( tf , p = 0.007 ; dd level , p < 0.0001 ) . moreover , when multivariate analysis was performed for ccc and dd level , both represented significant risk factors ( ccc , p = 0.014 ; dd level , p < 0.0001 ) . we saw no significant difference in histology in our examination of the respective impacts of histology and tf on vte development before the start of treatment of early- and advanced - stage diseases . in advanced - stage diseases , tf expression significantly increased the risk of vte development ( p = 0.021 ) , suggesting that the impact of tf expression on vte development is more evident in advanced - stage diseases . tissue factor is involved in the early stages of thrombus formation via the extrinsic coagulation process and can be regarded as the cause of vte . d - dimer is a degradation product remaining after fibrin formation14 and can be regarded as merely a result of thrombus formation . accordingly , the production of tf by eoc can be considered an essential risk factor for the pretreatment development of vte . we were able to confirm that tf is particularly markedly expressed in ccc compared with other histological types of eoc . in the report by uno et al,8 the 15 nac - treated patients accounted for nearly half of the total 32 patients , and the possibility that the effects of anticancer agents in the analyzed tissue specimens had biased the results of tf immunostaining was thus unable to be excluded . no pretreatment biopsies were taken from patients with eoc undergoing nac at our hospital if cancer of the ovaries , fallopian tubes , or peritoneum was suspected on image diagnosis ; if cells consistent with a malignant surface epithelial - stromal tumor were seen in an aspiration cytology of the tumor , pleural effusion , or ascites ; or if biopsy and advanced - stage diagnosis by microscopy during nac could be skipped because the patient met the criteria of ca125 of greater than 200 u / ml and carcinoembryonic antigen of less than 20 ng / ml.9,17,18 this study ruled out such potential bias by excluding patients who had received nac and studying only specimens obtained before the administration of anticancer agents . we thus consider that the present report has greater credibility with regard to the evaluation of tf expression in patients with eoc . in summary , this study confirmed that ( 1 ) tf expression in tumors represents an independent , significant risk factor for the pretreatment development of vte , with a stronger influence in advanced stages ; ( 2 ) increases in itfe are followed by an increased incidence of vte development ; ( 3 ) both the incidence and itfe are higher in ccc than in non - ccc ; and ( 4 ) ccc shows a higher incidence of pretreatment vte development compared with non - ccc . these findings strongly suggest that the high ability of ccc to produce tf is involved in the pretreatment development of vte in ccc .

objectivesour 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor ( tf ) in the development of venous thromboembolism ( vte ) before treatment , especially in clear cell carcinoma ( ccc ) . this follow - up study further investigated this possibility in a larger cohort.methodswe investigated the intensity of tf expression ( itfe ) and other variables for associations with vte using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between november 2004 and december 2010 , none of whom had received neoadjuvant chemotherapy . before starting treatment , all patients were ultrasonographically screened for vte . the itfe was graded based on immunostaining of surgical specimens.resultshistological types were serous carcinoma ( n = 42 ) , ccc ( n = 12 ) , endometrioid carcinoma ( n = 15 ) , mucinous carcinoma ( n = 53 ) , and undifferentiated carcinoma ( n = 6 ) . the prevalence of vte was significantly higher in ccc ( 34% ) than in non - ccc ( 17% , p = 0.03 ) . as itfe increased , the frequencies of ccc and vte increased significantly ( p < 0.001 and p = 0.014 , respectively ) . multivariate analysis identified tf expression and pretreatment dimerized plasmin fragment d level as significant independent risk factors for vte development . these factors showed particularly strong impacts on advanced - stage disease ( p = 0.021).conclusionsthe 2007 cohort was small , preventing multivariate analysis . this study of a larger cohort yielded stronger evidence that the development of vte in epithelial ovarian cancer may involve tf expression in cancer tissues .

MATERIALS AND METHODS Study Population Immunohistochemistry Detection of Deep Vein Thrombosis Detection of Pulmonary Thromboembolism Statistical Analysis RESULTS Subject Characteristics TF Expression in Cancer Tissues Risk Factors for the Development of VTE Before Starting Treatment DISCUSSION

participants were composed of 128 patients who underwent initial therapy at the study hospital between november 2004 and december 2010 and had been pathologically given a diagnosis of eoc . informed consent was obtained from all patients for the use of surgical specimens for research purposes . although 181 patients were treated during the study period , 53 patients who underwent nac were excluded considering the impact of anticancer drug exposure on immunostaining in tumor cells . the clinical stages of these 53 patients were stage ii in 2 patients , stage iii in 33 patients , and stage iv in 18 patients . the histological types were serous carcinoma in 45 patients , ccc in 5 patients , mucinous carcinoma in 2 patients , and undifferentiated carcinoma in 1 patient . twelve patients in whom nac was not expected to lead to the total elimination of cancer in the initial surgery , 23 patients in whom surgery needed to be cut short because of complications including vte or a poor general condition , and 18 patients in the nac group who took part in a phase iii trial of upfront debulking surgery versus nac for stage iii / iv ovarian , tubal , and peritoneal cancers9 were ultimately chosen for treatment , bringing the total number of patients included in this study to 128 . the intensity of tf expression ( itfe ) was graded into the following 4 levels based on the proportion of all cell populations that stained positively for tf : negative , no apparent positive tumor cells ; weakly positive , less than 50% positive tumor cells ; moderately positive , greater than or equal to 50% positive tumor cells with weak intensity ; and strongly positive , greater than or equal to 50% positive tumor cells with strong intensity ( fig . examination for vte was performed using the same method we have reported elsewhere.4,8 leg vein ultrasonography was performed on all patients using an atl hdi5000 system ( philips medical systems , bothell , wa ) with a 3- to 7.5-mhz transducer , to detect deep vein thrombosis ( dvt ) . however , the absence of a response to the valsalva maneuver would raise the suspicion of a blood flow disorder in the proximal vein , so thrombi in the pelvic veins were diagnosed based on the results of contrast - enhanced computed tomography ( ct ) . all patients underwent extensive imaging using ct and magnetic resonance imaging to detect the spread of pelvic tumors and blood clots in the iliac veins and inferior vena cava . the development of vte was investigated using univariate and multivariate analyses with a logistic model . age , body mass index ( bmi ) , stage , pretreatment dimerized plasmin fragment d ( d - dimer or dd ) level , histological type , and itfe were used as variables in the univariate analysis to identify potential risk factors for vte development . variables showing strong confounding suggestive of multicollinearity as well as a low p value were selected and included in the multivariate analysis model . all analyses were performed using sas version 9.4 software ( sas institute , cary , nc ) , and values of p < 0.05 were considered statistically significant . participants were composed of 128 patients who underwent initial therapy at the study hospital between november 2004 and december 2010 and had been pathologically given a diagnosis of eoc . informed consent was obtained from all patients for the use of surgical specimens for research purposes . although 181 patients were treated during the study period , 53 patients who underwent nac were excluded considering the impact of anticancer drug exposure on immunostaining in tumor cells . the clinical stages of these 53 patients were stage ii in 2 patients , stage iii in 33 patients , and stage iv in 18 patients . the histological types were serous carcinoma in 45 patients , ccc in 5 patients , mucinous carcinoma in 2 patients , and undifferentiated carcinoma in 1 patient . twelve patients in whom nac was not expected to lead to the total elimination of cancer in the initial surgery , 23 patients in whom surgery needed to be cut short because of complications including vte or a poor general condition , and 18 patients in the nac group who took part in a phase iii trial of upfront debulking surgery versus nac for stage iii / iv ovarian , tubal , and peritoneal cancers9 were ultimately chosen for treatment , bringing the total number of patients included in this study to 128 . the intensity of tf expression ( itfe ) was graded into the following 4 levels based on the proportion of all cell populations that stained positively for tf : negative , no apparent positive tumor cells ; weakly positive , less than 50% positive tumor cells ; moderately positive , greater than or equal to 50% positive tumor cells with weak intensity ; and strongly positive , greater than or equal to 50% positive tumor cells with strong intensity ( fig . examination for vte was performed using the same method we have reported elsewhere.4,8 leg vein ultrasonography was performed on all patients using an atl hdi5000 system ( philips medical systems , bothell , wa ) with a 3- to 7.5-mhz transducer , to detect deep vein thrombosis ( dvt ) . however , the absence of a response to the valsalva maneuver would raise the suspicion of a blood flow disorder in the proximal vein , so thrombi in the pelvic veins were diagnosed based on the results of contrast - enhanced computed tomography ( ct ) . all patients underwent extensive imaging using ct and magnetic resonance imaging to detect the spread of pelvic tumors and blood clots in the iliac veins and inferior vena cava . the development of vte was investigated using univariate and multivariate analyses with a logistic model . age , body mass index ( bmi ) , stage , pretreatment dimerized plasmin fragment d ( d - dimer or dd ) level , histological type , and itfe were used as variables in the univariate analysis to identify potential risk factors for vte development . variables showing strong confounding suggestive of multicollinearity as well as a low p value were selected and included in the multivariate analysis model . all analyses were performed using sas version 9.4 software ( sas institute , cary , nc ) , and values of p < 0.05 were considered statistically significant . at the time of initial treatment , mean age was 56.6 years ( range , 3188 years ) , and mean bmi was 22.4 kg / m ( range , 13.335.4 kg / m ) . for the 128 patients , stage was i in 55 patients , ii in 22 patients , iii in 37 patients , and iv in 14 patients , whereas the histological type was serous in 42 patients , mucinous in 12 patients , endometrioid in 15 patients , ccc in 53 patients , and undifferentiated in 6 patients ( table 1 ) . patient characteristics ( n = 128 ) tissue factor was expressed in 71 patients ( weakly positive , n = 23 ; moderately positive , n = 32 ; strongly positive , n = 16 ) , representing 55.5% of the 128 patients . according to stage , itfe was 81.8% for stage i and 45.4% for stage ii but was significantly lower for stages iii and iv , at 29.7% and 35.7% , respectively ( p < 0.001 ) . incidences according to histological type were 94.3% ( 50/53 ) for ccc , 23.8% ( 10/42 ) for serous carcinoma , 26.7% ( 4/15 ) for endometrioid carcinoma , 58.3% ( 7/12 ) for mucinous carcinoma , and 0% ( 0/6 ) for undifferentiated carcinoma . the itfe was significantly higher in ccc than in non - ccc ( p < 0.001 ) and was also stronger in ccc ( table 2 ) . as itfe increased from negative to mild , moderate , and strong , the incidence of vte increased significantly ( ptrend = 0.014 ) to 16% , 17% , 38% , and 38% , respectively ( table 3 ) . patient characteristics according to itfe in cancer tissues incidence of vte for each itfe venous thromboembolism developed before starting treatment in 31 patients ( 24.2% ) ( dvt alone , n = 20 ; dvt with pe , n = 8 ; pe alone , n = 3 ) , and the rate of subclinical disease was 96.8% . mean ( sd ) dd level was 7.4 ( 6.4 ) g / ml in patients with vte , which is significantly higher than in patients without vte ( 3.2 [ 5.0 ] g / ml ; p < 0.001 ) . venous thromboembolism developed in 34.0% ( 18/53 ) of the patients with ccc , which is significantly higher than the incidence of 17.3% ( 13/75 ) among patients with non - ccc ( p = 0.03 ) . tissue factor positive patients developed vte at an incidence of 31.0% ( 22/71 ) , which is significantly higher than the incidence of 15.8% ( 9/57 ) in tf - negative patients ( p = 0.0496 ) . comparison of the negative / weakly positive patient group with the moderately / strongly positive patient group showed a significant difference in vte incidences , at 16.3% ( 13/80 ) and 37.5% ( 18/48 ; p = 0.01 ) , respectively . the itfe was therefore entered into the multivariate analysis using those 2 groups ( negative / weakly positive vs moderately / strongly positive ) . a strong correlation was found between ccc and itfe , and because multicollinearity was seen ( spearman correlation coefficient , 0.66 ) , simultaneous incorporation of ccc and itfe in the multivariate analysis model was considered statistically inappropriate . accordingly , when itfe was selected as the factor with the lower p value and multivariate analysis was performed , the results showed that pretreatment dd level and itfe represented significant independent risk factors for the development of vte before the start of treatment ( table 4 ) . we also examined the impact of histology and tf on vte development before the start of treatment by early- and advanced - stage diseases . although no significant difference was seen between ccc and non - ccc in either early- or advanced - stage diseases , a tendency was seen for vte development to increase in ccc . when tf was moderately / strongly positive , no significant difference was seen in early - stage diseases , but a tendency toward an increased risk of vte development was seen . in advanced - stage diseases , the risk of vte development was significantly increased ( p = 0.021 ; table 5 ) . logistic regression analysis for vte effect of tf and histological type on the risk of vte in early- and advanced - stage diseases at the time of initial treatment , mean age was 56.6 years ( range , 3188 years ) , and mean bmi was 22.4 kg / m ( range , 13.335.4 kg / m ) . for the 128 patients , stage was i in 55 patients , ii in 22 patients , iii in 37 patients , and iv in 14 patients , whereas the histological type was serous in 42 patients , mucinous in 12 patients , endometrioid in 15 patients , ccc in 53 patients , and undifferentiated in 6 patients ( table 1 ) . tissue factor was expressed in 71 patients ( weakly positive , n = 23 ; moderately positive , n = 32 ; strongly positive , n = 16 ) , representing 55.5% of the 128 patients . according to stage , itfe was 81.8% for stage i and 45.4% for stage ii but was significantly lower for stages iii and iv , at 29.7% and 35.7% , respectively ( p < 0.001 ) . incidences according to histological type were 94.3% ( 50/53 ) for ccc , 23.8% ( 10/42 ) for serous carcinoma , 26.7% ( 4/15 ) for endometrioid carcinoma , 58.3% ( 7/12 ) for mucinous carcinoma , and 0% ( 0/6 ) for undifferentiated carcinoma . the itfe was significantly higher in ccc than in non - ccc ( p < 0.001 ) and was also stronger in ccc ( table 2 ) . as itfe increased from negative to mild , moderate , and strong , the incidence of vte increased significantly ( ptrend = 0.014 ) to 16% , 17% , 38% , and 38% , respectively ( table 3 ) . venous thromboembolism developed before starting treatment in 31 patients ( 24.2% ) ( dvt alone , n = 20 ; dvt with pe , n = 8 ; pe alone , n = 3 ) , and the rate of subclinical disease was 96.8% . mean ( sd ) dd level was 7.4 ( 6.4 ) g / ml in patients with vte , which is significantly higher than in patients without vte ( 3.2 [ 5.0 ] g / ml ; p < 0.001 ) . venous thromboembolism developed in 34.0% ( 18/53 ) of the patients with ccc , which is significantly higher than the incidence of 17.3% ( 13/75 ) among patients with non - ccc ( p = 0.03 ) . tissue factor positive patients developed vte at an incidence of 31.0% ( 22/71 ) , which is significantly higher than the incidence of 15.8% ( 9/57 ) in tf - negative patients ( p = 0.0496 ) . comparison of the negative / weakly positive patient group with the moderately / strongly positive patient group showed a significant difference in vte incidences , at 16.3% ( 13/80 ) and 37.5% ( 18/48 ; p = 0.01 ) , respectively . the itfe was therefore entered into the multivariate analysis using those 2 groups ( negative / weakly positive vs moderately / strongly positive ) . a strong correlation was found between ccc and itfe , and because multicollinearity was seen ( spearman correlation coefficient , 0.66 ) , simultaneous incorporation of ccc and itfe in the multivariate analysis model was considered statistically inappropriate . accordingly , when itfe was selected as the factor with the lower p value and multivariate analysis was performed , the results showed that pretreatment dd level and itfe represented significant independent risk factors for the development of vte before the start of treatment ( table 4 ) . we also examined the impact of histology and tf on vte development before the start of treatment by early- and advanced - stage diseases . although no significant difference was seen between ccc and non - ccc in either early- or advanced - stage diseases , a tendency was seen for vte development to increase in ccc . when tf was moderately / strongly positive , no significant difference was seen in early - stage diseases , but a tendency toward an increased risk of vte development was seen . in advanced - stage diseases , the risk of vte development was significantly increased ( p = 0.021 ; table 5 ) . logistic regression analysis for vte effect of tf and histological type on the risk of vte in early- and advanced - stage diseases this study was able to confirm that tf is involved in the pretreatment development of vte in eoc , particularly in ccc . uno et al8 studied 32 patients with ovarian cancer and revealed a relationship between tf expression in tumor tissues and the development of vte before the start of treatment . we were also able to verify that the incidence of vte development before the start of treatment increased significantly as itfe increased . tissue factor binds to blood coagulation factor vii , which is released on damage to the vascular endothelial cells and others and promotes extrinsic coagulation.13 we surmised that the mechanism involves tf present in tumors being released into the blood vessels and leading to an increased incidence of vte development . uno et al8 reported pretreatment vte development in 45.5% ( 5/11 ) of patients with ccc , which is significantly higher than the frequency in patients with non - ccc . some subsequent studies found development rates of 15% to 42%,1416 which are higher than those in patients with non - ccc . likewise , this study found pretreatment vte development in 33.9% ( 18/53 ) of patients with ccc and confirmed that the rate was significantly elevated . the possibility was suggested that the histological type of ovarian cancer represents a confounding factor that skews tf expression and vte development . for that reason , this study applied multivariate analysis using 2 factors : itfe with a lower p value and pretreatment dd level . that analysis confirmed both factors as significant risk factors ( tf , p = 0.007 ; dd level , p < 0.0001 ) . moreover , when multivariate analysis was performed for ccc and dd level , both represented significant risk factors ( ccc , p = 0.014 ; dd level , p < 0.0001 ) . we saw no significant difference in histology in our examination of the respective impacts of histology and tf on vte development before the start of treatment of early- and advanced - stage diseases . in advanced - stage diseases , tf expression significantly increased the risk of vte development ( p = 0.021 ) , suggesting that the impact of tf expression on vte development is more evident in advanced - stage diseases . tissue factor is involved in the early stages of thrombus formation via the extrinsic coagulation process and can be regarded as the cause of vte . accordingly , the production of tf by eoc can be considered an essential risk factor for the pretreatment development of vte . in the report by uno et al,8 the 15 nac - treated patients accounted for nearly half of the total 32 patients , and the possibility that the effects of anticancer agents in the analyzed tissue specimens had biased the results of tf immunostaining was thus unable to be excluded . no pretreatment biopsies were taken from patients with eoc undergoing nac at our hospital if cancer of the ovaries , fallopian tubes , or peritoneum was suspected on image diagnosis ; if cells consistent with a malignant surface epithelial - stromal tumor were seen in an aspiration cytology of the tumor , pleural effusion , or ascites ; or if biopsy and advanced - stage diagnosis by microscopy during nac could be skipped because the patient met the criteria of ca125 of greater than 200 u / ml and carcinoembryonic antigen of less than 20 ng / ml.9,17,18 this study ruled out such potential bias by excluding patients who had received nac and studying only specimens obtained before the administration of anticancer agents . we thus consider that the present report has greater credibility with regard to the evaluation of tf expression in patients with eoc . in summary , this study confirmed that ( 1 ) tf expression in tumors represents an independent , significant risk factor for the pretreatment development of vte , with a stronger influence in advanced stages ; ( 2 ) increases in itfe are followed by an increased incidence of vte development ; ( 3 ) both the incidence and itfe are higher in ccc than in non - ccc ; and ( 4 ) ccc shows a higher incidence of pretreatment vte development compared with non - ccc . these findings strongly suggest that the high ability of ccc to produce tf is involved in the pretreatment development of vte in ccc .

nigeria is a major producer and exporter of crude petroleum oil and also an important agricultural nation in the west african sub - region ( agbogidi et al . as crude oil comes from the well , it contains mixture of hydrocarbon compounds and relatively small quantities of other materials such as oxygen , nitrogen , sulphur , salt , water and some trace metals . in the refinery , most of these non - hydrocarbon substances are removed and the oil is broken down into useful products ( nwaichi et al . the soil is very important to human existence for various reasons , especially for agriculture and has been subjected to various abuses including spillage of petroleum ( crude oil ) and petroleum - by products , dumping of waste and other contaminating activities ( osam 2011 ) . soil contamination has been a growing concern since it can be a source of groundwater ( drinking water ) contamination and can also reduce the usability of land for development . elevated levels of some heavy metals in different parts of the globe have increased the interest for environmentalists and ecotoxicologists in toxicity and environmental degradation . humans and ecosystem may be exposed to chemical hazards such as heavy metals through direct ingestion of contaminated soil , consumption of crops and vegetables grown on the contaminated lands or drinking water that has percolated through such soils ( mclaughlin et al . these pollutants may cause long- or short - term damage by changing the growth rate of plant or animal species , or by interfering with human amenities , comfort , health or property values ( tietenberg 2006 ) . the definition of crude oil and gas pollution in this study embraces oil spillages on crop farms , areas of crop farms occupied by flow stations , oil wells , gas flaring sites , pipeline laying sites , borrow pits and other oil exploration , exploitation and related activities . within the european community , 11 elements of highest concern are arsenic , cadmium , cobalt , chromium , copper , mercury , manganese , nickel , lead , tin and thallium ( mepprm 2014 ) ; the emissions of which are regulated in waste incinerators . some of these elements are actually necessary for humans in minute amounts ( cobalt , copper , chromium , manganese , nickel ) while others are carcinogenic or toxic , affecting , among others , the central nervous system ( manganese , mercury , lead , arsenic ) , the kidneys or liver ( mercury , lead , cadmium , copper ) or skin , bones or teeth ( nickel , cadmium , copper , chromium ) ( zevenhoven and kilpinen 2001 ) . cadmium , lead and zinc are also released in tiny particulates as dust from rubber tyres on busy road surfaces ; the small size allows these toxic metals to rise in the wind to be inhaled , or transported onto topsoil or edible plants through precipitation of their compounds or by ion exchange into soils and muds . heavy metal pollutants can localize and lay dormant , and this can have multiple effects on the environment . polycyclic aromatic hydrocarbons ( pahs ) are produced from incomplete combustion of organic materials , fossil fuels , petroleum product spillage and various domestic and industrial activities ( johnsen et al . 2005 ) . based on their ecotoxicity , the united states environmental protection agency has prioritized 16 pahs as environmental pollutants ( nwaichi et al . 2010 ) . total petroleum hydrocarbons ( tph ) are the measurable amount of petroleum - based hydrocarbons in an environmental media ( rauckyte et al . ogoniland is situated in an area 1,000 km east of port harcourt in rivers state , nigeria . the area has a tragic history of pollution from oil spills ; oil well fires , environmental incidents , such as spills and uncontrolled flares ( unep , united nations environment programme 2011 ) . it lies on the coastal lowland in the south eastern part of rivers state and is characterised by high rainfall ( 2,0002,500 mm / yr ) , high temperature and high humidity . the control area , umuchichi village in osisioma lga , is a coastal plain located on the southern part of abia state , nigeria and lies 440 and 614 n and 710 and 8 e. it is a non - polluted area with less industry presence . this study therefore seeks to evaluate the distribution of some heavy metals , tph and pahs in selected regularly - consumed food crops and soils from an oil - polluted active agricultural farmland and making comparisons with a view to health implications . soil samples ( in random replicates of three ) were taken from oil - polluted active agricultural farmlands in gokana ( test ) and non - oil - polluted active agricultural farmlands in umuchichi ( control ) . leafy vegetables ( bitterleaf or vernonia amygdelina and waterleaf or talinum triangulare ) and tuber crops ( cassava or manihot esculenta and cocoyam or xanthotosoma sagittifolium ) were freshly harvested from these locations and were collected following standard environmental sampling protocols ( us . soil samples were air dried , crushed and sieved ( 2 mm screen ) . for heavy metals , 5 g of each sample was weighed into a clean , dry silica dish , covered and ignited in a furnace for 6 h at 500 c until a grey white ash was obtained . the cover of the dish was opened to allow for escape of gases . to cool ash samples , 5 ml of 10 % hcl was added to enhance dissolution and 5 ml of 10 % hno3 was added thereafter and set on a water bath to dissolve completely . the solution was transferred into a clean dry 50 ml standard volumetric flask and marked up with distilled water ( khan et al . the concentrations of fe , mn , zn , cu , cd and pb in the filtrate were determined by atomic absorption spectrometry ( contraa 300 , analytik jena , germany ) . the blank reagent and standard reference soil materials were included in each sample batch to verify the accuracy and precision of the digestion procedure and also for subsequent analyses . for tphs and pahs , 1 g sample was weighed into a clean extraction container and 10 ml dichloromethane ( extraction solvent ) was added . the mixture was carefully filtered into extraction bottle using clean filter paper fitted in a buchner funnel , and the extract was concentrated to 2 ml and then transferred for separation in a hp gas chromatograph 5890 series ii . about 810 ml of the eluent / extract was collected and labelled aromatics ( api , american petroleum institute 1994 ) . using a hypodermic syringe , 1 l of the concentrated aromatic fraction separation occurred as the vapour constituent s partition between the gas and liquid phases and detection was possible with fid . on the other hand , samples of various vegetables and tubers were washed with distilled water to remove loose particles . vegetable samples and chopped tubers were sun dried for 4 days and ground in a high speed plastic blender ( son binatone ) for several minutes until they became homogenous . data were validated by reviewing for completeness , holding times , calibrations ( initial and continuing ) , specific blank analysis , gc tuning and system performance , surrogate recoveries , field replication precision , compound quantitation and detection limits . obtained data were subjected to one - way analysis of variance ( anova ) using statistica vs 10 , and test of significance was done at 95 % confidence level . soil samples ( in random replicates of three ) were taken from oil - polluted active agricultural farmlands in gokana ( test ) and non - oil - polluted active agricultural farmlands in umuchichi ( control ) . leafy vegetables ( bitterleaf or vernonia amygdelina and waterleaf or talinum triangulare ) and tuber crops ( cassava or manihot esculenta and cocoyam or xanthotosoma sagittifolium ) were freshly harvested from these locations and were collected following standard environmental sampling protocols ( us . soil samples were air dried , crushed and sieved ( 2 mm screen ) . for heavy metals , 5 g of each sample was weighed into a clean , dry silica dish , covered and ignited in a furnace for 6 h at 500 c until a grey white ash was obtained . the cover of the dish was opened to allow for escape of gases . to cool ash samples , 5 ml of 10 % hcl was added to enhance dissolution and 5 ml of 10 % hno3 was added thereafter and set on a water bath to dissolve completely . the solution was transferred into a clean dry 50 ml standard volumetric flask and marked up with distilled water ( khan et al . the concentrations of fe , mn , zn , cu , cd and pb in the filtrate were determined by atomic absorption spectrometry ( contraa 300 , analytik jena , germany ) . the blank reagent and standard reference soil materials were included in each sample batch to verify the accuracy and precision of the digestion procedure and also for subsequent analyses . for tphs and pahs , 1 g sample was weighed into a clean extraction container and 10 ml dichloromethane ( extraction solvent ) was added . the mixture was carefully filtered into extraction bottle using clean filter paper fitted in a buchner funnel , and the extract was concentrated to 2 ml and then transferred for separation in a hp gas chromatograph 5890 series ii . about 810 ml of the eluent / extract was collected and labelled aromatics ( api , american petroleum institute 1994 ) . using a hypodermic syringe , 1 l of the concentrated aromatic fraction was injected through a rubber septum into the column . separation occurred as the vapour constituent s partition between the gas and liquid phases and detection was possible with fid . on the other hand , samples of various vegetables and tubers were washed with distilled water to remove loose particles . vegetable samples and chopped tubers were sun dried for 4 days and ground in a high speed plastic blender ( son binatone ) for several minutes until they became homogenous . data were validated by reviewing for completeness , holding times , calibrations ( initial and continuing ) , specific blank analysis , gc tuning and system performance , surrogate recoveries , field replication precision , compound quantitation and detection limits . obtained data were subjected to one - way analysis of variance ( anova ) using statistica vs 10 , and test of significance was done at 95 % confidence level . data were analysed to determine if any statistically marked differences existed between the datasets for the soils and plant tissue types . while impacted vegetation presented with brownish to yellowish coloration and stunted growth , those located 50 m away from the impacted site appeared greenish and healthy . from tables 1 and 2 , average individual concentrations of tphs ranged from below detection limit of 0.00010.58 0.001 mgkg dw for control samples , with highest concentration recorded for c-21 in talinum triangulare which differed marginally from 0.11 to 0.00 for c-31 in vernonia amygdelina . cumulative tphs distribution , however , gave the pattern v. amygdelina > t. triangulare > x. sagittifolium > m. esculenta > soil ( table 1 ) , as all soil values fell below the detection limit of the analytical instrument . according to deq ( 2000 ) , gasoline and condensate range organics ( gro ) generally include c4 through c9 hydrocarbons , diesel range organics ( dro ) generally include c10 through c24 , while crude oil , in general , includes c5 through c34 hydrocarbons . it is therefore presumed that meagre concentrations observed in control tissue samples could be attributed to neighbouring gas flaring activities . for the test samples , however , observed data ranged from below detection limit to 2,280 2.89 mgkg dw and this highest fractional mean tph concentration was recorded in soil for c-14 components . as indicated in table 1 , gc analysis revealed the presence of pristane ( c19h40 ) and phytane ( c20h42 ) in all the polluted samples ( table 2 ) . the disappearance of the lighter molecular weight hydrocarbon numbers below c12 in most of the test samples may indicate that the oil was slightly weathered after spill incident and hence , greater susceptibility to secondary processes of evaporative weathering and biodegradation . the plant samples similarly accumulated tphs in the order : v. amygdelina > t. triangulare > x. sagittifolium > m. esculenta , but were markedly ( p 0.05 ) low compared to soil total concentrations . it is noteworthy to mention that recorded cumulative tphs levels ( 3,830 19.6 mgkg dw ) in test soil far exceeded the maximum permissible limit of 50 mgkg dw by department of petroleum resources ( dpr ) . total mean soil levels in control samples were significantly lower ( with values : < 0.0001 , 0.58 0.04 , 0.49 0.02 , 0.14 0.01 and 0.05 0.004 mgkg dw ) than those of test ( with values : 3824.12 19.6 , 11.34 0.07 , 9.67 0.08 , 3.99 0.09 and 2.17 0.06 mgkg dw ) for all samples . up to 35.2 mgkg dw phythane was observed for test soil in contrast to non - detectable levels in control . secondary evaluation ( table 3 ) for such soil , with multiple contaminants gave value for the sum of the individual ri values ( ri ) > 1 . this implies further evaluation , potentially through site - specific risk assessment.table 1a mean tph ( mgkg ) levels in control area ( umuchichi)componentssoilbitterleafwaterleafcocoyamcassavac-8<0.001<0.001<0.001<0.001<0.001c-9<0.001<0.001<0.001<0.001<0.001c-10<0.001<0.001<0.001<0.001<0.001c-11<0.001<0.001<0.001<0.001<0.001c-12<0.001<0.001<0.001<0.001<0.001c-13<0.001<0.001<0.001<0.001<0.001c-14<0.0010.03 0.00 < 0.001<0.001<0.001c-15<0.0010.01 0.00 < 0.001<0.001<0.001c-16<0.0010.01 0.00 < 0.001<0.001<0.001c-17<0.0010.01 0.00 < 0.001<0.001<0.001pristane<0.0010.05 0.00 < 0.001<0.001<0.001c-18<0.0010.01 0.00 < 0.001<0.001<0.001phythane<0.0010.02 0.00 < 0.001<0.001<0.001c-19<0.0010.02 0.00 < 0.001<0.001<0.001c-20<0.0010.02 0.00 0.01 0.00 0.01 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \pm 0.00 $ $ \end{document } < 0.001c-21<0.0010.02 0.00 0.12 0.00 < 0.001<0.001c-22<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-23<0.0010.01 0.00 < 0.001<0.001<0.001componentssoilbitterleafwaterleafcocoyamcassavac-24<0.0010.01 0.00 < 0.001<0.001<0.001c-25<0.0010.01 0.00 < 0.001<0.001<0.001c-26<0.0010.02 0.00 < 0.001<0.001<0.001c-27<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-28<0.0010.02 0.00 0.02 0.00 < 0.001<0.001c-29<0.0010.03 0.00 0.01 0.00 < 0.001<0.001c-30<0.0010.02 0.00 0.01 0.00 < 0.001<0.001c-31<0.0010.11 0.00a0.01 0.00 0.01 0.00 0.00 0.02 0.00 < 0.001c-33<0.0010.04 0.00 0.09 0.00 0.01 0.00 < 0.001c-34<0.0010.03 0.00 0.05 0.00 0.05 0.00 0.01 0.00 c-35<0.0010.03 0.00 0.03 0.00 0.01 0.00 < 0.001c-36<0.0010.02 0.00 0.03 0.00 0.01 0.00 0.01 0.00 c-37<0.0010.01 0.00 0.02 0.00 0.01 0.00 < 0.001c-38<0.0010.01 0.00 < 0.001<0.001<0.001c-39<0.001<0.0010.03 0.00 < 0.001<0.001c-40<0.0010.02 0.00 < 0.001<0.001<0.001total<0.0010.58 0.04 0.49 0.02 0.14 0.01 0.05 0.00 values with different superscript letters ( a , b , c , d , e ) in the same row are significantly different at 0.05 level ( p 0.05)table 2a mean level of tph ( mgkg ) in test ( gokana ) areacomponentssoilbitterleafwaterleafcocoyamcassavac-8<0.001<0.001<0.001<0.001<0.001c-9<0.001<0.0010.01 0.00 < 0.001<0.001c-10<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-11<0.0010.01 0.00 0.02 0.00 < 0.001<0.001c-12<0.0010.02 0.00 0.01 0.00 < 0.001<0.001c-13224 7.77 0.53 0.00 0.23 0.00 0.02 0.00 0.01 0.00 c-142,280 2.89 0.35 0.00 0.07 0.00 0.14 0.00 0.07 0.00 c-1547.1 26.5 4.00 0.06 2.73 0.06 0.31 0.00 0.43 0.00 c-1660.2 0.06 0.10 0.00 0.20 0.00 0.09 0.00 0.08 0.00 c-17289 1.53 < 0.0010.01 0.00 0.03 0.00 0.01 0.01 pristane<0.0010.22 0.01 0.21 0.00 0.04 0.00 0.05 0.02 c-18189 1.15 0.03 0.00 0.07 0.00 0.22 0.00 0.03 0.00 phythane35.2 1.34 0.20 0.00 < 0.0010.02 0.00 0.04 0.00 c-1920.8 0.12 1.23 0.01 0.31 0.00 0.03 0.00 0.03 0.00 c-20297 1.15 0.11 0.00 0.11 0.00 0.05 0.00 0.17 0.00 c-2126.9 0.23 0.17 0.00 0.18 0.00 0.04 0.00 0.03 0.00 c-2232.3 0.10 1.39 0.09 0.15 0.00 0.06 0.00 0.02 0.00 c-23219 2.31 0.01 0.00 1.90 0.02 2.12 0.01 0.02 0.00 total3720.58.386.223.170.99componentssoilbitterleafwaterleafcocoyamcassavac-249.55 0.01 1.36 0.04 0.24 0.00 0.12 0.00 0.16 0.00 c-251.25 0.01 0.09 0.00 0.21 0.00 0.18 0.02 0.30 0.00 c-2611.7 0.15 0.30 0.00 0.20 0.00 0.04 0.00 0.03 0.00 c-276.31 0.01 0.05 0.02 0.04 0.01 0.04 0.02 0.30 0.01 c-285.33 0.01 0.08 0.00 0.16 0.00 0.04 0.00 0.07 0.00 c-291.28 0.02 0.03 0.00 0.01 0.00 < 0.001<0.001c-3011.0 0.12 0.03 0.00 0.01 0.00 0.03 0.00 0.03 0.00 c-3118.8 0.35 < 0.0010.06 0.00 < 0.0010.03 0.00 c-322.54 0.01 < 0.0010.02 0.00 0.02 0.00 0.01 0.00 c-331.81 0.02 < 0.0010.02 0.00 0.06 0.00 0.05 0.00 c-348.91 0.03 0.01 0.00 0.67 0.00 0.01 0.00 0.05 0.00 c-356.91 0.01 0.66 0.00 0.51 0.00 0.02 0.00 0.06 0.00 c-362.03 0.01 < 0.001<0.001<0.0010.06 0.00 c-374.46 0.01 0.08 0.00 0.01 0.00 < 0.0010.02 0.00 c-384.49 0.01 0.04 0.00 0.05 0.00 0.01 0.00 0.03 0.00 c-391.66 0.01 0.12 0.00 0.02 0.00 < 0.0010.01 0.00 c-400.97 0.01 0.14 0.00 1.25 0.01 0.14 0.02 0.12 0.00 total3,830 19.6 11.3 0.07 9.67 0.08 3.99 0.09 2.17 0.06 table 3evaluation of tph ( mgkg ) contaminants in study soilsourcechemical contaminantsoil ctviciri(ci / ctvi)further evaluation needed?(is ri > 1)testcontroltestcontroltestcontrolgasolinetph groa2.80e+01383e+031.0e03136.763.57e5yesnodiesel or crude oiltph dro2.30e+034.69e+031.0e031.674.35e7yesnori138.433.61e5yesno a is based on fate and transport evaluation for protection of groundwater . ri c / ctvi where : ri is the risk posed by contaminant i , ci is the maximum concentration of contaminant i in soil , in mgkg and ctvi is the soil cleanup level provided on the cleanup level look - up table , in mgkg ( mdnr and michigan department of natural resources 1993 ; deq 2000 ) . for multiple contaminants in soil , if the sum of the individual ri values ( ri ) > 1 , further evaluation would be required , potentially through site - specific risk assessment a mean tph ( mgkg ) levels in control area ( umuchichi ) values with different superscript letters ( a , b , c , d , e ) in the same row are significantly different at 0.05 level ( p 0.05 ) a mean level of tph ( mgkg ) in test ( gokana ) area evaluation of tph ( mgkg ) contaminants in study soil a is based on fate and transport evaluation for protection of groundwater . ri c / ctvi where : ri is the risk posed by contaminant i , ci is the maximum concentration of contaminant i in soil , in mgkg and ctvi is the soil cleanup level provided on the cleanup level look - up table , in mgkg ( mdnr and michigan department of natural resources 1993 ; deq 2000 ) . for multiple contaminants in soil , if the sum of the individual ri values ( ri ) > 1 , further evaluation would be required , potentially through site - specific risk assessment mean individual component concentrations of pahs in all control samples ( table 4 ) ranged from below detection limit of < 0.0001 to 18.4 2.3 mgkg dw and from < 0.0001 to 4,690 2.71 mgkg dw for test samples . it is noteworthy that these upper limits were recorded for carcinogenic benzo ( b ) fluoranthene in both control and test and showed similar trend in plant tissues especially for bitterleaf and waterleaf ( fig . 1 ) and this is attributable to several oil spills and other exploration - related activities in the area . its persistence in the environment and poor degradability is probably due to its high molecular weight ( giridhar , p. & krishna , p 2010 ) . their presence in crude oil makes differentiation of sources possible ( osuji and onojake 2006 ) using useful ratios . all pahs were below detection limit ( < 0.0001 ) in control soil but gave significant values ( p < 0.05 ) in corresponding plant samples . potential danger is inevitable as elevated levels of strongly carcinogenic benzo ( a ) pyrene exceeded dpr intervention limit of 0.01 mgkg dw for food ( table 5 ) and intensely exceeded common regulatory target cleanup levels for pahs and b(a)p - te ( 0.10.66 mgkg ) as reported by bradley et al . he presented urban surface soil results ( upper 95 % confidence interval of 3.3 and 12.4 mgkg for total b(a)p - te and total pahs , respectively ) that were much below the background concentrations of these compounds in test ( gokana ) soils . the total mean concentrations of pahs of 6,950 gkg in soil from study area ( gokana ) exceeded the department of petroleum resources permissible limit of 1 mgkg ( 1,000 gkg ) ( dpr 1991 ) and were significant in comparison to control . however , the levels in crop samples did not exceed the eu limit of 0.2 mgkg ( 200 gkg ) ( bfr 2010 ) for consumer products . no wonder , the natives complain of rashes , blisters , coughing , throat irritation , red , watery and itchy eyes and these signs and more , have been reported by aguilera et al . owing to their low solubility and a high tendency to interact with non - aqueous phases , high molecular weight pahs become potentially unavailable for degradation by microbes , which tend to favourably degrade compounds dissolved in water ( miller et al . 0.01 0.01 0.01 0.01 0.01 0.01 0.01 fluorene<0.001<0.001<0.001<0.001<0.001phenanthrene<0.0010.17 0.04 < 0.001<0.001<0.001anthracene<0.001<0.0010.29 0.05 < 0.001<0.001floranthene<0.001<0.001<0.001<0.001<0.001pyrene<0.001<0.001<0.0010.03 0.02 < 0.001benz(a)anthracene<0.001<0.001<0.001<0.001<0.001chrysene<0.001<0.001<0.001<0.001<0.001benzo(b)fluoranthene<0.00118.4 2.31 15.1 1.77 8.69 2.42 2.03 0.03 benzo(k)fluoranthene<0.001<0.0010.01 0.01 0.01 0.01 0.01 0.01 benzo(a)pyrene<0.001<0.0010.03 0.02 < 0.001<0.001indeno(1,2,3-cd)pyrene<0.0010.10 0.00 0.28 0.03 0.08 0.00 0.05 0.00 dibenz(a , h)anthracene<0.001<0.0010.01 0.01 < 0.001<0.001benzo(g , h , i)perylene<0.001<0.001<0.001<0.001<0.001total<0.00118.6 0.02 15.8 0.10 8.83 0.02 2.11 0.02 fig . 1comparative significance of carcinogenic bezo(b)fluoranthene distribution ; s soil , b bitterleaf , w waterleaf , co cocoyam , ca cassava , c contaminated , c trl control . vertical bars denote 0.95 confidence intervalstable 5mean levels of pahs ( mgkg ) in test areacomponentssoilbitterleafwaterleafcocoyamcassavanaphthalene<0.0010.01 0.00 < 0.001<0.001<0.001acenaphthylene<0.0010.03 0.00 0.06 0.00 0.02 0.00 0.01 0.00 acenaphthene297 1.43 0.34 0.00 < 0.001<0.001<0.001fluorene290 1.51 0.06 0.00 0.01 0.00 < 0.001<0.001phenanthrene278 1.78 0.03 0.00 0.01 0.00 < 0.001<0.001anthracene323 2.89 1.48 0.04 0.65 0.00 < 0.001<0.001floranthene110 2.67 0.03 0.01 < 0.001<0.001<0.001pyrene57.1 3.22 0.09 0.03 < 0.001<0.001<0.001benz(a)anthracene102 2.25 0.01 0.01 < 0.001<0.001<0.001chrysene99.1 6.21 0.04 0.00 < 0.001<0.001<0.001benzo(b)fluoranthene4,690 2.71 183 1.56 112 1.03 37.8 0.77 18.0 0.91 benzo(k)fluoranthene47.0 4.91 0.17 0.00 0.04 0.00 < 0.0010.02 0.00 benzo(a)pyrene47.4 3.89 0.40 0.05 0.07 0.00 0.15 0.00 0.01 0.00 indeno(1,2,3-cd)pyrene694 23.6 0.13 0.03 0.75 0.05 0.39 0.06 0.61 0.05 dibenz(a , h)anthracene0.55 0.13 0.01 0.00 0.01 0.00 < 0.001<0.001benzo(g , h , i)perylene4.14 1.17 0.01 0.01 < 0.001<0.001<0.001total6,950 68.3 186 1.74 114 4.06 18.7 0.77 18.7 0.92 mean levels of pahs ( mgkg ) in control area comparative significance of carcinogenic bezo(b)fluoranthene distribution ; s soil , b bitterleaf , w waterleaf , co cocoyam , ca cassava , c contaminated , c trl control . vertical bars denote 0.95 confidence intervals mean levels of pahs ( mgkg ) in test area secondary evaluation of carcinogenic pahs ( table 6 ) for such soil with multiple contaminants showed that the sum of the individual ri values ( ri ) > 1 , implying further evaluation , potentially through site - specific risk assessment . heavy metal distribution in test and control samples indicated serious threats as observed cr uptake levels for test and control and pb in test only exceeded given eu ( cec 2006 ) and who limits for such soil ( tables 7 and 8) . as would be expected , mn and zn ( as micronutrients ) levels in control were lower than those in test soils while abundant fe levels were typical of soils in the niger delta . toxic effects begin to occur at doses above 1020 mgkg of elemental iron , and ingestions of more than 50 mgkg of elemental iron are associated with severe toxicity ( iron poisoning . the first indication of iron poisoning by ingestion is a pain in the stomach , as the stomach lining becomes ulcerated . tissue pb levels for bitterleaf and cocoyam samples exceeded eu set limit of 0.3 mgkg while cr level exceeded who set limit of 0.05 mgkg for bitterleaf but fell below acute oral toxicity range of between 1,900 and 3,300 gkg risk ( monica et al . discrepancies in mn , zn and cr levels among the different samples analysed in control area may be attributed to accumulation of heavy metals from other sources including dump sites , use of fertilisers and aerial depositions . measured cd levels were below 0.1 mgkg limit set by codex ( 2007 ) . heavy metals uptake in plants varied directly with soil concentrations and agreed with the findings of lecoultre ( 2001 ) that uptake was higher in soil areas with higher concentrations . elevated cr levels in control samples may have arisen from flooding , which could mobilise heavy metals from soils particularly when readily oxidizable organic nutrients are available . this is possible as records of annual rainfall exceeded 2,0002,500 mm / year in the area . non - nutritive pb gave marked ( p 0.05 ) elevated levels ( above who limits of 0.3 mgkg ) in all test samples except for cassava samples . lead interferes with a variety of body processes and is toxic to many organs and tissues including the heart , bones , intestines , kidneys , reproductive and nervous systems . it interferes with the development of the nervous system and is therefore particularly toxic to children , causing potentially permanent learning and behaviour disorders . symptoms include abdominal pain , confusion , headache , anaemia , irritability and in severe cases , seizures , coma and death . a foetus developing in the womb of a woman who has elevated blood lead level is also susceptible to lead poisoning and is at greater risk of being born prematurely or with a low birth weight ( woolf et al . 2007 ) . children are more at risk for lead poisoning because their smaller bodies are in a continuous state of growth and development ( landrigan et al . 2002 ) and a threatening situation may result in study area in the nearest future . many people , therefore , could be at risk of singly or combined adverse effects resulting from consumption of garden vegetables or tubers cultivated from oil - polluted soil and beyond , as supplies of these commodities are not limited.table 6evaluation of carcinogenic pahs ( mgkg ) in study soilschemical contaminantsoil ctviciri(ci / ctvi)further evaluation needed?(is ri > 1)testcontroltestcontroltestcontrolbenz(a)anthracene1.50e011.02e+021.0e036800.007yesnobenzo(b)fluoranthene3.30e014.69e+031.0e0314,2120.003yesnobenzo(k)fluoranthene3.30e014.70e+011.0e03142.40.003yesnodibenz(a , h)anthracene3.30e015.50e011.0e03166.70.003yesnoindeno(1,2,3-cd)pyrene3.30e016.94e+021.0e032,1030.003yesnochrysene3.30e019.91e+011.0e03300.30.003yesnobenzo(a)pyrene1.50e024.74e+011.0e033,1600.067yesnori20,7640.089yesnotable 7mean levels ( mgkg ) of studied metals in control area ( umuchichi)soilbitterleafwaterleafcocoyamcassavawho limitspb0.14 0.00 0.24 0.00 0.22 0.00 0.11 0.00 < 0.00010.30cd0.01 0.00 0.08 0.00 0.07 0.00 0.02 0.00 < 0.00010.10cr0.04 0.00 0.20 0.01 0.11 0.00 0.02 0.00 < 0.00010.05mn4.07 0.00 3.05 0.01 1.92 0.00 0.67 0.00 0.89 0.01 fe256 0.26 0.82 0.00 1.35 0.00 10.5 0.01 < 0.0001zn1.22 0.01 0.21 0.01 0.13 0.00 1.58 0.06 0.10 0.00 100table 8mean levels ( mgkg ) of studied metals in test area ( gokana)soilbitterleafwaterleafcocoyamcassavawho limitspb0.39 0.00 0.48 0.05 0.29 0.00 0.37 0.04 0.20 0.00 0.30cd0.02 0.00 0.04 0.00 0.03 0.00 0.03 0.00 0.02 0.00 0.10cr0.01 0.00 0.07 0.00 0.04 0.00 < 0.0001<0.00010.05mn0.78 0.01 1.18 0.03 1.19 0.02 2.73 0.02 0.37 0.00 fe284 0.60 6.54 0.02 4.74 0.04 23.6 0.07 16.0 0.03 zn0.90 0.00 2.32 0.00 1.81 0.00 2.94 0.00 0.98 0.00 100 evaluation of carcinogenic pahs ( mgkg ) in study soils mean levels ( mgkg ) of studied metals in control area ( umuchichi ) mean levels ( mgkg ) of studied metals in test area ( gokana ) potentially carcinogenic hydrocarbons and heavy metals pollution in gokana could be attributed to anthropogenic heavy metals enrichment of the oil - rich industrialized state , as well as oil exploration and related ill practices sighted during the study . humans and grazing animals may be in serious danger due to exposure along the food chain , and urgent need for remediation strategies and management of these contaminated zones is implied . in this study , 24 plant samples , besides soils , were collected from environmentally disturbed and undisturbed communities and analysed for pahs , tph and some metals . the results of the statistical analyses show that with respect to pahs , the eight datasets are significantly different ( p 0.05 ) and can be considered dissimilar dataset representative of disturbed and undisturbed environments . farmers can suffer direct exposure from such contaminated soils besides ingestion of food items , which is boundless , and immediate action , therefore , is needed following the results obtained from secondary evaluation of carcinogenic hydrocarbons distribution in study soils .

owing to the importance of clean and fertile agricultural soil for the continued existence of man , this study investigated the concentrations of total petroleum hydrocarbons ( tphs ) , polycyclic aromatic hydrocarbons ( pahs ) and some heavy metals in soils and selected commonly consumed vegetables and tubers from oil - polluted active agricultural farmland in gokana of ogoniland , rivers state , nigeria . samples from umuchichi , osisioma local government area in abia state , nigeria , a non - oil - polluted area constituted the control . in test and control , up to 3,830 19.6 mgkg1 dw and 6,950 68.3 mgkg1 dw ( exceeding dpr set limits ) and 11.3 0.04 mgkg1 dw and 186 0.02 mgkg1 dw for tph and pahs , respectively , were recorded in test soil and plant samples , respectively . among the metals studied ( pb , cd , cr , mn , fe and zn ) , pb and cr uptake exceeded who set limits for crops in test samples . combined sources of pollution were evident from our studies . bitterleaf and waterleaf could be tried as bioindicators owing to expressed contaminants uptake pattern .

Introduction Materials and methods Sample sourcing Sample preparation and analysis Statistical analysis Results and discussion Conclusions

the control area , umuchichi village in osisioma lga , is a coastal plain located on the southern part of abia state , nigeria and lies 440 and 614 n and 710 and 8 e. it is a non - polluted area with less industry presence . this study therefore seeks to evaluate the distribution of some heavy metals , tph and pahs in selected regularly - consumed food crops and soils from an oil - polluted active agricultural farmland and making comparisons with a view to health implications . soil samples ( in random replicates of three ) were taken from oil - polluted active agricultural farmlands in gokana ( test ) and non - oil - polluted active agricultural farmlands in umuchichi ( control ) . soil samples ( in random replicates of three ) were taken from oil - polluted active agricultural farmlands in gokana ( test ) and non - oil - polluted active agricultural farmlands in umuchichi ( control ) . this implies further evaluation , potentially through site - specific risk assessment.table 1a mean tph ( mgkg ) levels in control area ( umuchichi)componentssoilbitterleafwaterleafcocoyamcassavac-8<0.001<0.001<0.001<0.001<0.001c-9<0.001<0.001<0.001<0.001<0.001c-10<0.001<0.001<0.001<0.001<0.001c-11<0.001<0.001<0.001<0.001<0.001c-12<0.001<0.001<0.001<0.001<0.001c-13<0.001<0.001<0.001<0.001<0.001c-14<0.0010.03 0.00 < 0.001<0.001<0.001c-15<0.0010.01 0.00 < 0.001<0.001<0.001c-16<0.0010.01 0.00 < 0.001<0.001<0.001c-17<0.0010.01 0.00 < 0.001<0.001<0.001pristane<0.0010.05 0.00 < 0.001<0.001<0.001c-18<0.0010.01 0.00 < 0.001<0.001<0.001phythane<0.0010.02 0.00 < 0.001<0.001<0.001c-19<0.0010.02 0.00 < 0.001<0.001<0.001c-20<0.0010.02 0.00 0.01 0.00 0.01 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \pm 0.00 $ $ \end{document } < 0.001c-21<0.0010.02 0.00 0.12 0.00 < 0.001<0.001c-22<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-23<0.0010.01 0.00 < 0.001<0.001<0.001componentssoilbitterleafwaterleafcocoyamcassavac-24<0.0010.01 0.00 < 0.001<0.001<0.001c-25<0.0010.01 0.00 < 0.001<0.001<0.001c-26<0.0010.02 0.00 < 0.001<0.001<0.001c-27<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-28<0.0010.02 0.00 0.02 0.00 < 0.001<0.001c-29<0.0010.03 0.00 0.01 0.00 < 0.001<0.001c-30<0.0010.02 0.00 0.01 0.00 < 0.001<0.001c-31<0.0010.11 0.00a0.01 0.00 0.01 0.00 0.00 0.02 0.00 < 0.001c-33<0.0010.04 0.00 0.09 0.00 0.01 0.00 < 0.001c-34<0.0010.03 0.00 0.05 0.00 0.05 0.00 0.01 0.00 c-35<0.0010.03 0.00 0.03 0.00 0.01 0.00 < 0.001c-36<0.0010.02 0.00 0.03 0.00 0.01 0.00 0.01 0.00 c-37<0.0010.01 0.00 0.02 0.00 0.01 0.00 < 0.001c-38<0.0010.01 0.00 < 0.001<0.001<0.001c-39<0.001<0.0010.03 0.00 < 0.001<0.001c-40<0.0010.02 0.00 < 0.001<0.001<0.001total<0.0010.58 0.04 0.49 0.02 0.14 0.01 0.05 0.00 values with different superscript letters ( a , b , c , d , e ) in the same row are significantly different at 0.05 level ( p 0.05)table 2a mean level of tph ( mgkg ) in test ( gokana ) areacomponentssoilbitterleafwaterleafcocoyamcassavac-8<0.001<0.001<0.001<0.001<0.001c-9<0.001<0.0010.01 0.00 < 0.001<0.001c-10<0.0010.01 0.00 0.01 0.00 < 0.001<0.001c-11<0.0010.01 0.00 0.02 0.00 < 0.001<0.001c-12<0.0010.02 0.00 0.01 0.00 < 0.001<0.001c-13224 7.77 0.53 0.00 0.23 0.00 0.02 0.00 0.01 0.00 c-142,280 2.89 0.35 0.00 0.07 0.00 0.14 0.00 0.07 0.00 c-1547.1 26.5 4.00 0.06 2.73 0.06 0.31 0.00 0.43 0.00 c-1660.2 0.06 0.10 0.00 0.20 0.00 0.09 0.00 0.08 0.00 c-17289 1.53 < 0.0010.01 0.00 0.03 0.00 0.01 0.01 pristane<0.0010.22 0.01 0.21 0.00 0.04 0.00 0.05 0.02 c-18189 1.15 0.03 0.00 0.07 0.00 0.22 0.00 0.03 0.00 phythane35.2 1.34 0.20 0.00 < 0.0010.02 0.00 0.04 0.00 c-1920.8 0.12 1.23 0.01 0.31 0.00 0.03 0.00 0.03 0.00 c-20297 1.15 0.11 0.00 0.11 0.00 0.05 0.00 0.17 0.00 c-2126.9 0.23 0.17 0.00 0.18 0.00 0.04 0.00 0.03 0.00 c-2232.3 0.10 1.39 0.09 0.15 0.00 0.06 0.00 0.02 0.00 c-23219 2.31 0.01 0.00 1.90 0.02 2.12 0.01 0.02 0.00 total3720.58.386.223.170.99componentssoilbitterleafwaterleafcocoyamcassavac-249.55 0.01 1.36 0.04 0.24 0.00 0.12 0.00 0.16 0.00 c-251.25 0.01 0.09 0.00 0.21 0.00 0.18 0.02 0.30 0.00 c-2611.7 0.15 0.30 0.00 0.20 0.00 0.04 0.00 0.03 0.00 c-276.31 0.01 0.05 0.02 0.04 0.01 0.04 0.02 0.30 0.01 c-285.33 0.01 0.08 0.00 0.16 0.00 0.04 0.00 0.07 0.00 c-291.28 0.02 0.03 0.00 0.01 0.00 < 0.001<0.001c-3011.0 0.12 0.03 0.00 0.01 0.00 0.03 0.00 0.03 0.00 c-3118.8 0.35 < 0.0010.06 0.00 < 0.0010.03 0.00 c-322.54 0.01 < 0.0010.02 0.00 0.02 0.00 0.01 0.00 c-331.81 0.02 < 0.0010.02 0.00 0.06 0.00 0.05 0.00 c-348.91 0.03 0.01 0.00 0.67 0.00 0.01 0.00 0.05 0.00 c-356.91 0.01 0.66 0.00 0.51 0.00 0.02 0.00 0.06 0.00 c-362.03 0.01 < 0.001<0.001<0.0010.06 0.00 c-374.46 0.01 0.08 0.00 0.01 0.00 < 0.0010.02 0.00 c-384.49 0.01 0.04 0.00 0.05 0.00 0.01 0.00 0.03 0.00 c-391.66 0.01 0.12 0.00 0.02 0.00 < 0.0010.01 0.00 c-400.97 0.01 0.14 0.00 1.25 0.01 0.14 0.02 0.12 0.00 total3,830 19.6 11.3 0.07 9.67 0.08 3.99 0.09 2.17 0.06 table 3evaluation of tph ( mgkg ) contaminants in study soilsourcechemical contaminantsoil ctviciri(ci / ctvi)further evaluation needed? (is ri > 1)testcontroltestcontroltestcontrolbenz(a)anthracene1.50e011.02e+021.0e036800.007yesnobenzo(b)fluoranthene3.30e014.69e+031.0e0314,2120.003yesnobenzo(k)fluoranthene3.30e014.70e+011.0e03142.40.003yesnodibenz(a , h)anthracene3.30e015.50e011.0e03166.70.003yesnoindeno(1,2,3-cd)pyrene3.30e016.94e+021.0e032,1030.003yesnochrysene3.30e019.91e+011.0e03300.30.003yesnobenzo(a)pyrene1.50e024.74e+011.0e033,1600.067yesnori20,7640.089yesnotable 7mean levels ( mgkg ) of studied metals in control area ( umuchichi)soilbitterleafwaterleafcocoyamcassavawho limitspb0.14 0.00 0.24 0.00 0.22 0.00 0.11 0.00 < 0.00010.30cd0.01 0.00 0.08 0.00 0.07 0.00 0.02 0.00 < 0.00010.10cr0.04 0.00 0.20 0.01 0.11 0.00 0.02 0.00 < 0.00010.05mn4.07 0.00 3.05 0.01 1.92 0.00 0.67 0.00 0.89 0.01 fe256 0.26 0.82 0.00 1.35 0.00 10.5 0.01 < 0.0001zn1.22 0.01 0.21 0.01 0.13 0.00 1.58 0.06 0.10 0.00 100table 8mean levels ( mgkg ) of studied metals in test area ( gokana)soilbitterleafwaterleafcocoyamcassavawho limitspb0.39 0.00 0.48 0.05 0.29 0.00 0.37 0.04 0.20 0.00 0.30cd0.02 0.00 0.04 0.00 0.03 0.00 0.03 0.00 0.02 0.00 0.10cr0.01 0.00 0.07 0.00 0.04 0.00 < 0.0001<0.00010.05mn0.78 0.01 1.18 0.03 1.19 0.02 2.73 0.02 0.37 0.00 fe284 0.60 6.54 0.02 4.74 0.04 23.6 0.07 16.0 0.03 zn0.90 0.00 2.32 0.00 1.81 0.00 2.94 0.00 0.98 0.00 100 evaluation of carcinogenic pahs ( mgkg ) in study soils mean levels ( mgkg ) of studied metals in control area ( umuchichi ) mean levels ( mgkg ) of studied metals in test area ( gokana ) potentially carcinogenic hydrocarbons and heavy metals pollution in gokana could be attributed to anthropogenic heavy metals enrichment of the oil - rich industrialized state , as well as oil exploration and related ill practices sighted during the study . in this study , 24 plant samples , besides soils , were collected from environmentally disturbed and undisturbed communities and analysed for pahs , tph and some metals .

such a dramatic situation is due , at least in part , to the ability of the airborne bacillus to resist killing by , and to parasitize host alveolar macrophages ( ms ; reference 1 ) . protective anti - mycobacterial immune response involves mainly t lymphocytes that activate the m microbicidal functions through the release of interferon ( 2 , 3 ) . priming of naive t lymphocytes against mycobacterial antigens is thought to occur in the proximal lns and to rely on a particular subset of phagocytic cells , the dendritic cells ( dcs ) . indeed , dcs exhibit the unique ability to activate naive lymphocytes after migration from infection sites , where they capture antigens , to the lns where they express high amounts of presentation molecules , such as mhc - ii , and costimulatory molecules , such as cd80 and cd86 ( 4 ) . the early interaction between the dcs present as a dense network in the airway mucosa ( 5 ) and m. tuberculosis is thus likely to be critical for mounting a protective anti - mycobacterial immune response ( 3 , 69 ) . however , m. tuberculosis interactions with dcs are poorly understood at the molecular level . in particular , the ability of m. tuberculosis to replicate in dcs , relative to ms , remains controversial ( 7 , 9 , 10 ) , and the receptor(s ) used by m. tuberculosis to bind and to enter dcs are still unknown , whereas those involved in the parasitism of ms have been well characterized in vitro . mycobacterial binding to ms occurs in cholesterol - rich domains of the host cell plasma membrane ( 11 ) and involves cr3 , together with other molecules like mr , cr1 , cr4 , cd14 , surfactant protein ( sp)-a receptors , as well as scavenger receptors ( 12 , 13 ) . other surface molecules , such as toll - like receptors ( tlrs ) , are also essential for mycobacterial interactions with phagocytic cells ( 14 ) , though their role in mycobacterial entry remains to be evaluated . some of these receptors ( e.g. , cr3 , mr ) are present on dcs and may be involved in the binding and entry of mycobacteria into these cells . these additional receptors include the recently identified dc - sign ( 15 ) , a calcium - dependent ( c - type ) lectin , containing a carbohydrate recognition domain ( crd ) at its extracellular cooh - terminal end , that recognizes mannose - rich molecules ( 16 ) . dc - sign was initially described as a receptor for icam-3 at the surface of t cells , triggering the formation of the immunological synapse between dcs and naive t lymphocytes . interestingly , dc - sign binds to hiv and simian immunodeficiency viruses , and is involved in the trans - infection of cd4 t lymphocytes by hiv- or siv - infected dcs ( 17 ) . dc - sign has also been recently involved in leishmania pifanoi binding to dcs ( 18 ) . here we show that m. tuberculosis infects dcs via ligation of dc - sign by the mycobacterial surface - exposed lipoglycan lipoarabinomannan ( lam ) . freshly isolated ldcs were found to express dc - sign , and m. tuberculosis derived material was detected in dc - signcd14hla - dr cells in lns from patients with tuberculosis . hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ) ( 19 ) , were cultured and infected in rpmi-1640 ( gibco brl / invitrogen ) supplemented with 10% heat - inactivated fcs ( dutscher ) . mononuclear cells were isolated from the blood of healthy volunteers ( etablissement franais du sang ) by ficoll - paque centrifugation . t , b , and nk cells were depleted using m-450 pan t / cd2 and m-450 pan b / cd19 dynabeads ( dynal ) . the recovered cells , referred to as monocytes , were seeded in 6-well plates at 2 10 cells / well in 3 ml rpmi-1640 , 10% fcs , l - glutamine , granulocyte / m-colony stimulating factor ( 10 ng / ml ) , and interleukin 4 ( 20 ng / ml ; both from r&d systems ) . gfp - expressing strains of m. tuberculosis h37rv and m. bovis bcg were generated by transformation with the gfp - encoding plasmid pegfp and propagated in medium containing 50 g / ml hygromycin b ( boehringer ) . human lung dcs ( ldcs ) were isolated as described ( 20 , 21 ) . lung samples were from surgical specimen distant from primary carcinoma , obtained with the patients ' consent , and used according to institutional guidelines . in brief , after treating the lung fragments with collagenase , cells were separated on a ficoll - paque gradient to obtain pulmonary mononuclear cells , which were cultured in petri dishes for 1 h before removing nonadherent cells . loosely adherent mononuclear cells released after three rinses in saline were separated into ldcs and autofluorescent alveolar ms with a facstar ( becton dickinson ) according to the presence or absence of autofluorescent inclusions , using a 488 nm wavelength for excitation and a 588 nm filter for emission . in contrast to alveolar ms , the latter cells are potent stimulators of allogeneic t lymphocytes ( data not depicted , and reference 20 ) . lymph nodes were referred to the laboratory of pathology at the saint - louis hospital ( paris , france ) for the purpose of tuberculosis diagnosis with the patients ' consent and used according to institutional guidelines . human tissues were previously fixed in afa ( carlo erba ) , a mix of 2% formalin ( vol / vol ) , 5% acetic acid ( vol / vol ) , 75% ethanol ( vol / vol ) , and 18% water ( vol / vol ) , and then embedded in paraffin for histopathological diagnosis . smear positive and/or culture positive ( in less than 12 d ) biopsies were selected from the collection between may 1995 and december 2001 , and 10 sections per biopsy were used for immunostaining . cells were infected at the indicated multiplicity of infection ( moi ) for 4 h at 4c in rpmi-1640 , 10% fcs , extensively washed in rpmi-1640 , and analyzed by flow cytometry . fluorescence was assessed on a total of 2 10 cells per sample using a facscalibur and cellquest software ( becton dickinson ) . in some experiments , the same samples were also plated out onto agar medium and cfus were scored after 3 wk at 37c . alternatively , infections were performed in the presence of 10% complete human serum , in order to opsonize bacteria with complement , as indicated ( 22 ) . the following mabs were used in binding inhibition experiments : anti - cr3/cd11b ( clone m1/70 ; bd biosciences ) , -cr3 ( 2lpm19c ; dako ) , -mr ( clone 15/2 ; hycult biotechnology ) , -cd40 ( clone 5c3 ; bd biosciences ) , and -dc - sign ( clones 120507 ; r&d systems ) . 1b10 ( igg2a- ) were produced as follows : balb / c mice were immunized with 293 t cells transfected with cdna encoding dc - sign ( cloned from human monocyte - derived dcs ) . hybridoma supernatants were screened for the ability to recognize dc - sign expressing hela cells , and were purified from bulk cultures . 1b10 neutralizes hiv gp120 binding to dc - sign and prevents trans - infection of cd4 t cells by hiv - pulsed dc - sign cell lines ( a. amara , personal communication ) . binding inhibition experiments were performed by preincubating the cells with the indicated mabs at different concentrations ( see legends to figures ) for 1 h at 4c before the binding assay . for confocal microscopy , dc - sign was detected using clone 120507 mab and a cy3-conjugated secondary mab ( amersham biosciences ) . for flow cytometry , dc - sign , cr3/cd11b , and mr were detected using phycoerythrin - conjugated mabs from clones 120507 , m1/70 , and 3.29b1.10 ( immunotech ) , respectively ; cd14 was detected using an allophycocyanin - conjugated mab ( clone m5e2 , bd biosciences ) ; hla - dr was detected using fluorescein isothiocyanate - conjugated mab ( clone h279 ; immunotech ) . for immunohistochemistry , cd3 , cd20 , dc - sign , cd14 , hla - dr , and m. tuberculosis were detected using polyclonal rabbit serum ( dako ) , mabs from clone h1(fb1 ; dako ) , from clone 1b10 , from clone 7 ( novocastra ) , from clone cr3/43 ( dako ) , and an anti m . hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ) ( 19 ) , were cultured and infected in rpmi-1640 ( gibco brl / invitrogen ) supplemented with 10% heat - inactivated fcs ( dutscher ) . mononuclear cells were isolated from the blood of healthy volunteers ( etablissement franais du sang ) by ficoll - paque centrifugation . t , b , and nk cells were depleted using m-450 pan t / cd2 and m-450 pan b / cd19 dynabeads ( dynal ) . the recovered cells , referred to as monocytes , were seeded in 6-well plates at 2 10 cells / well in 3 ml rpmi-1640 , 10% fcs , l - glutamine , granulocyte / m-colony stimulating factor ( 10 ng / ml ) , and interleukin 4 ( 20 ng / ml ; both from r&d systems ) . gfp - expressing strains of m. tuberculosis h37rv and m. bovis bcg were generated by transformation with the gfp - encoding plasmid pegfp and propagated in medium containing 50 g / ml hygromycin b ( boehringer ) . human lung dcs ( ldcs ) were isolated as described ( 20 , 21 ) . lung samples were from surgical specimen distant from primary carcinoma , obtained with the patients ' consent , and used according to institutional guidelines . in brief , after treating the lung fragments with collagenase , cells were separated on a ficoll - paque gradient to obtain pulmonary mononuclear cells , which were cultured in petri dishes for 1 h before removing nonadherent cells . loosely adherent mononuclear cells released after three rinses in saline were separated into ldcs and autofluorescent alveolar ms with a facstar ( becton dickinson ) according to the presence or absence of autofluorescent inclusions , using a 488 nm wavelength for excitation and a 588 nm filter for emission . in contrast to alveolar ms , the latter cells are potent stimulators of allogeneic t lymphocytes ( data not depicted , and reference 20 ) . lymph nodes were referred to the laboratory of pathology at the saint - louis hospital ( paris , france ) for the purpose of tuberculosis diagnosis with the patients ' consent and used according to institutional guidelines . human tissues were previously fixed in afa ( carlo erba ) , a mix of 2% formalin ( vol / vol ) , 5% acetic acid ( vol / vol ) , 75% ethanol ( vol / vol ) , and 18% water ( vol / vol ) , and then embedded in paraffin for histopathological diagnosis . smear positive and/or culture positive ( in less than 12 d ) biopsies were selected from the collection between may 1995 and december 2001 , and 10 sections per biopsy were used for immunostaining . cells were infected at the indicated multiplicity of infection ( moi ) for 4 h at 4c in rpmi-1640 , 10% fcs , extensively washed in rpmi-1640 , and analyzed by flow cytometry . fluorescence was assessed on a total of 2 10 cells per sample using a facscalibur and cellquest software ( becton dickinson ) . in some experiments , the same samples were also plated out onto agar medium and cfus were scored after 3 wk at 37c . alternatively , infections were performed in the presence of 10% complete human serum , in order to opsonize bacteria with complement , as indicated ( 22 ) . the following mabs were used in binding inhibition experiments : anti - cr3/cd11b ( clone m1/70 ; bd biosciences ) , -cr3 ( 2lpm19c ; dako ) , -mr ( clone 15/2 ; hycult biotechnology ) , -cd40 ( clone 5c3 ; bd biosciences ) , and -dc - sign ( clones 120507 ; r&d systems ) . 1b10 ( igg2a- ) were produced as follows : balb / c mice were immunized with 293 t cells transfected with cdna encoding dc - sign ( cloned from human monocyte - derived dcs ) . hybridoma supernatants were screened for the ability to recognize dc - sign expressing hela cells , and were purified from bulk cultures . 1b10 neutralizes hiv gp120 binding to dc - sign and prevents trans - infection of cd4 t cells by hiv - pulsed dc - sign cell lines ( a. amara , personal communication ) . binding inhibition experiments were performed by preincubating the cells with the indicated mabs at different concentrations ( see legends to figures ) for 1 h at 4c before the binding assay . for confocal microscopy , dc - sign was detected using clone 120507 mab and a cy3-conjugated secondary mab ( amersham biosciences ) . for flow cytometry , dc - sign , cr3/cd11b , and mr were detected using phycoerythrin - conjugated mabs from clones 120507 , m1/70 , and 3.29b1.10 ( immunotech ) , respectively ; cd14 was detected using an allophycocyanin - conjugated mab ( clone m5e2 , bd biosciences ) ; hla - dr was detected using fluorescein isothiocyanate - conjugated mab ( clone h279 ; immunotech ) . for immunohistochemistry , cd3 , cd20 , dc - sign , cd14 , hla - dr , and m. tuberculosis were detected using polyclonal rabbit serum ( dako ) , mabs from clone h1(fb1 ; dako ) , from clone 1b10 , from clone 7 ( novocastra ) , from clone cr3/43 ( dako ) , and an anti m . given the unique richness of the mycobacterial envelope in poly - mannosylated materials ( 23 ) , we asked whether m. tuberculosis interacts with dc - sign on the surface of dcs . we first compared the binding of virulent gfp - expressing m. tuberculosis h37rv , the most commonly used reference m. tuberculosis strain , to hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ; reference 19 ) . we found that m. tuberculosis bound to p4-dc cells up to 25 times more than to p4 cells ( fig . 1 a ) . similar results were obtained with m. tuberculosis clinical isolate mt103 ( data not shown ) , indicating that binding to dc - sign was not restricted to laboratory mycobacterial strains . we then studied the binding of m. tuberculosis to dcs , and the role of dc - sign in this process , as compared with cr3 and mr . as reported ( 24 ) , human monocyte - derived dcs ( mddcs ; reference 25 ) expressed high levels of dc - sign together with cr3 and mr . we performed a binding assay with mddcs that had been preincubated or not with different mabs . dc - sign mabs inhibited attachment of m. tuberculosis up to 90% ( fig . 1 b ) . interestingly , preincubation with two anti - cr3 mabs , used in combination , an anti - mr mab , or an irrelevant ( i.e. , directed against a non - mycobacteria - binding protein ) anti - cd40 mab , had only minor effects on the binding of m. tuberculosis to mddcs ( fig . mabs inhibited m. tuberculosis binding to monocyte - derived ms ( mdms ) by 50 and 45% , respectively ( data not shown ) , thus confirming previous reports that crs and mr are major m. tuberculosis receptors on mdms ( 22 ) . it was also important to assess whether dc - sign was still the predominant m. tuberculosis receptor on dcs in the presence of a complement source , a condition that might be expected in vivo . to this end , we performed a binding inhibition experiment in the presence of complete human serum . anti - dc - sign mabs were then still able to inhibit up to 90% mycobacterial binding to dcs ( fig . 1 b ) . under the same conditions , anti - cr3 and -mr mabs inhibited mycobacterial binding to mdms by 60 and 20% , respectively ( data not shown ) . thus , our results indicate that dc - sign acts as the major m. tuberculosis receptor on human mddcs , even in the presence of complement ( a ) epithelial hela - derived p4 cells expressing or not dc - sign ( p4-dc and p4 , respectively ) were infected with gfp expressing ( gfp+ ) m. tuberculosis h37rv at different mois . bacteria binding was evaluated by flow cytometry ( left panel ) and cfu counts ( right panel ) . tuberculosis h37rv at an moi of 1 bacterium per cell in the presence or not of 10% complete human serum , either directly ( control ) or after preincubation with 10 g / ml of mabs directed against cr3/cd11b , mr , cd40 , or dc - sign . preincubation with the corresponding isotype controls led to no significant inhibition of mycobacteria binding ( not shown ) . data were expressed as percentages of binding relative to control values ( 100% , no mab ) , and means ( sd ) of three independent experiments are shown . bovis bcg , salmonella typhimurium ( clinical isolate ) , or listeria monocytogenes ( clinical isolate ) and subjected to the binding assay . in experiments using s. typhimurium and l. monocytogenes , infected cells were plated out onto agar medium and cfus were scored after 24 h at 37c . data are expressed as in b. ( d ) m. tuberculosis binding to dc - sign is inhibited by lam . cells were pretreated for 1 h at 4c with 10 g / ml mannan as control , or with 10 g / ml lam , and subjected to the binding assay . data are expressed as in b. we next examined whether dc - sign contributes to the attachment of other intracellular bacterial species to mddcs . binding of the vaccine strain mycobacterium bovis bacillus calmette - gurin ( bcg ) , which belongs to the tuberculosis complex , was also found to be mediated by dc - sign ( fig . 1 c ) . however , pretreatment with anti dc - sign mabs had no effect on the binding of either the gram - positive listeria monocytogenes or the gram - negative salmonella typhimurium species ( fig . major structural differences exist at the surface of mycobacteria compared with gram and gram species , which may explain differences in binding to dc - sign . in particular , lam is an abundant poly - mannosylated lipoglycan , specific to the mycobacterial envelope ( 26 ) , and it has been shown to bind to various human c - type lectins on ms , such as surfactant protein d ( 27 ) . inasmuch as dc - sign is a c - type lectin that recognizes mannose - rich molecules ( 16 ) , we investigated whether m. tuberculosis binding to the lectin was mannosyl - defined . binding of m. tuberculosis to both p4-dc and mddcs was inhibited up to 90% by yeast mannan as well as by m. tuberculosis h37rv - derived lam ( fig . by contrast , preincubation of cells with lps derived from escherichia coli or with dextran had no effect on the binding process ( data not shown ) . these findings suggest that lam may constitute a privileged mycobacterial ligand for dc - sign , even though other mycobacterial components may also bind to the lectin . interestingly , mycobacterium smegmatis derived lam , that is devoid of mannose capping residues , was found to only moderately inhibit m. tuberculosis binding to dc - sign ( unpublished data ) . after attaching to the cell surface , pathogenic mycobacteria are taken up by phagocytic cells and reside in phagosomes that do not fuse with host cell late endosomes and lysosomes , but take part in the recycling pathway ( 1 ) . to investigate dc - sign trafficking in m. tuberculosis - infected cells , we performed confocal microscopy analysis of mddcs infected with gfp - expressing mycobacteria . during the first hour of phagocytosis , most bacilli were detected as either attached extracellularly to the cells ( fig . 2 a , top panel ) , or colocalized with dc - sign in nascent phagosomes ( fig . however , dc - sign staining was not detected on phagosomes that had detached from the plasma membrane , indicating that it was excluded from the vacuoles very soon after phagocytosis ( fig . these data indicate that dc - sign is present in m. tuberculosis vacuoles during the early steps of bacterial uptake , and is then rapidly expelled from the phagosome ( fig . 2 b ) , possibly as a result of recycling to the cell plasma membrane . we also examined whether m. tuberculosis infection could modify dc - sign expression at the surface of infected cells . as reported ( 6 , 7 ) , infection was found to induce cell maturation , as illustrated by up - regulation of cd83 ( fig . 2 c ) , cd86 , and hla - dr ( data not shown ) . dc - sign expression was only slightly down - modulated in mature infected cells , even 48 h after infection ( fig . tuberculosis , washed extensively in rpmi-1640 , and chased at 37c for the indicated periods of time . ( a ) the two top panels shows cells representative of early phagocytosis events ; dc - sign was detected both at the cell surface and in intracellular vesicles , but due to the strong surface staining , the red signal had to be reduced . each panel shows a representative cell . ( b ) kinetics of dc - sign colocalization with m. tuberculosis ( m. tb ) : a minimum of 100 bacteria were scored at each time point . extracellular bacteria that were found attached to the cells ( early time points , 15 and 30 min ) were scored as colocalizing with dc - sign . ( c ) surface expression of cd83 ( left panel ) and dc - sign ( right panel ) on mddcs was assessed by flow cytometry 48 h after infection with gfp - m . dotted line : isotype control labeling ; plain lines : uninfected cells ; bold lines : infected cells . it was then important to determine whether m. tuberculosis could interact with dc - sign cells in vivo . we first evaluated the presence of the lectin on human interstitial ldcs as compared with in vitro 3 a ) and cd14 ( data not shown ) , a phenotype shared by mddcs . like mddcs , ldcs expressed surface dc - sign , cr3 , and mr ( fig . although we can not formally rule out the possibility that ldc preparations were devoid of contaminant cells of other type(s ) ( e.g. , activated macrophages ) , it is tempting to suggest that m. tuberculosis may encounter and interact with dc - sign dcs during the natural course of infection . however , too few cells could be recovered from surgical samples to allow us to perform binding experiments to test this hypothesis . to further investigate dc - sign possible involvement in the interactions of m. tuberculosis with dcs in vivo , we reasoned that if dc - sign dcs take up m. tuberculosis in the lungs , and since dc - sign expression is only slightly reduced by infection - associated maturation of the cells , then we should be able to detect mycobacteria - derived material in dc - sign dcs in lns from patients with tuberculosis . ln paraffin - embedded sections from seven patients were stained with anti - mycobacterial and anti dc - sign mabs ( fig . the selected lns were rich in granulomas , characterized by caseous centers surrounded by rings of t and b lymphocytes ( fig . as reported ( 15 , 28 ) , dc - sign was detected mostly in intergerminal t cell zones and not in germinal centers . in addition , dc - sign cells were located in granulomatous structures , and were cd14 and hla - dr ( fig . 3 b , middle and right panels , respectively ) . mycobacteria and mycobacteria - derived antigens were immunodetected in samples from 5 out of 7 patients , in both granulomatous ( fig . 3 c , left and right panels ) , and nongranulomatous regions of the lns , including subcapsular sinuses ( fig . 3 c , middle panel ) . in most cases ( 80100% ) , mycobacteria - specific signal was detected within dc - sign cells ( fig . these findings indicate that m. tuberculosis interacts with dc - sign in vivo and that dc - sign cells , possibly dcs , may carry mycobacteria or mycobacteria - derived material from the lungs to the lns during their maturation process . dc - sign expression on lung dcs ( ldcs ) and in lymph nodes ( lns ) . ( a ) ldcs are hla - dr and express dc - sign , cr3 , and mr . surface expression of hla - dr , dc - sign , cr3/cd11b , and mr on ldcs from a noninfected patient was assessed by flow cytometry using the appropriate cytochrome - conjugated mabs . ( b ) dc - sign expression in the ln from a patient with tuberculosis ( g , granuloma ) . left panel , cd3 ( blue ) and cd20 ( red ) ; middle panel , dc - sign ( blue ) and cd14 ( red ) ; right panel , dc - sign ( blue ) and hla - dr ( red ) . ( c ) localization of m. tuberculosis derived antigens in dc - sign cells in lns from two patients with tuberculosis . dc - sign ( blue ) and m. tuberculosis ( red ) were immunodetected both in granulomas ( g ; left and right panels ) and in nongranulomatous regions , including subcapsular sinuses ( scs ; middle panel ) . in b and c , bars represent 0.5 mm and squares represent areas shown at higher magnification at the single cell level in the insets . staining of the samples with igg2a ( 1b10 isotype control ) or with a naive rabbit serum led to no detectable signal ( data not depicted ) . altogether , our results demonstrate that dc - sign / cd209 is the predominant m. tuberculosis receptor on human dcs , whereas the mycobacterial m receptors , cr3 and mr , appear to play a minor role , if any , in this binding . this exclusivity may be due to dc - sign abundance on dcs relative to cr3 and mr , and/or to the affinity of the lectin for its ligand(s ) . affinity for dc - sign seems to be fairly restrictive among bacteria , as neither gram l. monocytogenes nor gram s. typhimurium species bound to the lectin . this is not surprising , as the envelopes of gram and gram bacteria are very poor in poly - mannosylated material . unique characteristics of the mycobacterial cell wall , which is the most complex of all bacterial cell surfaces ( 23 ) , might thus account for the affinity for dc - sign . our finding that lam , like mannan , which contain common mannosyl motifs , can block dc - sign mediated attachment of m. tuberculosis to dcs and to p4-dc cells is consistent with the high affinity of the lectin for mannose - rich molecules ( 16 ) , and suggests that lam may be one of its major mycobacterial ligands . ligation of dc - sign by mycobacteria is likely to have important effects on the immunological and pathological events associated with m. tuberculosis infection . differential receptor usage by m. tuberculosis on dcs and ms may account for the different survival ability and trafficking patterns of mycobacteria in the two cell types , which is still a matter of debate ( 7 , 9 , 10 ) . dc - sign has been detected on alveolar ms ( 28 , 29 ) , that constitute the privileged cell targets of m. tuberculosis during the early steps of infection . it will be important to evaluate whether the lectin is also a predominant mycobacterial receptor on this cell population . it will also be of great interest to investigate what type of pro- or antiinflammatory cytokines are induced or repressed upon dc - sign ligation by mycobacteria ( 30 ) , as compared with ligation of other signal transducers , such as the tlrs ( 14 ) . dcs are also involved in the early activation of non - mhc restricted and cd1-restricted t cells specific for various mycobacterial glycolipids , including lam ( 31 ) . the intracellular trafficking pattern of dc - sign in m. tuberculosis - infected dcs suggests that dc - sign may carry mycobacterial glycolipids from the bacterial vacuole to the cell plasma membrane and/or to various subcellular compartments , where glycolipids could be loaded onto cd1 molecules for presentation to cd1-restricted lymphocytes ( 32 ) . in the lungs , submucosal and interstitial ldcs are thought to play a key role in immune surveillance of the respiratory tract ( 5 ) . in particular , interactions of dcs present in the alveolar septal walls with m. tuberculosis could be crucial for initiating an efficient anti - mycobacterial immune response . our finding that ldcs express dc - sign suggests that dc - sign is likely to interact with m. tuberculosis in vivo . this is strengthened by detection of mycobacteria - derived material in dc - sign dcs in lns from patients with tuberculosis . it is interesting that both m. tuberculosis and hiv can bind to dc - sign . dc - sign expressing cells may carry either intracellular or surface - attached m. tuberculosis during their migration from the site of infection to the lns , and could thus constitute a mycobacterial reservoir . this has also been suggested for hiv ( 17 ) , and might account for several pathological and immunological aspects of m. tuberculosis infection , e.g. , mediastinal adenitis , the formation of secondary granulomas in the lns , and the chronic stimulation of the immune system that is required to maintain the latency period of the disease .

early interactions between lung dendritic cells ( ldcs ) and mycobacterium tuberculosis , the etiological agent of tuberculosis , are thought to be critical for mounting a protective anti - mycobacterial immune response and for determining the outcome of infection . however , these interactions are poorly understood , at least at the molecular level . here we show that m. tuberculosis enters human monocyte - derived dcs after binding to the recently identified lectin dc - specific intercellular adhesion molecule-3 grabbing nonintegrin ( dc - sign ) . by contrast , complement receptor ( cr)3 and mannose receptor ( mr ) , which are the main m. tuberculosis receptors on macrophages ( ms ) , appeared to play a minor role , if any , in mycobacterial binding to dcs . the mycobacteria - specific lipoglycan lipoarabinomannan ( lam ) was identified as a key ligand of dc - sign . freshly isolated human ldcs were found to express dc - sign , and m. tuberculosis derived material was detected in cd14hla - dr+dc - sign+ cells in lymph nodes ( lns ) from patients with tuberculosis . thus , as for human immunodeficiency virus ( hiv ) , which is captured by the same receptor , dc - sign mediated entry of m. tuberculosis in dcs in vivo is likely to influence bacterial persistence and host immunity .

Introduction Materials and Methods Cells and Bacteria. Lymph Node Samples. Binding Assay. Antibodies. Results and Discussion

such a dramatic situation is due , at least in part , to the ability of the airborne bacillus to resist killing by , and to parasitize host alveolar macrophages ( ms ; reference 1 ) . protective anti - mycobacterial immune response involves mainly t lymphocytes that activate the m microbicidal functions through the release of interferon ( 2 , 3 ) . priming of naive t lymphocytes against mycobacterial antigens is thought to occur in the proximal lns and to rely on a particular subset of phagocytic cells , the dendritic cells ( dcs ) . the early interaction between the dcs present as a dense network in the airway mucosa ( 5 ) and m. tuberculosis is thus likely to be critical for mounting a protective anti - mycobacterial immune response ( 3 , 69 ) . however , m. tuberculosis interactions with dcs are poorly understood at the molecular level . in particular , the ability of m. tuberculosis to replicate in dcs , relative to ms , remains controversial ( 7 , 9 , 10 ) , and the receptor(s ) used by m. tuberculosis to bind and to enter dcs are still unknown , whereas those involved in the parasitism of ms have been well characterized in vitro . mycobacterial binding to ms occurs in cholesterol - rich domains of the host cell plasma membrane ( 11 ) and involves cr3 , together with other molecules like mr , cr1 , cr4 , cd14 , surfactant protein ( sp)-a receptors , as well as scavenger receptors ( 12 , 13 ) . other surface molecules , such as toll - like receptors ( tlrs ) , are also essential for mycobacterial interactions with phagocytic cells ( 14 ) , though their role in mycobacterial entry remains to be evaluated . these additional receptors include the recently identified dc - sign ( 15 ) , a calcium - dependent ( c - type ) lectin , containing a carbohydrate recognition domain ( crd ) at its extracellular cooh - terminal end , that recognizes mannose - rich molecules ( 16 ) . dc - sign was initially described as a receptor for icam-3 at the surface of t cells , triggering the formation of the immunological synapse between dcs and naive t lymphocytes . interestingly , dc - sign binds to hiv and simian immunodeficiency viruses , and is involved in the trans - infection of cd4 t lymphocytes by hiv- or siv - infected dcs ( 17 ) . dc - sign has also been recently involved in leishmania pifanoi binding to dcs ( 18 ) . here we show that m. tuberculosis infects dcs via ligation of dc - sign by the mycobacterial surface - exposed lipoglycan lipoarabinomannan ( lam ) . freshly isolated ldcs were found to express dc - sign , and m. tuberculosis derived material was detected in dc - signcd14hla - dr cells in lns from patients with tuberculosis . hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ) ( 19 ) , were cultured and infected in rpmi-1640 ( gibco brl / invitrogen ) supplemented with 10% heat - inactivated fcs ( dutscher ) . gfp - expressing strains of m. tuberculosis h37rv and m. bovis bcg were generated by transformation with the gfp - encoding plasmid pegfp and propagated in medium containing 50 g / ml hygromycin b ( boehringer ) . lymph nodes were referred to the laboratory of pathology at the saint - louis hospital ( paris , france ) for the purpose of tuberculosis diagnosis with the patients ' consent and used according to institutional guidelines . cells were infected at the indicated multiplicity of infection ( moi ) for 4 h at 4c in rpmi-1640 , 10% fcs , extensively washed in rpmi-1640 , and analyzed by flow cytometry . the following mabs were used in binding inhibition experiments : anti - cr3/cd11b ( clone m1/70 ; bd biosciences ) , -cr3 ( 2lpm19c ; dako ) , -mr ( clone 15/2 ; hycult biotechnology ) , -cd40 ( clone 5c3 ; bd biosciences ) , and -dc - sign ( clones 120507 ; r&d systems ) . 1b10 ( igg2a- ) were produced as follows : balb / c mice were immunized with 293 t cells transfected with cdna encoding dc - sign ( cloned from human monocyte - derived dcs ) . hybridoma supernatants were screened for the ability to recognize dc - sign expressing hela cells , and were purified from bulk cultures . for confocal microscopy , dc - sign was detected using clone 120507 mab and a cy3-conjugated secondary mab ( amersham biosciences ) . for flow cytometry , dc - sign , cr3/cd11b , and mr were detected using phycoerythrin - conjugated mabs from clones 120507 , m1/70 , and 3.29b1.10 ( immunotech ) , respectively ; cd14 was detected using an allophycocyanin - conjugated mab ( clone m5e2 , bd biosciences ) ; hla - dr was detected using fluorescein isothiocyanate - conjugated mab ( clone h279 ; immunotech ) . for immunohistochemistry , cd3 , cd20 , dc - sign , cd14 , hla - dr , and m. tuberculosis were detected using polyclonal rabbit serum ( dako ) , mabs from clone h1(fb1 ; dako ) , from clone 1b10 , from clone 7 ( novocastra ) , from clone cr3/43 ( dako ) , and an anti m . hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ) ( 19 ) , were cultured and infected in rpmi-1640 ( gibco brl / invitrogen ) supplemented with 10% heat - inactivated fcs ( dutscher ) . gfp - expressing strains of m. tuberculosis h37rv and m. bovis bcg were generated by transformation with the gfp - encoding plasmid pegfp and propagated in medium containing 50 g / ml hygromycin b ( boehringer ) . lymph nodes were referred to the laboratory of pathology at the saint - louis hospital ( paris , france ) for the purpose of tuberculosis diagnosis with the patients ' consent and used according to institutional guidelines . the following mabs were used in binding inhibition experiments : anti - cr3/cd11b ( clone m1/70 ; bd biosciences ) , -cr3 ( 2lpm19c ; dako ) , -mr ( clone 15/2 ; hycult biotechnology ) , -cd40 ( clone 5c3 ; bd biosciences ) , and -dc - sign ( clones 120507 ; r&d systems ) . 1b10 ( igg2a- ) were produced as follows : balb / c mice were immunized with 293 t cells transfected with cdna encoding dc - sign ( cloned from human monocyte - derived dcs ) . hybridoma supernatants were screened for the ability to recognize dc - sign expressing hela cells , and were purified from bulk cultures . for confocal microscopy , dc - sign was detected using clone 120507 mab and a cy3-conjugated secondary mab ( amersham biosciences ) . for flow cytometry , dc - sign , cr3/cd11b , and mr were detected using phycoerythrin - conjugated mabs from clones 120507 , m1/70 , and 3.29b1.10 ( immunotech ) , respectively ; cd14 was detected using an allophycocyanin - conjugated mab ( clone m5e2 , bd biosciences ) ; hla - dr was detected using fluorescein isothiocyanate - conjugated mab ( clone h279 ; immunotech ) . for immunohistochemistry , cd3 , cd20 , dc - sign , cd14 , hla - dr , and m. tuberculosis were detected using polyclonal rabbit serum ( dako ) , mabs from clone h1(fb1 ; dako ) , from clone 1b10 , from clone 7 ( novocastra ) , from clone cr3/43 ( dako ) , and an anti m . given the unique richness of the mycobacterial envelope in poly - mannosylated materials ( 23 ) , we asked whether m. tuberculosis interacts with dc - sign on the surface of dcs . we first compared the binding of virulent gfp - expressing m. tuberculosis h37rv , the most commonly used reference m. tuberculosis strain , to hela - derived cells expressing or not dc - sign ( p4-dc and p4 , respectively ; reference 19 ) . similar results were obtained with m. tuberculosis clinical isolate mt103 ( data not shown ) , indicating that binding to dc - sign was not restricted to laboratory mycobacterial strains . we then studied the binding of m. tuberculosis to dcs , and the role of dc - sign in this process , as compared with cr3 and mr . as reported ( 24 ) , human monocyte - derived dcs ( mddcs ; reference 25 ) expressed high levels of dc - sign together with cr3 and mr . dc - sign mabs inhibited attachment of m. tuberculosis up to 90% ( fig . , directed against a non - mycobacteria - binding protein ) anti - cd40 mab , had only minor effects on the binding of m. tuberculosis to mddcs ( fig . mabs inhibited m. tuberculosis binding to monocyte - derived ms ( mdms ) by 50 and 45% , respectively ( data not shown ) , thus confirming previous reports that crs and mr are major m. tuberculosis receptors on mdms ( 22 ) . it was also important to assess whether dc - sign was still the predominant m. tuberculosis receptor on dcs in the presence of a complement source , a condition that might be expected in vivo . anti - dc - sign mabs were then still able to inhibit up to 90% mycobacterial binding to dcs ( fig . under the same conditions , anti - cr3 and -mr mabs inhibited mycobacterial binding to mdms by 60 and 20% , respectively ( data not shown ) . thus , our results indicate that dc - sign acts as the major m. tuberculosis receptor on human mddcs , even in the presence of complement ( a ) epithelial hela - derived p4 cells expressing or not dc - sign ( p4-dc and p4 , respectively ) were infected with gfp expressing ( gfp+ ) m. tuberculosis h37rv at different mois . bovis bcg , salmonella typhimurium ( clinical isolate ) , or listeria monocytogenes ( clinical isolate ) and subjected to the binding assay . ( d ) m. tuberculosis binding to dc - sign is inhibited by lam . data are expressed as in b. we next examined whether dc - sign contributes to the attachment of other intracellular bacterial species to mddcs . binding of the vaccine strain mycobacterium bovis bacillus calmette - gurin ( bcg ) , which belongs to the tuberculosis complex , was also found to be mediated by dc - sign ( fig . major structural differences exist at the surface of mycobacteria compared with gram and gram species , which may explain differences in binding to dc - sign . inasmuch as dc - sign is a c - type lectin that recognizes mannose - rich molecules ( 16 ) , we investigated whether m. tuberculosis binding to the lectin was mannosyl - defined . binding of m. tuberculosis to both p4-dc and mddcs was inhibited up to 90% by yeast mannan as well as by m. tuberculosis h37rv - derived lam ( fig . these findings suggest that lam may constitute a privileged mycobacterial ligand for dc - sign , even though other mycobacterial components may also bind to the lectin . interestingly , mycobacterium smegmatis derived lam , that is devoid of mannose capping residues , was found to only moderately inhibit m. tuberculosis binding to dc - sign ( unpublished data ) . however , dc - sign staining was not detected on phagosomes that had detached from the plasma membrane , indicating that it was excluded from the vacuoles very soon after phagocytosis ( fig . these data indicate that dc - sign is present in m. tuberculosis vacuoles during the early steps of bacterial uptake , and is then rapidly expelled from the phagosome ( fig . 2 b ) , possibly as a result of recycling to the cell plasma membrane . we also examined whether m. tuberculosis infection could modify dc - sign expression at the surface of infected cells . ( a ) the two top panels shows cells representative of early phagocytosis events ; dc - sign was detected both at the cell surface and in intracellular vesicles , but due to the strong surface staining , the red signal had to be reduced . ( b ) kinetics of dc - sign colocalization with m. tuberculosis ( m. tb ) : a minimum of 100 bacteria were scored at each time point . extracellular bacteria that were found attached to the cells ( early time points , 15 and 30 min ) were scored as colocalizing with dc - sign . it was then important to determine whether m. tuberculosis could interact with dc - sign cells in vivo . like mddcs , ldcs expressed surface dc - sign , cr3 , and mr ( fig . , activated macrophages ) , it is tempting to suggest that m. tuberculosis may encounter and interact with dc - sign dcs during the natural course of infection . to further investigate dc - sign possible involvement in the interactions of m. tuberculosis with dcs in vivo , we reasoned that if dc - sign dcs take up m. tuberculosis in the lungs , and since dc - sign expression is only slightly reduced by infection - associated maturation of the cells , then we should be able to detect mycobacteria - derived material in dc - sign dcs in lns from patients with tuberculosis . ln paraffin - embedded sections from seven patients were stained with anti - mycobacterial and anti dc - sign mabs ( fig . as reported ( 15 , 28 ) , dc - sign was detected mostly in intergerminal t cell zones and not in germinal centers . in addition , dc - sign cells were located in granulomatous structures , and were cd14 and hla - dr ( fig . in most cases ( 80100% ) , mycobacteria - specific signal was detected within dc - sign cells ( fig . these findings indicate that m. tuberculosis interacts with dc - sign in vivo and that dc - sign cells , possibly dcs , may carry mycobacteria or mycobacteria - derived material from the lungs to the lns during their maturation process . dc - sign expression on lung dcs ( ldcs ) and in lymph nodes ( lns ) . ( a ) ldcs are hla - dr and express dc - sign , cr3 , and mr . surface expression of hla - dr , dc - sign , cr3/cd11b , and mr on ldcs from a noninfected patient was assessed by flow cytometry using the appropriate cytochrome - conjugated mabs . left panel , cd3 ( blue ) and cd20 ( red ) ; middle panel , dc - sign ( blue ) and cd14 ( red ) ; right panel , dc - sign ( blue ) and hla - dr ( red ) . ( c ) localization of m. tuberculosis derived antigens in dc - sign cells in lns from two patients with tuberculosis . dc - sign ( blue ) and m. tuberculosis ( red ) were immunodetected both in granulomas ( g ; left and right panels ) and in nongranulomatous regions , including subcapsular sinuses ( scs ; middle panel ) . altogether , our results demonstrate that dc - sign / cd209 is the predominant m. tuberculosis receptor on human dcs , whereas the mycobacterial m receptors , cr3 and mr , appear to play a minor role , if any , in this binding . affinity for dc - sign seems to be fairly restrictive among bacteria , as neither gram l. monocytogenes nor gram s. typhimurium species bound to the lectin . unique characteristics of the mycobacterial cell wall , which is the most complex of all bacterial cell surfaces ( 23 ) , might thus account for the affinity for dc - sign . our finding that lam , like mannan , which contain common mannosyl motifs , can block dc - sign mediated attachment of m. tuberculosis to dcs and to p4-dc cells is consistent with the high affinity of the lectin for mannose - rich molecules ( 16 ) , and suggests that lam may be one of its major mycobacterial ligands . ligation of dc - sign by mycobacteria is likely to have important effects on the immunological and pathological events associated with m. tuberculosis infection . differential receptor usage by m. tuberculosis on dcs and ms may account for the different survival ability and trafficking patterns of mycobacteria in the two cell types , which is still a matter of debate ( 7 , 9 , 10 ) . dc - sign has been detected on alveolar ms ( 28 , 29 ) , that constitute the privileged cell targets of m. tuberculosis during the early steps of infection . it will also be of great interest to investigate what type of pro- or antiinflammatory cytokines are induced or repressed upon dc - sign ligation by mycobacteria ( 30 ) , as compared with ligation of other signal transducers , such as the tlrs ( 14 ) . the intracellular trafficking pattern of dc - sign in m. tuberculosis - infected dcs suggests that dc - sign may carry mycobacterial glycolipids from the bacterial vacuole to the cell plasma membrane and/or to various subcellular compartments , where glycolipids could be loaded onto cd1 molecules for presentation to cd1-restricted lymphocytes ( 32 ) . in the lungs , submucosal and interstitial ldcs are thought to play a key role in immune surveillance of the respiratory tract ( 5 ) . in particular , interactions of dcs present in the alveolar septal walls with m. tuberculosis could be crucial for initiating an efficient anti - mycobacterial immune response . our finding that ldcs express dc - sign suggests that dc - sign is likely to interact with m. tuberculosis in vivo . this is strengthened by detection of mycobacteria - derived material in dc - sign dcs in lns from patients with tuberculosis . dc - sign expressing cells may carry either intracellular or surface - attached m. tuberculosis during their migration from the site of infection to the lns , and could thus constitute a mycobacterial reservoir . this has also been suggested for hiv ( 17 ) , and might account for several pathological and immunological aspects of m. tuberculosis infection , e.g.

the corpus callosum ( cc ) connects the cerebral hemispheres and provides for interhemispheric integration and transfer of information . ever since electrophysiological recording from callosal fibers showed somatosensory receptive fields in the anterior portion of the cat commissure [ 1 , 2 ] and visual inputs to the splenium [ 3 , 4 ] , it was hypothesized that the cc was endowed with a topographical organization . subsequent electrophysiological and neuroanatomical findings [ 6 , 7 ] obtained from nonhuman primates after selective cortical ablation or tracing injections , plus a vast body of data ranging from postmortem investigations to studies of patients with cc lesions or callosal resection ( split - brain subjects ; ; see [ 1012 ] for a review ) , lent further support to the notion . such organization seems to result in modality - specific regions , where the anterior callosal fibers interconnecting the frontal lobes transfer motor information and posterior fibers , which connect the parietal , temporal , and occipital lobes bilaterally , are responsible for the integration of somatosensory ( posterior midbody ) , auditory ( isthmus ) , and visual ( splenium ) information . . functional magnetic resonance imaging ( fmri ) investigations of split - brain patients by our group [ 12 , 14 , 15 ] provided evidence that interhemispheric transfer in the tactile modality is likely mediated by fibers running through the posterior part of the callosal body , thus confirming that the posterior midbody is the tactual channel . a recent study of nonepileptic patients with transection of different portions of the anterior cc performed to remove cysts provided further confirmation demonstrating that the middle part of the genu is involved in motor coordination and the anterior portion of the body in the transfer of simple somesthetic information . investigations of other sensory modalities have shown that the splenium is crucial for the interhemispheric transfer of visual ( see data and literature in ) as well as auditory information [ 18 , 19 ] . the advent of functional mri subsequently made it possible to study the intact brain noninvasively . in recent years an increasing number of fmri studies have described white matter ( wm ) activation in the anterior cc during behavioral tasks involving interhemispheric transfer [ 2023 ] as well as during voluntary swallowing , which is not a specific interhemispheric transfer task . a bold ( blood oxygenation level dependent ) effect was also detected in the posterior callosal region ( isthmus and splenium ) during an interhemispheric transfer task based on the crossed nature of the visual and motor systems which assumed that information must cross the cc to elicit a behavioral response ( crossed condition ; ) . the anterior cc has been seen to be involved in transferring information between prefrontal and premotor regions , and the posterior cc in information transfer between parietal , occipital , and temporal cortices [ 2528 ] . these reports , documenting callosal functional activation during behavioral tasks , prompted a review of our published and unpublished fmri data obtained from studies of the cortical representation of gustatory , tactile , auditory , and visual sensitivity and of motor activation in normal subjects to establish whether the bold effect could be detected in callosal wm and whether the activation foci evoked by a range of simple sensory stimuli and motor tasks were consistent with a topographical organization . indeed activation foci were consistently present in discrete cc regions : anterior ( taste stimuli ) , central ( motor tasks ) , central and posterior ( tactile stimuli ) , isthmus ( auditory stimuli ) and splenium ( visual stimuli ) , demonstrating that the functional topography of the cc can be explored with fmri . very recently we administered the peripheral sensory stimulation protocols previously applied to study normal subjects ( described in ) to patients with partial callosotomy ( polonara et al . , the results were analyzed for evidence of a specific bold effect in the extant callosal portions , further to document the notion of a functional cc map . diffusion tensor imaging ( dti ) data were acquired in control subjects and in patients and analyzed to establish whether cc activation was colocalized with tracts seeded from activated clusters in cortical areas involved by specific sensory stimuli . in this paper , the results obtained in normal subjects and in callosotomy patients are presented together to provide an overview of the functional organization of the cc . the data were collected from 36 normal volunteers ( age range 2251 years , 20 women ; table 1 ) and 6 callosotomy patients ( age age range 2251 years , 2 women ; table 2 ) during studies of gustatory , tactile , auditory , visual , and motor cortical representation . the callosotomy , performed to treat drug - resistant epilepsy , involved the anterior cc in 4 subjects ( l.m . , r.v . , and d.b . ) , the posterior cc in one ( m.c . ) and the central cc in the last patient ( p.f . ; table 2 ) . all subjects ( controls and patients ) gave their informed consent to participate in the study , whose experimental protocol was approved by the local ethics committee . tactile stimulation was applied to 22 normal subjects and to all 6 patients , gustatory stimulation to 13 controls and 4 patients , and visual stimulation to 14 control subjects and 5 patients ; one control subject and 3 patients also received auditory stimulation . all stimuli were presented according to a block - design protocol that alternated periods of rest and of stimulation . taste and touch stimuli were applied to the left or right side in different sessions ; visual stimuli were presented to the left or right visual field ( lvf ; rvf ) in the same session , or to the central visual field ( cvf ) in a separate session . auditory stimuli were presented alternatively to the left and right ear in the same session ( 2 patients ) or bilaterally to both ears ( the other patient and the control subject ) . for all investigations , subjects were placed in a 1.5 tesla ( t ) scanner ( signa excite nv / i cv / i , general electric medical system , milwaukee , wi , usa ) equipped with 50 mt / m gradients , with their head restrained within a circularly polarized head coil . they were instructed to keep their eyes closed and find a comfortable position and relax , avoiding even minimal movement ; their ears were plugged . an identical 4-step experimental procedure was applied in all cases . in the first step , an anatomical three - plane localizer ( 2d spgr , tr 120 ms , te 15 ms , flip angle 70 , fov 23 23 cm , slice thickness 5 mm , matrix 256 256 the second step entailed acquisition of a 3d data set ( ir prep fast spgr ; tr 15.2 ms , te 6.9 ms , ti 500 ms , flip angle 15 , fov 29 29 cm , slice thickness 1 mm , matrix 288 288 , 1 nex , and scan time 8 : 20 min ) . the third involved acquisition of 10 ( 20 in more recent studies ) contiguous 5-mm - thick axial or oblique functional images with a single - shot t2 * -weighted gradient - echo epi sequence ( tr 3000 ms , te 60 ms , flip angle 90 , fov 28 21 cm , matrix 96 64 , 1 nex , scan time 5 : 12 min ) . in the fourth step high - resolution axial ( or oblique ) anatomical images were acquired from 10 selected planes ( 2d spgr , tr 100 ms , te 12 ms , flip angle 70 , fov 28 21 cm , thickness 5 mm , matrix 256 256 , 1 nex , scan time 3 : 17 min for 10 images ) so that functional activation images could be superimposed onto the anatomical landmarks , to show blood vessels which are possible sources of bold signal . in more recent studies one thousand ( or 2000 ) axial or oblique functional images ( 100 per section , 1 image/3 s ) were acquired during the stimulation cycle from the 10 contiguous 5-mm - thick axial sections obtained from the 10 ( or 20 ) previously selected planes . consecutive images from each section were examined in cine mode for head movements ( see for a review ) . the axial planes were orthogonal to both the sagittal and the coronal planes and allowed the identification of the central sulcus , postcentral gyrus , sylvian sulcus , frontal and parietal operculum , calcarine fissure , and primary visual cortex ( vi ) . when dti became available at our institution a fifth step was added . data were acquired from 17 control subjects and all 6 patients to study the connections between activated cortical areas and establish whether cc regions crossed by interhemispheric fibers reconstructed by diffusion tensor tracking ( dtt ) were linked to areas where the bold effect had been observed . for the dti study a series of oblique axial images was obtained using a single shot spin - echo echo - planar sequence with a diffusion - sensitizing gradient . acquisition parameters were tr 6500 ms , te 76.2 ms , matrix 128 128 , fov 26 26 cm , slice thickness 5.0 mm , interslice gap 1.0 mm , nex 2 , and scan time 5 : 51 min . tastants were 1 m nacl for the salty stimulus ( 10 subjects ) , 10% sucrose for the sweet stimulus ( 6 subjects ) , and 0.002 m quinine chloride for the bitter stimulus ( 3 subjects ) . a neutral stimulus ( distilled water ) was also applied in 7 subjects , twice or 4 times as appropriate . there were two taste stimulation protocols , each lasting 5 min : each sapid stimulus was presented twice under protocol 1 ( 60 s rest , 30 s stimulation , 90 s rest , 30-s stimulation , 90 s rest ) and 4 times under protocol 2 ( 30 s rest , 15 s stimulation , 45 s rest , 15 s stimulation , 45 s rest ) . in the 4 patients receiving gustatory stimuli 1 m nacl solution was applied to each side of the tongue in different scans , according to protocol 2 . seven subjects were scanned while performing motor tasks ( two block - design protocols envisaging 10 alternate 30-s periods of rest and stimulation ; table 1 ) that consisted of alternate flexion and distension of the fingers of one hand ( motor protocol 1 ) or in simultaneous haptic manipulation of an object held in both hands ( motor protocol 2 ) . stimuli were applied to the right or left body side , or to both sides ( table 1 ) by rubbing the skin with a soft cotton pad ( trunk ) , a soft sponge ( face ) , or a rough sponge ( hand , foot , and limbs ) at a frequency of 1 hz ( table 1 ) . touch protocol 1 was used to stimulate a single body region and was consisted of ten 30-s alternating periods of rest and stimulation . touch protocol 2 envisaged stimulation of two body regions in the same scanning session and included 20 alternating periods of rest and stimulation , each lasting 15 s ; during stimulation periods , one of the two body regions was stimulated alternately ( e.g. , hand and foot ) , enabling a larger number of regions to be stimulated . , m.s . , and lu.a . ) also participated in a study of taste sensitivity and received tactile stimulation of the tongue for comparison with taste stimulation . in this case , tactile stimuli were applied with the same timing of taste protocol 2 . as regards patients , tactile stimuli were applied to the left and/or right hand ( 6 patients ; table 2 ) by rubbing the palm with a rough sponge at a frequency of 1 hz ( table 1 ) using a 5 min protocol consisting of ten 30-s alternating periods of rest and stimulation . auditory stimulation involved 1 control and 3 patients ( tables 1 and 2 ) . for 2 patients the stimuli were pieces of classical music played alternately to the left ( l ) or right ( r ) ear , according to a block - design experimental paradigm consisting of 20 alternating periods of rest ( 15 s ) and stimulation ( 15 s ) . for the third patient and the control subject auditory stimuli were italian words spoken to both ears at the same time , according to a block - design experimental paradigm consisting of 10 alternating periods of rest ( 30 s ) and stimulation ( 30 s ) . stimuli were administered by means of fmri - compatible headphones ( resonance technology inc . , stimuli were generated using an in - house - developed software and projected into an fmri - compatible goggle system ( resonance technology inc . , the block - design experimental paradigm consisted of 20 alternating periods of rest ( 15 s ) and stimulation ( 15 s ) . a black and white checkerboard ( amplitude : 6 ; virtual cartesian distance from viewer 's eyes : 75 cm ) was flashed ( 1 hz ) to the cvf ( 9 subjects ) or to the lateral vf ( 5 subjects ) , alternately to the left ( l ) or right ( r ) periphery ( polar distance from the center of the display : 12 ) . in 2 subjects the same types of stimuli were also presented to the 5 callosotomy patients : to the cvf in 3 and to the lateral vf in 5 patients ( table 2 ) , alternately to the lvf and rvf periphery . all subjects were asked to fixate a cross in the centre of the display during functional image acquisition ; eye movements were monitored with an internal camera . after each experimental session images were transferred to a unix workstation ( general electric advantage windows 4.2 ) and then to a personal computer . data were analyzed with the brainvoyager qx ( bv qx ) software ( brain innovation , maastricht , the netherlands ) . the first two images of each functional series were discarded to take into account signal intensity variations due to progressive saturation . slice scan time correction and head motion correction were applied to the functional data of each subject . 3d anatomical data were preprocessed with intensity inhomogeneity correction and spatial transformation , and then transformed to talairach standard space . coregistration of functional and anatomical data resulted in a normalized 4d volume time course data , which allowed the transformation of functional time series into talairach space and identification of the position of activated areas using the talairach coordinate system . statistical analysis was performed for each subject using the general linear model ( glm ) . this model aims to predict the variation of a dependent variable ( the fmri time course ) in terms of linear combination . the predictor time course was convolved with a standard hemodynamic response function ( hrf ) to account for the hemodynamic delay . activation foci observed in callosal wm were studied by selecting regions of interest ( rois ) in the cc portions harboring activation foci . when the signal was significantly greater than the baseline ( p < 0.05 ) and it correlated temporally with the stimulation pattern , activation was assumed to be evoked by the peripheral stimulus and the focus was included in the counts . only foci whose voxels were all superimposed on the cc were counted as callosal . however , if foci were detected both in the anterior and posterior cc , data were assigned to different data groups . anterior and those whose y coordinate was equal to or less than 10 were considered posterior . the value of 10 was equally distant from the most anterior and the most posterior foci . averaged time courses were calculated within each roi to show the mean bold signal change due to the stimulus . the wilcoxon test was applied to the y coordinates of the callosal wm foci evoked by the different tasks . for dti data analysis , images were transferred to the unix workstation for postprocessing with functool 3.1.22 ( general electric medical systems , milwaukee , wi , usa ) . diffusion eigenvectors and eigenvalues calculated from the diffusion tensor represented the main direction of diffusion and the associated diffusivity . the fibertrak option allows functool to create 2d color orientation maps , 2d color eigenvector maps , and 3d tractography maps . the 3d volume viewer enables areas of high fa to be displayed as 3d images . for tractography , cc fiber tracts were reconstructed starting from voxels with an fa > 0.18 in the different axial planes , according to the main vector , up to those with an fa < 0.18 , or up to a maximum step size of 160 m ( the length threshold of the fibers generated by the tracking ) . rois were selected in brain regions including activated cortical areas to track the nerve fibers arising from individual rois . rois selected in different cortical regions had different sizes ( gustatory cortex , 330 mm ; somatosensory cortex , 160 mm ; visual cortex , 105 mm ) due to the different size of the activation foci . as a control , rois measuring 2650 mm were placed in cc portions displaying a bold effect , to establish which cortical areas were connected by the fibers coursing through them and whether they were the same areas activated by peripheral sensory stimulation . all rois were defined manually on color - coded maps of the main diffusion directions . taste stimulation ( 13 normal subjects and 4 callosotomy patients ) unilateral taste stimulation applied to each side of the tongue in separate scanning sessions ( table 1 ) induced bilateral activation of the primary gustatory area ( gi ) in the frontoparietal operculum in all 13 subjects . the same stimuli also evoked one or two callosal foci that were found most frequently in the anterior cc ( figures 1(a)1(d ) ) ; foci were sometimes detected also in the posterior cc ( figure 1(c)1 ) . both anterior and posterior foci were seen after sweet stimulation in one subject and after bitter stimulation in two ( table 3 ) . the anteroposterior talairach coordinate ( y ) of the posterior foci ranged from 31 to 40 , and that of the anterior foci from 9 to 23 . the mean y values of the anterior foci activated by the sweet stimulus appeared to be significantly more anterior than those evoked by salty and bitter tastants ( table 3 ) . in these subjects interhemispheric fibers crossing through the anterior cc were visible ( figure 1(e ) ) , as detailed below . also in the 4 patients a salty stimulus applied to each side of the tongue in separate scanning sessions ( table 2 ) induced bilateral activation of area gi . in the 2 subjects ) the same stimulus also evoked a callosal focus in the anterior cc ( figure 2(a)1 ) . the mean y coordinate of the callosal foci was 22.5 ( table 3 ; p < 0.05 ) . in these 2 patients interhemispheric fibers crossing through the spared anterior cc were visible ( figure 2(a)2 ) , as detailed below . in the other 2 patients and in p.f . an activation focus was also noted in the posterior cc ( mean y coordinate : 35.33 ; table 3 ; figures 2(b ) and 2(c ) ) . the coordinates of both anterior and posterior foci evoked by taste stimulation were similar to those observed in control subjects . motor activation ( 7 normal subjects ) alternate flexion and distension of the fingers of a hand and object manipulation with both hands evoked foci in cortical motor areas and in the middle portion of the cc ( figure 3(a)2)where anterior foci evoked by tactile hand and foot stimulation were also observed in all 7 subjects ( table 3 ) . the values of the y coordinate ( 1 to 23 ) overlapped with those of the anterior somatosensory foci . the interhemispheric fibers linking the activated motor cortical areas crossed through the middle part of the cc ( figures 3(b)1 and 3(b)2 ) at sites harboring the bold foci . tactile stimulation ( 22 normal subjects and 6 callosotomy patients ) unilateral tactile stimuli applied to left or right body regions ( table 1 ) activated the primary ( si ; [ 35 , 36 ] ) and secondary ( sii ; ) somatosensory areas in the parietal cortex in all 22 subjects . they also evoked callosal foci , more frequently in the middle cc and in the posterior callosal body ( figure 4 ) . stimuli applied to the hand and foot evoked a bold focus in the middle of the cc ( figure 4(a ) ) , while stimulation of proximal body regions ( trunk , shoulder , and thigh ) activated fibers in more posterior regions ( figure 4(b ) ) . stimulation of the arm , foot , and leg also activated posterior fibers ( figure 4(c ) ) that were not , however , clearly distinguishable from those activated by the stimulation of proximal body areas . in some cases , multiple foci were elicited by stimulation of a single region ( e.g. , hand , 4 subjects ; tongue , 1 subject ; foot , 1 subject ; table 3 ) . the y talairach coordinate ranged from 8 to 9 for anterior foci and from 21 to 36 for posterior foci . interhemispheric fibers crossing through the cc ( figure 4(a)3 ) were also detected at sites harboring cc foci . unilateral tactile stimuli applied to the left or right hand ( table 2 ) activated both si and sii in the contralateral parietal cortex in all 6 patients , as in control subjects . in addition , the 3 patients whose pcb was spared exhibited different activation patterns that included bilateral activation of the posterior parietal ( pp ) cortex and sii ( l.m . ; figure 5(a)1 ) ; bilateral pp cortex activation ( o.t . ) ; bilateral sii activation ( p.f . ; tactile stimuli also evoked callosal foci , more frequently in the pcb , in patients in whom the central cc region was spared ( l.m . the y coordinate ranged from 19 to 26 ( see mean values in table 2 ) , very similar to that observed in control subjects ( see above ; ) . in the patients with anterior callosotomy interhemispheric fibers crossing through the cc ( figures 5(a)3 and 5(b)3 ) o.t . exhibited additional foci , one in the anterior callosal midbody ( not shown ) and another in the splenium , while l.m . and p.f . showed a focus in the splenium ( figure 5(c ) ; table 4 ) . auditory stimulation ( 1 normal subject and 3 callosotomy patients ) monaural ( music ) or binaural ( words ) stimuli induced activation in the primary auditory cortex and in the posterior cc ( isthmus / splenium ) in all participants ( figure 6 ) . the values of the y coordinate were 36 in the control subject , and ranged from 35 to 38 in patients . mean control values are reported in table 3 , and mean patient values in table 4 . interhemispheric fibers connecting the activated auditory areas crossed through the isthmus in the control subject and the spared splenium in patients ( figure 6 ) at the same sites harboring the bold foci . visual stimulation ( 14 normal subjects and 5 callosotomy patients ) unilateral visual stimuli , presented to the lvf , rvf , or cvf ( table 1 ) , elicited activation foci in the primary visual cortex and in the splenium in all 14 subjects ( figure 7(a ) for cvfs , figure 7(b ) for peripheral vf ) . foci evoked in the splenium by cvf stimulation seemed to be slightly more anterior than those evoked by lvf stimulation , but the difference was not significant . the interhemispheric fibers linking the activated visual areas crossed through the splenium ( figures 7(a)3 and 7(b)3 ) at sites harboring the bold foci . also in patients unilateral visual stimuli presented to the lvf , rvf , or cvf ( table 2 ) induced activation foci in vi in all 5 patients ( figure 8(a ) for cvf stimulation and figures 8(b ) and 8(c ) for peripheral vf stimulation ) . vi activation was usually bilateral after cvf stimulation , and contralateral after peripheral vf stimulation . in patients whose splenium was spared an activation focus was seen in this region ( figure 8 , central column ) . the value of the y coordinate ranged from 32 to 38 ( mean values in table 3 ) , consistent with control values ( mean 35 ; ) . the interhemispheric fibers linking the activated visual areas crossed through the extant splenium ( figure 8 , right column ) at sites harboring the bold foci . multiple foci were quite often elicited by peripheral stimulation in control subjects ( table 3 ) , namely , in 4 subjects receiving tactile hand stimulation , in one receiving tactile stimulation of the tongue , in one receiving foot stimulation , in 2 subjects receiving bitter taste stimulation , and in one subject receiving the sweet stimulus . subjects showing double foci for tactile and sweet stimulation of the tongue also displayed multiple activations after tactile stimulation of the hand . these multiple foci are shown in table 3 and figures 1 ( taste ) and 5 ( touch ) . two foci could also be observed in patients , particularly after taste and touch stimulation ; one usually lays at the site corresponding to the sensory stimulus applied ( i.e. , in the anterior cc after taste stimulation , in the mid - posterior cc after touch stimulation ) , whereas the other was found in the splenium ( figures 2(b ) , 2(c ) , and 5(c ) ) . statistical analysis with wilcoxon 's test showed that the y coordinates of the foci evoked by visual stimulation were significantly different ( p 0.01 ) from those elicited by all the other modalities , sensory as well as motor . the foci evoked by tactile hand stimulation were different ( p = 0.01 ) from those evoked by taste activation . hand motor stimuli evoked foci at cc sites that were significantly different from those evoked by hand touch stimuli in the posterior cc , but not from those elicited in the anterior cc . the anterior and posterior gustatory foci , if considered separately , were both significantly different from those elicited by cc motor activation ( p 0.05 ) . data for dti and dtt processing were obtained from 17 control subjects and from all 6 patients . fa values in the cc regions crossed by fibers interconnecting activated areas were similar to those reported in previous studies [ 3840 ] and were not significantly different among callosal regions . in particular , fa was 0.72 in the genu of control subjects and 0.68 in the spared genu of patients ; 0.69 in the pcb of normal subjects and 0.61 in patients ' pcb ; 0.68 in the splenium of controls and 0.73 in the extant splenium of patients [ 39 , 40 ] . the fa difference between the same callosal regions in controls and patients was never significant . callosal fibers arising from individual cortical rois examination of the fibers arising from a cortical roi selected in area gi showed that they crossed through the anterior part of the cc ( genu ) , that is , the region activated by taste stimuli , both in controls ( figure 1(e ) ) and in patients with intact genu ( m.c . and p.f . no fibers from area gi were seen to cross through the cc at the level of the splenium . fibers from an roi selected in the mediolateral frontal cortex activated by motor stimulation of the hand crossed through the cc slightly more posteriorly ( figure 3(b ) ) . fibers from an roi selected in the mediolateral parietal cortex activated by tactile stimulation of the hand crossed through the cc even more posterior in controls ( figure 4(a)3 ) and in patients with this callosal region preserved ( l.m . analysis of an roi selected in the primary auditory area showed fibers crossing through the isthmus / splenium , at a site activated by auditory stimulation , both in the unique control subject and in patients ( figure 6 ) . finally , analysis of an roi selected in the activated region of vi showed fibers crossing through the splenium , at a site activated by visual stimuli , both in controls ( figure 7 ) and in patients ( figure 8) . when rois were selected in the genu , anterior body , pcb , isthmus , and splenium harboring activation foci , the callosal fibers were seen to interconnect , respectively , the frontoparietal opercula and prerolandic , parietal , temporal , and occipital regions , which harbored foci activated by taste stimulation of the tongue , hand motor tasks , tactile stimulation of the hand and trunk , and visual stimulation . these findings regarded both control subjects and patients . even though activation foci were seen in the splenium of some patients after taste or touch stimulation , no interhemispheric fibers from gustatory or somatosensory cortical areas were seen to cross through the cc at this level ; similarly , no callosal fibers coursed from the foci in the splenium to cortical regions other than occipital or temporal areas . finally , no correlation was observed between the fa values of the different callosal regions and the occurrence of the activation foci . in this paper we review our data showing the callosal activation evoked by a variety of peripheral sensory stimuli in a group of normal subjects ( controls ) and in 6 partial callosotomy patients . altogether these findings show that ( i ) a callosal bold effect can be evoked by peripheral sensory stimulation and by motor tasks other than interhemispheric transfer tasks , ( ii ) cc activation foci occupy consistent locations that are related to the sensory or motor stimulus applied , and ( iii ) the topographical map of the cc thus obtained is in line with human postmortem data , with the investigations of patients with cc injury or surgical resection [ 9 , 14 , 15 , 4246 ] see ( [ 10 , 11 ] , for a review ) , and with electrophysiological recording and neuroanatomical animal studies [ 13 , 57 ] . to date , a callosal bold effect has been described in relation to visual and motor stimulation [ 2024 , 47 ] and , recently , in response to simple sensory stimuli . energy - dependent processes also take place in wm , since they are often axon conduction mediated ( at the level of the nodes of ranvier ) by adenosine triphosphate - dependent na - k ion pumps that restore ionic gradients across the neuron membrane after action potential propagation [ 48 , 49 ] . moreover , it has been shown that the inhibition of voltage - dependent na channels suppresses the fmri response to forepaw somatosensory activation . in addition , according to recent evidence , spiking activity is also correlated with fmri activation [ 5052 ] . it has tentatively been explained with the involvement of astrocytes [ 53 , 54 ] acting on vessel dilation to meet the greater metabolic demand from the heightened activity , which in turn results in increased neurotransmitter release in the extracellular environment , in raised k levels in the extracellular medium due to augmented neural activity , and/or in increased cytoplasmic ca [ 53 , 55 ] . both astrocytes and capillaries are found in the commissure , and since callosal axon fibers need energy to conduct action potentials , the mechanism is likely active in cc fibers too . according to another purely physical hypothesis the heat generated by the increased axonal metabolic activity would be sufficient per se to produce callosal microvessel vasodilation ( lebihan , 2009 , personal communication ) . the foci evoked by different types of sensory stimuli and by motor stimulation occupied different locations along the commissure . apart from some exceptions that will be addressed below , the topographical distribution of the callosal activation foci was consistent with the functional organization of the commissure as emerging from anatomical and neuropsychological studies . in our subjects foci induced by taste stimuli lay mainly in the anterior cc , that is , the genu and anterior body , which also harbored foci elicited by tactile stimulation of the tongue . the overlap is likely due to the complex nature of taste , which includes a tactile component . the foci evoked by hand motor stimulation and by tactile stimuli of different body regions were seen in the anterior and posterior body and in the isthmus , respectively . in particular , proximal body representations seem to be connected by callosal fibers running through the posterior isthmus and anterior splenium ( figure 9 ) . a dorsoventral topographical organization could not be recognized . as expected , foci evoked by auditory and visual stimuli were found in the callosal region corresponding to the isthmus / splenium and splenium , respectively . callosal foci activated by central or peripheral vf stimulation lay at sites whose coordinates were not significantly different . unlike previous investigations of the callosal bold effect , the sensory stimuli and motor tasks employed in our studies did not require interhemispheric transfer . cc activation was nonetheless observed , suggesting that all information reaching a cortical area is likely transferred to the opposite hemisphere and used to build a continuous representation of the external world . this is the case of a posterior focus , which was often evoked by taste stimuli in some controls and patients in addition to the anterior foci seen in all subjects . similarly , tactile stimulation of the hand and foot elicited a focus in the anterior part of the posterior body in the cc regions activated by the hand motor tasks ; in addition , stimulation of the hand produced a focus in the splenium in some control subjects and in some patients . the first explanation that comes to mind , that which the available data can not , however , rule out , is that the ectopic however , since their presence does not appear to be accidental , they are in fact probably related to the activity evoked by peripheral stimuli . thus , the anterior focus observed after tactile hand and foot stimulation could be associated with motor cortex activation , which is often induced by tactile stimulation of these regions ; the foci evoked in the splenium by taste and hand tactile stimuli might result from concurrent activation of high - order association areas , like the posterior parietal cortex ( ppc ) in the case of tactile stimuli , and/or temporal areas for both taste and tactile stimuli ; the fact the ppc and temporal areas are connected by fibers crossing though the splenium [ 58 , 59 ] may explain those findings . the functional callosal topography sketched by the foci described herein , apart from the exception mentioned above , is in line with the topographical organization of cc fibers described in previous anatomical studies [ 25 , 5861 ] . the fibers connecting the prefrontal cortical areas were seen to cross through the anterior part of the cc , those connecting the premotor and motor cortical areas crossed at the level of the central callosal body ( see also ) , those connecting the parietal cortical areas crossed through the posterior callosal body , and the fibers linking the occipital and temporal areas crossed at the level of the splenium [ 38 , 63 ] . in all our tests and subjects , the fibers arising from the cortical areas activated by each stimulus type crossed through the cc in a region corresponding to the one harboring the relevant callosal focus / i . the coincidence of callosal bold activation and crossing point of the dtt - reconstructed interhemispheric bundles ( figure 9 ) strongly suggests that the cc foci evoked by sensory stimulation and motor tasks may be due to the activation of the fibers connecting the activated areas to the corresponding contralateral areas and carrying specific information . in all control subjects and in patients the presence of callosal foci in the spared callosal regions broadly corresponded with bilateral activation of the cortical areas reached by specific unilateral peripheral sensory input . although an exact correspondence can not at present be demonstrated due to technical limitations ( i.e. , bold and dti data can not be coregistered and shown in the same image ) , we are convinced that it does exist , at least based on the consistency of the observations . the present outline of cc topography is based on the study of fibers arising from different sensory areas activated by relevant peripheral stimuli . with regard to taste we analyzed different submodalities ( tastants ) ; in the touch modality we studied discrete body representations , and in the visual modality we explored the activation evoked by peripheral and central visual field stimulation . with regard to auditory stimulation our data are still confined to a small number of subjects and stimuli , while motor activation was only analyzed in controls . we decided to include the data from all , even small samples , because together with those from larger samples they may help provide a more comprehensive picture of callosal functional topography . the main difference between previous human after mortem and dti studies and monkey neuroanatomical tracing investigations is that the topographical map outlined by our data is less precise . in brief , we have two sets of data one of which agrees with several studies from other laboratories ; the other set is ostensibly in contrast with previous knowledge . often , multiple foci ( usually two ) were activated by the stimulation of a single body region . this happened for tactile hand stimulation and sometimes also for taste and tactile foot stimulation . the multiple foci contrast with the single callosal sites identified in previous dtt studies [ 5862 ] . the discrepancy might be due to the fact that the natural peripheral stimulation applied in our fmri studies may have involved a larger number of areas than primary sensory and/or motor cortical ones , whereas dti and monkey neuroanatomical studies address the trajectories of fibers arising in circumscribed cortical areas . in particular , tactile stimulation of the hand or foot evokes activation in the primary motor cortex , which could anticipate an action of the limb receiving the tactile stimulus , explaining a subsequent callosal activation ( as in interhemispheric transmission aimed at motor output coordination ) . splenium activation could also account for the good performance of callosotomy patients in whom only the splenium survives in tasks involving interhemispheric transfer of tactile information , a commonly reported observation [ 12 , 64 , 65 ] . it may be therefore hypothesized that the splenium is involved in the transfer of touch information , providing for a degree of plasticity in patients with callosal partial resection . further evidence from neuropsychological studies of taste sensitivity in callosotomy patients [ 66 , 67 ] points to a role for the splenium in transferring taste information . the splenial callosal foci elicited by unilateral taste stimulation in our studies lend support to this notion . an involvement of the splenium in the transfer of information other than visual sensory data could be due to the prominent role of the visual representation of the external environment , typical of humans , where sensory experience is usually associated with a visual component . previous papers describing callosal activation reported a bold effect in the anterior portion of the cc ( see data and literature in ) . fibers interconnecting prefrontal and motor cortical areas course through this region and are likely involved in behavioral visuomotor interhemispheric transfer tasks , like those evoking callosal activation in those studies . anterior cc activation has been hypothesized to be related to the involvement of fibers carrying interhemispheric information between these cortical areas . the data reviewed in the present study were obtained from simple sensory stimulation that did not involve a motor output , and from simple motor tasks . this may explain the wm activation seen in regions where sensory or motor fibers cross through the commissure . in the present paper we have reported the data collected in normal subjects ( controls ) and in some callosotomy patients during fmri studies on peripheral sensory stimulation . it has been shown that a bold effect can be evoked in the corpus callosum by peripheral sensory stimulation and by motor tasks and that cc activation foci occupy consistent locations related to the sensory or motor stimulus applied ; it has thus emerged that a functional topographical map of the cc is in line with previous investigations . further studies , however , combining fmri and dti , are needed to provide a better understanding of the topography of callosal activation and to confirm or exclude its correspondence with fiber crossing . it would be also interesting to clarify if callosal fibres carrying information about different submodality or different regions of the sensory periphery also follow any particular organization . as pointed out by pandya and seltzer the understanding of the precise topography for commissural fibers allows one to assess the nature of functional deficits following selective commissural lesions and to predict the localization of functions within cerebral commissural systems .

the concept of a topographical map of the corpus callosum ( cc ) has emerged from human lesion studies and from electrophysiological and anatomical tracing investigations in other mammals . over the last few years a rising number of researchers have been reporting functional magnetic resonance imaging ( fmri ) activation in white matter , particularly the cc . in this study the scope for describing cc topography with fmri was explored by evoking activation through simple sensory stimulation and motor tasks . we reviewed our published and unpublished fmri and diffusion tensor imaging data on the cortical representation of tactile , gustatory , auditory , and visual sensitivity and of motor activation , obtained in 36 normal volunteers and in 6 patients with partial callosotomy . activation foci were consistently detected in discrete cc regions : anterior ( taste stimuli ) , central ( motor tasks ) , central and posterior ( tactile stimuli ) , and splenium ( auditory and visual stimuli ) . reconstruction of callosal fibers connecting activated primary gustatory , motor , somatosensory , auditory , and visual cortices by diffusion tensor tracking showed bundles crossing , respectively , through the genu , anterior and posterior body , and splenium , at sites harboring fmri foci . these data confirm that the cc commissure has a topographical organization and demonstrate that its functional topography can be explored with fmri .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

the corpus callosum ( cc ) connects the cerebral hemispheres and provides for interhemispheric integration and transfer of information . ever since electrophysiological recording from callosal fibers showed somatosensory receptive fields in the anterior portion of the cat commissure [ 1 , 2 ] and visual inputs to the splenium [ 3 , 4 ] , it was hypothesized that the cc was endowed with a topographical organization . subsequent electrophysiological and neuroanatomical findings [ 6 , 7 ] obtained from nonhuman primates after selective cortical ablation or tracing injections , plus a vast body of data ranging from postmortem investigations to studies of patients with cc lesions or callosal resection ( split - brain subjects ; ; see [ 1012 ] for a review ) , lent further support to the notion . such organization seems to result in modality - specific regions , where the anterior callosal fibers interconnecting the frontal lobes transfer motor information and posterior fibers , which connect the parietal , temporal , and occipital lobes bilaterally , are responsible for the integration of somatosensory ( posterior midbody ) , auditory ( isthmus ) , and visual ( splenium ) information . functional magnetic resonance imaging ( fmri ) investigations of split - brain patients by our group [ 12 , 14 , 15 ] provided evidence that interhemispheric transfer in the tactile modality is likely mediated by fibers running through the posterior part of the callosal body , thus confirming that the posterior midbody is the tactual channel . a recent study of nonepileptic patients with transection of different portions of the anterior cc performed to remove cysts provided further confirmation demonstrating that the middle part of the genu is involved in motor coordination and the anterior portion of the body in the transfer of simple somesthetic information . in recent years an increasing number of fmri studies have described white matter ( wm ) activation in the anterior cc during behavioral tasks involving interhemispheric transfer [ 2023 ] as well as during voluntary swallowing , which is not a specific interhemispheric transfer task . a bold ( blood oxygenation level dependent ) effect was also detected in the posterior callosal region ( isthmus and splenium ) during an interhemispheric transfer task based on the crossed nature of the visual and motor systems which assumed that information must cross the cc to elicit a behavioral response ( crossed condition ; ) . the anterior cc has been seen to be involved in transferring information between prefrontal and premotor regions , and the posterior cc in information transfer between parietal , occipital , and temporal cortices [ 2528 ] . these reports , documenting callosal functional activation during behavioral tasks , prompted a review of our published and unpublished fmri data obtained from studies of the cortical representation of gustatory , tactile , auditory , and visual sensitivity and of motor activation in normal subjects to establish whether the bold effect could be detected in callosal wm and whether the activation foci evoked by a range of simple sensory stimuli and motor tasks were consistent with a topographical organization . indeed activation foci were consistently present in discrete cc regions : anterior ( taste stimuli ) , central ( motor tasks ) , central and posterior ( tactile stimuli ) , isthmus ( auditory stimuli ) and splenium ( visual stimuli ) , demonstrating that the functional topography of the cc can be explored with fmri . very recently we administered the peripheral sensory stimulation protocols previously applied to study normal subjects ( described in ) to patients with partial callosotomy ( polonara et al . diffusion tensor imaging ( dti ) data were acquired in control subjects and in patients and analyzed to establish whether cc activation was colocalized with tracts seeded from activated clusters in cortical areas involved by specific sensory stimuli . in this paper , the results obtained in normal subjects and in callosotomy patients are presented together to provide an overview of the functional organization of the cc . the data were collected from 36 normal volunteers ( age range 2251 years , 20 women ; table 1 ) and 6 callosotomy patients ( age age range 2251 years , 2 women ; table 2 ) during studies of gustatory , tactile , auditory , visual , and motor cortical representation . tactile stimulation was applied to 22 normal subjects and to all 6 patients , gustatory stimulation to 13 controls and 4 patients , and visual stimulation to 14 control subjects and 5 patients ; one control subject and 3 patients also received auditory stimulation . data were acquired from 17 control subjects and all 6 patients to study the connections between activated cortical areas and establish whether cc regions crossed by interhemispheric fibers reconstructed by diffusion tensor tracking ( dtt ) were linked to areas where the bold effect had been observed . seven subjects were scanned while performing motor tasks ( two block - design protocols envisaging 10 alternate 30-s periods of rest and stimulation ; table 1 ) that consisted of alternate flexion and distension of the fingers of one hand ( motor protocol 1 ) or in simultaneous haptic manipulation of an object held in both hands ( motor protocol 2 ) . stimuli were applied to the right or left body side , or to both sides ( table 1 ) by rubbing the skin with a soft cotton pad ( trunk ) , a soft sponge ( face ) , or a rough sponge ( hand , foot , and limbs ) at a frequency of 1 hz ( table 1 ) . also participated in a study of taste sensitivity and received tactile stimulation of the tongue for comparison with taste stimulation . as regards patients , tactile stimuli were applied to the left and/or right hand ( 6 patients ; table 2 ) by rubbing the palm with a rough sponge at a frequency of 1 hz ( table 1 ) using a 5 min protocol consisting of ten 30-s alternating periods of rest and stimulation . a black and white checkerboard ( amplitude : 6 ; virtual cartesian distance from viewer 's eyes : 75 cm ) was flashed ( 1 hz ) to the cvf ( 9 subjects ) or to the lateral vf ( 5 subjects ) , alternately to the left ( l ) or right ( r ) periphery ( polar distance from the center of the display : 12 ) . activation foci observed in callosal wm were studied by selecting regions of interest ( rois ) in the cc portions harboring activation foci . only foci whose voxels were all superimposed on the cc were counted as callosal . however , if foci were detected both in the anterior and posterior cc , data were assigned to different data groups . both anterior and posterior foci were seen after sweet stimulation in one subject and after bitter stimulation in two ( table 3 ) . the anteroposterior talairach coordinate ( y ) of the posterior foci ranged from 31 to 40 , and that of the anterior foci from 9 to 23 . in these subjects interhemispheric fibers crossing through the anterior cc were visible ( figure 1(e ) ) , as detailed below . in these 2 patients interhemispheric fibers crossing through the spared anterior cc were visible ( figure 2(a)2 ) , as detailed below . the coordinates of both anterior and posterior foci evoked by taste stimulation were similar to those observed in control subjects . motor activation ( 7 normal subjects ) alternate flexion and distension of the fingers of a hand and object manipulation with both hands evoked foci in cortical motor areas and in the middle portion of the cc ( figure 3(a)2)where anterior foci evoked by tactile hand and foot stimulation were also observed in all 7 subjects ( table 3 ) . the interhemispheric fibers linking the activated motor cortical areas crossed through the middle part of the cc ( figures 3(b)1 and 3(b)2 ) at sites harboring the bold foci . stimuli applied to the hand and foot evoked a bold focus in the middle of the cc ( figure 4(a ) ) , while stimulation of proximal body regions ( trunk , shoulder , and thigh ) activated fibers in more posterior regions ( figure 4(b ) ) . stimulation of the arm , foot , and leg also activated posterior fibers ( figure 4(c ) ) that were not , however , clearly distinguishable from those activated by the stimulation of proximal body areas . in some cases , multiple foci were elicited by stimulation of a single region ( e.g. interhemispheric fibers crossing through the cc ( figure 4(a)3 ) were also detected at sites harboring cc foci . in the patients with anterior callosotomy interhemispheric fibers crossing through the cc ( figures 5(a)3 and 5(b)3 ) o.t . auditory stimulation ( 1 normal subject and 3 callosotomy patients ) monaural ( music ) or binaural ( words ) stimuli induced activation in the primary auditory cortex and in the posterior cc ( isthmus / splenium ) in all participants ( figure 6 ) . the values of the y coordinate were 36 in the control subject , and ranged from 35 to 38 in patients . interhemispheric fibers connecting the activated auditory areas crossed through the isthmus in the control subject and the spared splenium in patients ( figure 6 ) at the same sites harboring the bold foci . visual stimulation ( 14 normal subjects and 5 callosotomy patients ) unilateral visual stimuli , presented to the lvf , rvf , or cvf ( table 1 ) , elicited activation foci in the primary visual cortex and in the splenium in all 14 subjects ( figure 7(a ) for cvfs , figure 7(b ) for peripheral vf ) . the interhemispheric fibers linking the activated visual areas crossed through the splenium ( figures 7(a)3 and 7(b)3 ) at sites harboring the bold foci . also in patients unilateral visual stimuli presented to the lvf , rvf , or cvf ( table 2 ) induced activation foci in vi in all 5 patients ( figure 8(a ) for cvf stimulation and figures 8(b ) and 8(c ) for peripheral vf stimulation ) . in patients whose splenium was spared an activation focus was seen in this region ( figure 8 , central column ) . the interhemispheric fibers linking the activated visual areas crossed through the extant splenium ( figure 8 , right column ) at sites harboring the bold foci . multiple foci were quite often elicited by peripheral stimulation in control subjects ( table 3 ) , namely , in 4 subjects receiving tactile hand stimulation , in one receiving tactile stimulation of the tongue , in one receiving foot stimulation , in 2 subjects receiving bitter taste stimulation , and in one subject receiving the sweet stimulus . , in the anterior cc after taste stimulation , in the mid - posterior cc after touch stimulation ) , whereas the other was found in the splenium ( figures 2(b ) , 2(c ) , and 5(c ) ) . statistical analysis with wilcoxon 's test showed that the y coordinates of the foci evoked by visual stimulation were significantly different ( p 0.01 ) from those elicited by all the other modalities , sensory as well as motor . the anterior and posterior gustatory foci , if considered separately , were both significantly different from those elicited by cc motor activation ( p 0.05 ) . fa values in the cc regions crossed by fibers interconnecting activated areas were similar to those reported in previous studies [ 3840 ] and were not significantly different among callosal regions . callosal fibers arising from individual cortical rois examination of the fibers arising from a cortical roi selected in area gi showed that they crossed through the anterior part of the cc ( genu ) , that is , the region activated by taste stimuli , both in controls ( figure 1(e ) ) and in patients with intact genu ( m.c . no fibers from area gi were seen to cross through the cc at the level of the splenium . fibers from an roi selected in the mediolateral frontal cortex activated by motor stimulation of the hand crossed through the cc slightly more posteriorly ( figure 3(b ) ) . fibers from an roi selected in the mediolateral parietal cortex activated by tactile stimulation of the hand crossed through the cc even more posterior in controls ( figure 4(a)3 ) and in patients with this callosal region preserved ( l.m . analysis of an roi selected in the primary auditory area showed fibers crossing through the isthmus / splenium , at a site activated by auditory stimulation , both in the unique control subject and in patients ( figure 6 ) . finally , analysis of an roi selected in the activated region of vi showed fibers crossing through the splenium , at a site activated by visual stimuli , both in controls ( figure 7 ) and in patients ( figure 8) . when rois were selected in the genu , anterior body , pcb , isthmus , and splenium harboring activation foci , the callosal fibers were seen to interconnect , respectively , the frontoparietal opercula and prerolandic , parietal , temporal , and occipital regions , which harbored foci activated by taste stimulation of the tongue , hand motor tasks , tactile stimulation of the hand and trunk , and visual stimulation . even though activation foci were seen in the splenium of some patients after taste or touch stimulation , no interhemispheric fibers from gustatory or somatosensory cortical areas were seen to cross through the cc at this level ; similarly , no callosal fibers coursed from the foci in the splenium to cortical regions other than occipital or temporal areas . in this paper we review our data showing the callosal activation evoked by a variety of peripheral sensory stimuli in a group of normal subjects ( controls ) and in 6 partial callosotomy patients . altogether these findings show that ( i ) a callosal bold effect can be evoked by peripheral sensory stimulation and by motor tasks other than interhemispheric transfer tasks , ( ii ) cc activation foci occupy consistent locations that are related to the sensory or motor stimulus applied , and ( iii ) the topographical map of the cc thus obtained is in line with human postmortem data , with the investigations of patients with cc injury or surgical resection [ 9 , 14 , 15 , 4246 ] see ( [ 10 , 11 ] , for a review ) , and with electrophysiological recording and neuroanatomical animal studies [ 13 , 57 ] . in our subjects foci induced by taste stimuli lay mainly in the anterior cc , that is , the genu and anterior body , which also harbored foci elicited by tactile stimulation of the tongue . the foci evoked by hand motor stimulation and by tactile stimuli of different body regions were seen in the anterior and posterior body and in the isthmus , respectively . in particular , proximal body representations seem to be connected by callosal fibers running through the posterior isthmus and anterior splenium ( figure 9 ) . as expected , foci evoked by auditory and visual stimuli were found in the callosal region corresponding to the isthmus / splenium and splenium , respectively . unlike previous investigations of the callosal bold effect , the sensory stimuli and motor tasks employed in our studies did not require interhemispheric transfer . cc activation was nonetheless observed , suggesting that all information reaching a cortical area is likely transferred to the opposite hemisphere and used to build a continuous representation of the external world . this is the case of a posterior focus , which was often evoked by taste stimuli in some controls and patients in addition to the anterior foci seen in all subjects . similarly , tactile stimulation of the hand and foot elicited a focus in the anterior part of the posterior body in the cc regions activated by the hand motor tasks ; in addition , stimulation of the hand produced a focus in the splenium in some control subjects and in some patients . thus , the anterior focus observed after tactile hand and foot stimulation could be associated with motor cortex activation , which is often induced by tactile stimulation of these regions ; the foci evoked in the splenium by taste and hand tactile stimuli might result from concurrent activation of high - order association areas , like the posterior parietal cortex ( ppc ) in the case of tactile stimuli , and/or temporal areas for both taste and tactile stimuli ; the fact the ppc and temporal areas are connected by fibers crossing though the splenium [ 58 , 59 ] may explain those findings . the fibers connecting the prefrontal cortical areas were seen to cross through the anterior part of the cc , those connecting the premotor and motor cortical areas crossed at the level of the central callosal body ( see also ) , those connecting the parietal cortical areas crossed through the posterior callosal body , and the fibers linking the occipital and temporal areas crossed at the level of the splenium [ 38 , 63 ] . in all our tests and subjects , the fibers arising from the cortical areas activated by each stimulus type crossed through the cc in a region corresponding to the one harboring the relevant callosal focus / i . the coincidence of callosal bold activation and crossing point of the dtt - reconstructed interhemispheric bundles ( figure 9 ) strongly suggests that the cc foci evoked by sensory stimulation and motor tasks may be due to the activation of the fibers connecting the activated areas to the corresponding contralateral areas and carrying specific information . in all control subjects and in patients the presence of callosal foci in the spared callosal regions broadly corresponded with bilateral activation of the cortical areas reached by specific unilateral peripheral sensory input . , bold and dti data can not be coregistered and shown in the same image ) , we are convinced that it does exist , at least based on the consistency of the observations . with regard to auditory stimulation our data are still confined to a small number of subjects and stimuli , while motor activation was only analyzed in controls . the main difference between previous human after mortem and dti studies and monkey neuroanatomical tracing investigations is that the topographical map outlined by our data is less precise . in particular , tactile stimulation of the hand or foot evokes activation in the primary motor cortex , which could anticipate an action of the limb receiving the tactile stimulus , explaining a subsequent callosal activation ( as in interhemispheric transmission aimed at motor output coordination ) . it may be therefore hypothesized that the splenium is involved in the transfer of touch information , providing for a degree of plasticity in patients with callosal partial resection . an involvement of the splenium in the transfer of information other than visual sensory data could be due to the prominent role of the visual representation of the external environment , typical of humans , where sensory experience is usually associated with a visual component . previous papers describing callosal activation reported a bold effect in the anterior portion of the cc ( see data and literature in ) . fibers interconnecting prefrontal and motor cortical areas course through this region and are likely involved in behavioral visuomotor interhemispheric transfer tasks , like those evoking callosal activation in those studies . the data reviewed in the present study were obtained from simple sensory stimulation that did not involve a motor output , and from simple motor tasks . in the present paper we have reported the data collected in normal subjects ( controls ) and in some callosotomy patients during fmri studies on peripheral sensory stimulation . it has been shown that a bold effect can be evoked in the corpus callosum by peripheral sensory stimulation and by motor tasks and that cc activation foci occupy consistent locations related to the sensory or motor stimulus applied ; it has thus emerged that a functional topographical map of the cc is in line with previous investigations . further studies , however , combining fmri and dti , are needed to provide a better understanding of the topography of callosal activation and to confirm or exclude its correspondence with fiber crossing .

this was a population - based , observational study in central illinois representing a population of about 1.4 million persons . this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers , in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories . the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type . this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion . shared reporting of colonoscopies ( screening and surveillance ) in central illinois the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center , decatur digestive disease center , decatur memorial hospital , methodist medical center of illinois , osf saint francis medical center , pekin hospital , and proctor hospital in the central illinois . each site was responsible for abstracting data through their own screening and surveillance colonoscopies and then entering data into the central illinois colonoscopy access database and electronically transferring data to quality quest for health of illinois . the database is currently managed by the department of medicine in the university of illinois college of medicine at peoria . the information in the database which was used in this study includes age in years ( exam year ) , gender , previous and current procedure date , personal history of crc ( yes / no ) , family history of crc including first and second degree relatives ( yes / no ) , bowel preparation type , bowel preparation assessment ( excellent , good , fair , and poor ) , examination completion ( yes / no ) , american society of anesthesiology ( asa ) classification score with a range of 1 to 5 , and time of withdrawal after insertion ( minutes ) . this was a retrospective study where results would not change the course of patient care or current patient outcomes . no risk was involved in collecting patient data as information was the minimum necessary information for research purposes . also , this study was approved by the institutional review board ( irb ) at the university of illinois college of medicine at peoria . a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies . in order to examine the influence of bowel preparation type on the quality of prep , ordinal logistic regressions were used to estimate odds ratio ( or ) and 95% confidence interval ( 95% ci ) compared with its reference group . we employed a logistic regression model to analyze the association between exam completion and preparation quality . a log transformation was used for the time of withdrawal after insertion due to its skewed distribution . then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion . for all the above models , we also did multivariable analyses , which controlled age , gender , asa score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . a secondary analysis was performed to examine the impact of preparation quality on detecting any adenoma or advanced adenoma . advanced adenoma was defined as a villous / tubulovillous adenoma , severely / high - grade dysplastic polyp , or colorectal cancer based on the polyp histopathology . we excluded those had inadequate preparation ( poor ) from the secondary analysis because of its low exam completion . after univariate analysis , multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma , and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion . variables were reported as mean , standard deviation , median , and range for continuous variables , and percentage for categorical variables . a two - tailed p - value was calculated for all tests and p0.05 was considered as being of statistical significance . this was a population - based , observational study in central illinois representing a population of about 1.4 million persons . this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers , in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories . the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type . this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion . shared reporting of colonoscopies ( screening and surveillance ) in central illinois was established by the development of a quality of health index database in 2010 . the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center , decatur digestive disease center , decatur memorial hospital , methodist medical center of illinois , osf saint francis medical center , pekin hospital , and proctor hospital in the central illinois . each site was responsible for abstracting data through their own screening and surveillance colonoscopies and then entering data into the central illinois colonoscopy access database and electronically transferring data to quality quest for health of illinois . the database is currently managed by the department of medicine in the university of illinois college of medicine at peoria . the information in the database which was used in this study includes age in years ( exam year ) , gender , previous and current procedure date , personal history of crc ( yes / no ) , family history of crc including first and second degree relatives ( yes / no ) , bowel preparation type , bowel preparation assessment ( excellent , good , fair , and poor ) , examination completion ( yes / no ) , american society of anesthesiology ( asa ) classification score with a range of 1 to 5 , and time of withdrawal after insertion ( minutes ) . this was a retrospective study where results would not change the course of patient care or current patient outcomes . no risk was involved in collecting patient data as information was the minimum necessary information for research purposes . also , this study was approved by the institutional review board ( irb ) at the university of illinois college of medicine at peoria . a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies . in order to examine the influence of bowel preparation type on the quality of prep , ordinal logistic regressions were used to estimate odds ratio ( or ) and 95% confidence interval ( 95% ci ) compared with its reference group . we employed a logistic regression model to analyze the association between exam completion and preparation quality . a log transformation was used for the time of withdrawal after insertion due to its skewed distribution . then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion . for all the above models , we also did multivariable analyses , which controlled age , gender , asa score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . a secondary analysis was performed to examine the impact of preparation quality on detecting any adenoma or advanced adenoma . advanced adenoma was defined as a villous / tubulovillous adenoma , severely / high - grade dysplastic polyp , or colorectal cancer based on the polyp histopathology . we excluded those had inadequate preparation ( poor ) from the secondary analysis because of its low exam completion . after univariate analysis , multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma , and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion . all analyses were conducted with sas 9.4 ( by sas institute inc . , cary , nc , usa ) . variables were reported as mean , standard deviation , median , and range for continuous variables , and percentage for categorical variables . a two - tailed p - value was calculated for all tests and p0.05 was considered as being of statistical significance . a total of 28,368 colonoscopies ; half the patients were male , and the average age was 619 years . the majority ( 75% ) lived with mild - to - moderate medical conditions ( asa score=2 ) . the most popular bowel preparation type was peg - based preparations ( 70.2% ) , followed by sodium sulfate based preparations ( 21.4% ) , sodium phosphate based preparations ( 2.5% ) , and magnesium - based preparations ( 0.4% ) . patients who selected sodium phosphate based preparations were a little younger than others . around 21.5% of patients who chose magnesium - based preparation were in poor conditions ( asa>2 ) , which was higher than others . subgroups a : peg - based preparations , n=19,912 ; b : magnesium - based preparations , n=107 ; c : sodium phosphate based preparations , n=6,081 ; e : other preparations , n=322 ; f : not recorded , n=1,257 . american society of anesthesiology ( asa ) classification score was defined as five levels ( 1 = healthy , no comorbidities ; 2 = mild - to - moderate medical conditions controlled ; 3 = disease severely limits activities ; 4 = severe life - threatening disorders ; 5 = moribund ) . as it was shown in table 2 , sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparation was not as good as peg - based preps in the quality of preparations ( table 3 ) . influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . in peg - based preparation , moviprep was better than other peg - based preparations ( or=1.3 , 95% ci 1.21.4 ) . in magnesium - based preparation , no significant difference was found between magnesium citrate only and magnesium citrate with ducolax ( or=0.6 , 95% ci 0.31.6 ) . in sodium sulfate based preparation , the effect of visicol tabs was very similar with osmoprep ( or=0.9 , 95% ci 0.71.2 ) . a better bowel preparation significantly increased the rate of exam completion ( table 4 ) . only 88.4% completed exams when the bowel preparation was poor , whereas 99.5% completed exams when the bowel preparation was excellent . the rate of exam completion was also acceptable when the bowel preparation was good or fair . association between preparations quality and exam completion logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent ( 10.45.5 min ) , followed by good bowel preparation ( 11.06.0 min ) , poor bowel preparation ( 12.28.5 min ) , and fair bowel preparation ( 13.57.7 min ) . general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . our general polyp detection rate was 44.1% ( 12,525/28,386 ) , while adenoma detection rate in fair , good , and excellent preps was 51.7 , 58.3 , and 54.7 , respectively . although the differences of adenoma detection rate among them were not large , the good and excellent preps still increased the likelihood of adenoma detection than the fair prep ( or=1.1 , 95% ci 1.01.2 ; or=1.3 , 95% ci 1.214 , respectively ) . as shown in table 6 , we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma ( 5.0 , 3.6 , and 2.9% for excellent , good , and fair , respectively ) . association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio ( or ) , 95% confidence interval ( 95% ci ) , and predicted detection rate controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , adenoma detection during the last colonoscopy , and time of withdrawal after insertion . a total of 28,368 colonoscopies ; half the patients were male , and the average age was 619 years . the majority ( 75% ) lived with mild - to - moderate medical conditions ( asa score=2 ) . the most popular bowel preparation type was peg - based preparations ( 70.2% ) , followed by sodium sulfate based preparations ( 21.4% ) , sodium phosphate based preparations ( 2.5% ) , and magnesium - based preparations ( 0.4% ) . patients who selected sodium phosphate based preparations were a little younger than others . around 21.5% of patients who chose magnesium - based preparation were in poor conditions ( asa>2 ) , which was higher than others . subgroups a : peg - based preparations , n=19,912 ; b : magnesium - based preparations , n=107 ; c : sodium phosphate based preparations , n=6,081 ; e : other preparations , n=322 ; f : not recorded , n=1,257 . american society of anesthesiology ( asa ) classification score was defined as five levels ( 1 = healthy , no comorbidities ; 2 = mild - to - moderate medical conditions controlled ; 3 = disease severely limits activities ; 4 = severe life - threatening disorders ; 5 = moribund ) . as it was shown in table 2 , sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparation was not as good as peg - based preps in the quality of preparations ( table 3 ) . influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . in peg - based preparation , moviprep was better than other peg - based preparations ( or=1.3 , 95% ci 1.21.4 ) . in magnesium - based preparation , no significant difference was found between magnesium citrate only and magnesium citrate with ducolax ( or=0.6 , 95% ci 0.31.6 ) . in sodium sulfate based preparation , the effect of visicol tabs was very similar with osmoprep ( or=0.9 , 95% ci 0.71.2 ) . a better bowel preparation significantly increased the rate of exam completion ( table 4 ) . only 88.4% completed exams when the bowel preparation was poor , whereas 99.5% completed exams when the bowel preparation was excellent . the rate of exam completion was also acceptable when the bowel preparation was good or fair . association between preparations quality and exam completion logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent ( 10.45.5 min ) , followed by good bowel preparation ( 11.06.0 min ) , poor bowel preparation ( 12.28.5 min ) , and fair bowel preparation ( 13.57.7 min ) . general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . our general polyp detection rate was 44.1% ( 12,525/28,386 ) , while adenoma detection rate in fair , good , and excellent preps was 51.7 , 58.3 , and 54.7 , respectively . although the differences of adenoma detection rate among them were not large , the good and excellent preps still increased the likelihood of adenoma detection than the fair prep ( or=1.1 , 95% ci 1.01.2 ; or=1.3 , 95% ci 1.214 , respectively ) . as shown in table 6 , we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma ( 5.0 , 3.6 , and 2.9% for excellent , good , and fair , respectively ) . association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio ( or ) , 95% confidence interval ( 95% ci ) , and predicted detection rate controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , adenoma detection during the last colonoscopy , and time of withdrawal after insertion . this study compares the impact of bowel preparation type on the quality of colonoscopy in a large , population - based cohort of colonoscopies that were conducted in clinical practice . considering the fact that the national colorectal roundtable set a goal of increasing colorectal screening to 80% by 2018 , the use of screening colonoscopies will continue to increase . this will save valuable resources and also play an essential part in improving clinical outcomes and reducing the disease burden of colorectal cancer ( 11 , 16 ) . a meta - analysis of 104 studies from 1985 to 2010 showed no difference in the efficacy between sodium phosphate based preparations and peg ( or=0.82 , 95% ci 0.561.21 ; p=0.36 ) . in addition , peg - based preparations were found to provide a better cleaning of the proximal portion of the colon ( 12 , 17 ) . based preparations to be superior in terms of bowel preparation and rates of complete examination ( 12 , 18 ) . in addition to this , sodium- and magnesium - based preparations are slightly cheaper as compared with peg - containing preparations ( 19 ) . based preparations had six and two times better quality of bowel preparation when compared with peg - based preparation , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparations were found to be inferior to peg - based formulations ( or=0.6 , 95% ci 0.40.9 ) . peg- and sodium - based bowel preparations had acceptable levels of bowel cleanliness , mostly ranging from fair to good with a good response in 6070% of the patients , independent of the type of preparation being used . in our study , 19,912 patients used peg - based preparation and out of these only 14.5% had excellent preparations , where as 31.8 and 55.8% with sodium phosphate and sodium sulfate these results were consistent with several previous studies in which patients with sodium - based preparations had better results as well as superior completion rates ( 12 , 20 ) . excellent bowel preparation was found in 58% ( ci 4967% ) of the patients taking sodium phosphate tablets , 42.1% ( ci 3351% ) for sodium phosphate solution , and 33.7% ( ci 2641% ) for those who had used 4 l peg ( 20 ) . in a meta - analysis of seven randomized trials comparing sodium phosphate and peg solution , the relative risk of having an excellent preparation was 1.28 ( 95% ci 1.111.48 ) in favor of sodium phosphate ( nnt=10 ) ( 18 ) . similarly , other studies have shown that sodium phosphate is also superior to sodium picosulfate in terms of bowel purging activity with a similar side - effect profile ( 21 , 22 ) . completion rates of colonoscopy did not vary significantly between fair , good , and excellent preparations ( 99.5 , 99.4 , and 99.1% , respectively ) but had almost a 12% decline in patients with poor preparation ( 88.4% ) . these results are in contrast to a recent study in which completion rates were significantly lower in patients with fair and poor bowel preparations ( 75.4 and 72.1% , respectively ) as compared with those with good and excellent bowel preparations ( 99.7 and 99.9% , respectively ; p<0.001 ) ( 23 ) . this difference could possibly be because of smaller number of patients in their study ( 23 ) . other studies have reported a completion rate of 90% in people with intermediate and high - quality preparations while completion rates of 70% in those with low - quality bowel preparations ( 24 ) . our study clearly shows that withdrawal times were faster in patients with excellent and good preparation ( 10.45.5 and 11.06.0 min , respectively ) while those with fair and poor preparation had longer withdrawal times after insertion ( 13.57.7 and 12.2 8.5 , respectively ) . the longer withdrawal time in fair preparation could be due to higher completion rates and effort by physicians in patients with poor preparation of which only 88.4% underwent complete examination as compared with 99% of those with fair preparation who underwent complete exam . previous studies have shown that inadequate bowel preparation decreases the adenoma detection rate ( 2527 ) . other studies have also suggested that the adenoma detection rate is comparable in patients with fair - quality bowel preparation and those with adequate bowel preparation ( 25 , 28 ) . the overall adenoma detection rate in our study was 44.1% which is comparable with other studies and well above the 30% mark set by the american college of gastroenterology task force ( 29 ) . this supports the fact that the quality of colonoscopic examination in our study was consistent with national standards . we found better bowel preparations could increase the detection rate of adenoma , especially advanced adenoma . advanced adenoma detection rate was 2.9% in those with fair bowel prep as compared with 3.6 and 5% in those with good and excellent bowel preparations , respectively . our results clearly suggest that the odds of finding an advanced polyp in a patient with excellent bowel preparation was almost two times higher compared with those with fair prep ( or=1.8 , 95% ci 1.52.1 ) we did not record whether the patients received split - dose preparation or nightly preparations in the case of peg - based preparations . we did not measure true adenoma missed rates by performing follow - up colonoscopies in patients with poor or fair bowel preparation . other confounding factors that could have altered outcomes include concomitant laxative use , hydration variability , and differences in patient compliance across various groups and hospitals . in summary , while peg - based preparations continue to be used most commonly , the search for an ideal bowel purge , which is inexpensive , offers good outcomes with a high success rate , with relatively no side effects continues to be a goal for physicians . we recommend that sodium - based bowel preparations should be used whenever possible as sodium - based preparations appear superior to peg- and magnesium - based preparations according to our study results . further , the findings suggest that adequate bowel preparation not only improves withdrawal times but also enhances the adenoma detection rate in specifically advanced adenomas . in addition , our results also support the finding that patients with fair bowel preparation should also be screened earlier to enhance the chances of detecting any missed adenomas . with an increasing population of patients entering into colorectal cancer screening age , the volume of screening colonoscopies will increase . continued exploration for an optimal bowel preparation remains essential for continued reduction in colorectal cancer . all authors have disclosed that they have no significant relationships with or financial interests in any commercial companies related to this study or article . post presentation was at the digestive disease week ( ddw ) 2015 conference in washington , dc ( may 1619 , 2015 ) . the manuscript was drafted and revised by sw , jr , dm , ca , sd , za , and sp . statistical analysis was done by jr , and data were acquired by ca and jr .

backgroundhigh - quality bowel preparation is crucial for achieving the goals of colonoscopy . however , choosing a bowel preparation in clinical practice can be challenging because of the many formulations . this study aims to assess the impact the type of bowel preparation on the quality of colonoscopy in a community hospital setting.methodsa retrospective , observational study was conducted utilizing a colonoscopy screening / surveillance database in central illinois during the period of january 1 , 2010 , to march 31 , 2014 . patients without bowel preparation assessment were excluded from this study . controlling for the confounders , generalized linear models were used to estimate the adjusted impact [ odds ratio ( or ) ] of bowel preparation type on the quality of preparation ( excellent , good , fair , and poor ) , and on the detection of advanced adenoma . the association between the time of withdrawal after insertion and the quality of preparation was also examined using a linear model.resultsa total of 28,368 colonoscopies ; half the patients were male , and the average age was 619 years . polyethylene glycol ( peg ) was used in the majority ( 70.2% ) of bowel preparations , followed by sodium sulfate ( 21.4% ) , sodium phosphate ( 2.5% ) , magnesium sulfate ( 0.4% ) , and others . compared with peg , magnesium sulfate had a poorer quality of bowel preparations ( or=0.6 , 95% ci 0.40.9 ; p<0.05 ) , whereas the quality of bowel preparation was significantly improved by using sodium sulfate ( or=5.7 , 95% ci 5.46.1 ; p<0.001 ) and sodium phosphate ( or=2.1 , 95% ci 1.82.5 ; p<0.001 ) . for those who had adequate bowel preparation , the better quality of preparation significantly increased the detection rate of advanced adenoma ( 5.0 , 3.6 , and 2.9% for excellent , good , and fair , respectively).conclusionwhen possible , sodium sulfate based preparations should be recommended in the community setting for colonoscopy because of their high quality of bowel preparation .

Methods Study design Data collection Ethical issues Exclusion criteria Statistical analysis Results Demographics Bowel preparation type Exam completion Time of withdrawal after insertion Detection of adenoma and advanced adenoma Discussion Conflict of interest and funding Authors contributions

this was a population - based , observational study in central illinois representing a population of about 1.4 million persons . this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers , in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories . the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type . this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion . shared reporting of colonoscopies ( screening and surveillance ) in central illinois the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center , decatur digestive disease center , decatur memorial hospital , methodist medical center of illinois , osf saint francis medical center , pekin hospital , and proctor hospital in the central illinois . the information in the database which was used in this study includes age in years ( exam year ) , gender , previous and current procedure date , personal history of crc ( yes / no ) , family history of crc including first and second degree relatives ( yes / no ) , bowel preparation type , bowel preparation assessment ( excellent , good , fair , and poor ) , examination completion ( yes / no ) , american society of anesthesiology ( asa ) classification score with a range of 1 to 5 , and time of withdrawal after insertion ( minutes ) . a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies . in order to examine the influence of bowel preparation type on the quality of prep , ordinal logistic regressions were used to estimate odds ratio ( or ) and 95% confidence interval ( 95% ci ) compared with its reference group . a log transformation was used for the time of withdrawal after insertion due to its skewed distribution . then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion . we excluded those had inadequate preparation ( poor ) from the secondary analysis because of its low exam completion . after univariate analysis , multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma , and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion . this was a population - based , observational study in central illinois representing a population of about 1.4 million persons . this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers , in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories . the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type . this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion . shared reporting of colonoscopies ( screening and surveillance ) in central illinois was established by the development of a quality of health index database in 2010 . the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center , decatur digestive disease center , decatur memorial hospital , methodist medical center of illinois , osf saint francis medical center , pekin hospital , and proctor hospital in the central illinois . the information in the database which was used in this study includes age in years ( exam year ) , gender , previous and current procedure date , personal history of crc ( yes / no ) , family history of crc including first and second degree relatives ( yes / no ) , bowel preparation type , bowel preparation assessment ( excellent , good , fair , and poor ) , examination completion ( yes / no ) , american society of anesthesiology ( asa ) classification score with a range of 1 to 5 , and time of withdrawal after insertion ( minutes ) . a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies . in order to examine the influence of bowel preparation type on the quality of prep , ordinal logistic regressions were used to estimate odds ratio ( or ) and 95% confidence interval ( 95% ci ) compared with its reference group . a log transformation was used for the time of withdrawal after insertion due to its skewed distribution . then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion . a secondary analysis was performed to examine the impact of preparation quality on detecting any adenoma or advanced adenoma . we excluded those had inadequate preparation ( poor ) from the secondary analysis because of its low exam completion . after univariate analysis , multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma , and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion . a total of 28,368 colonoscopies ; half the patients were male , and the average age was 619 years . the most popular bowel preparation type was peg - based preparations ( 70.2% ) , followed by sodium sulfate based preparations ( 21.4% ) , sodium phosphate based preparations ( 2.5% ) , and magnesium - based preparations ( 0.4% ) . subgroups a : peg - based preparations , n=19,912 ; b : magnesium - based preparations , n=107 ; c : sodium phosphate based preparations , n=6,081 ; e : other preparations , n=322 ; f : not recorded , n=1,257 . as it was shown in table 2 , sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparation was not as good as peg - based preps in the quality of preparations ( table 3 ) . influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . in peg - based preparation , moviprep was better than other peg - based preparations ( or=1.3 , 95% ci 1.21.4 ) . in magnesium - based preparation , no significant difference was found between magnesium citrate only and magnesium citrate with ducolax ( or=0.6 , 95% ci 0.31.6 ) . in sodium sulfate based preparation , the effect of visicol tabs was very similar with osmoprep ( or=0.9 , 95% ci 0.71.2 ) . a better bowel preparation significantly increased the rate of exam completion ( table 4 ) . the rate of exam completion was also acceptable when the bowel preparation was good or fair . association between preparations quality and exam completion logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent ( 10.45.5 min ) , followed by good bowel preparation ( 11.06.0 min ) , poor bowel preparation ( 12.28.5 min ) , and fair bowel preparation ( 13.57.7 min ) . general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . our general polyp detection rate was 44.1% ( 12,525/28,386 ) , while adenoma detection rate in fair , good , and excellent preps was 51.7 , 58.3 , and 54.7 , respectively . although the differences of adenoma detection rate among them were not large , the good and excellent preps still increased the likelihood of adenoma detection than the fair prep ( or=1.1 , 95% ci 1.01.2 ; or=1.3 , 95% ci 1.214 , respectively ) . as shown in table 6 , we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma ( 5.0 , 3.6 , and 2.9% for excellent , good , and fair , respectively ) . association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio ( or ) , 95% confidence interval ( 95% ci ) , and predicted detection rate controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , adenoma detection during the last colonoscopy , and time of withdrawal after insertion . a total of 28,368 colonoscopies ; half the patients were male , and the average age was 619 years . the most popular bowel preparation type was peg - based preparations ( 70.2% ) , followed by sodium sulfate based preparations ( 21.4% ) , sodium phosphate based preparations ( 2.5% ) , and magnesium - based preparations ( 0.4% ) . subgroups a : peg - based preparations , n=19,912 ; b : magnesium - based preparations , n=107 ; c : sodium phosphate based preparations , n=6,081 ; e : other preparations , n=322 ; f : not recorded , n=1,257 . as it was shown in table 2 , sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparation was not as good as peg - based preps in the quality of preparations ( table 3 ) . influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . in peg - based preparation , moviprep was better than other peg - based preparations ( or=1.3 , 95% ci 1.21.4 ) . in magnesium - based preparation , no significant difference was found between magnesium citrate only and magnesium citrate with ducolax ( or=0.6 , 95% ci 0.31.6 ) . in sodium sulfate based preparation , the effect of visicol tabs was very similar with osmoprep ( or=0.9 , 95% ci 0.71.2 ) . a better bowel preparation significantly increased the rate of exam completion ( table 4 ) . the rate of exam completion was also acceptable when the bowel preparation was good or fair . association between preparations quality and exam completion logistic regression was used to calculate the odds ratio ( or ) and 95% confidence interval ( 95% ci ) controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent ( 10.45.5 min ) , followed by good bowel preparation ( 11.06.0 min ) , poor bowel preparation ( 12.28.5 min ) , and fair bowel preparation ( 13.57.7 min ) . general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , and adenoma detection during the last colonoscopy . our general polyp detection rate was 44.1% ( 12,525/28,386 ) , while adenoma detection rate in fair , good , and excellent preps was 51.7 , 58.3 , and 54.7 , respectively . although the differences of adenoma detection rate among them were not large , the good and excellent preps still increased the likelihood of adenoma detection than the fair prep ( or=1.1 , 95% ci 1.01.2 ; or=1.3 , 95% ci 1.214 , respectively ) . as shown in table 6 , we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma ( 5.0 , 3.6 , and 2.9% for excellent , good , and fair , respectively ) . association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio ( or ) , 95% confidence interval ( 95% ci ) , and predicted detection rate controlling for age , gender , american society of anesthesiology classification score , family history of colorectal cancer , personal history of colorectal cancer , adenoma detection during the last colonoscopy , and time of withdrawal after insertion . this study compares the impact of bowel preparation type on the quality of colonoscopy in a large , population - based cohort of colonoscopies that were conducted in clinical practice . a meta - analysis of 104 studies from 1985 to 2010 showed no difference in the efficacy between sodium phosphate based preparations and peg ( or=0.82 , 95% ci 0.561.21 ; p=0.36 ) . based preparations to be superior in terms of bowel preparation and rates of complete examination ( 12 , 18 ) . in addition to this , sodium- and magnesium - based preparations are slightly cheaper as compared with peg - containing preparations ( 19 ) . based preparations had six and two times better quality of bowel preparation when compared with peg - based preparation , respectively ( or=5.7 , 95% ci 5.46.1 ; or=2.1 , 95% ci 1.82.5 ) . magnesium - based preparations were found to be inferior to peg - based formulations ( or=0.6 , 95% ci 0.40.9 ) . peg- and sodium - based bowel preparations had acceptable levels of bowel cleanliness , mostly ranging from fair to good with a good response in 6070% of the patients , independent of the type of preparation being used . in our study , 19,912 patients used peg - based preparation and out of these only 14.5% had excellent preparations , where as 31.8 and 55.8% with sodium phosphate and sodium sulfate these results were consistent with several previous studies in which patients with sodium - based preparations had better results as well as superior completion rates ( 12 , 20 ) . excellent bowel preparation was found in 58% ( ci 4967% ) of the patients taking sodium phosphate tablets , 42.1% ( ci 3351% ) for sodium phosphate solution , and 33.7% ( ci 2641% ) for those who had used 4 l peg ( 20 ) . in a meta - analysis of seven randomized trials comparing sodium phosphate and peg solution , the relative risk of having an excellent preparation was 1.28 ( 95% ci 1.111.48 ) in favor of sodium phosphate ( nnt=10 ) ( 18 ) . similarly , other studies have shown that sodium phosphate is also superior to sodium picosulfate in terms of bowel purging activity with a similar side - effect profile ( 21 , 22 ) . completion rates of colonoscopy did not vary significantly between fair , good , and excellent preparations ( 99.5 , 99.4 , and 99.1% , respectively ) but had almost a 12% decline in patients with poor preparation ( 88.4% ) . these results are in contrast to a recent study in which completion rates were significantly lower in patients with fair and poor bowel preparations ( 75.4 and 72.1% , respectively ) as compared with those with good and excellent bowel preparations ( 99.7 and 99.9% , respectively ; p<0.001 ) ( 23 ) . other studies have reported a completion rate of 90% in people with intermediate and high - quality preparations while completion rates of 70% in those with low - quality bowel preparations ( 24 ) . our study clearly shows that withdrawal times were faster in patients with excellent and good preparation ( 10.45.5 and 11.06.0 min , respectively ) while those with fair and poor preparation had longer withdrawal times after insertion ( 13.57.7 and 12.2 8.5 , respectively ) . other studies have also suggested that the adenoma detection rate is comparable in patients with fair - quality bowel preparation and those with adequate bowel preparation ( 25 , 28 ) . this supports the fact that the quality of colonoscopic examination in our study was consistent with national standards . we found better bowel preparations could increase the detection rate of adenoma , especially advanced adenoma . advanced adenoma detection rate was 2.9% in those with fair bowel prep as compared with 3.6 and 5% in those with good and excellent bowel preparations , respectively . our results clearly suggest that the odds of finding an advanced polyp in a patient with excellent bowel preparation was almost two times higher compared with those with fair prep ( or=1.8 , 95% ci 1.52.1 ) we did not record whether the patients received split - dose preparation or nightly preparations in the case of peg - based preparations . we recommend that sodium - based bowel preparations should be used whenever possible as sodium - based preparations appear superior to peg- and magnesium - based preparations according to our study results . further , the findings suggest that adequate bowel preparation not only improves withdrawal times but also enhances the adenoma detection rate in specifically advanced adenomas .

gain- and loss - of - function experiments have shown laminar - specific effects on local cortical processing ( beltramo et al . , 2013 , olsen et al . , 2012 ) , but how the layers work together remains unclear . the synchrony of action potential ( ap ) firing across cortical layers is thought to be a fundamental aspect of translaminar processing and is determined by the strength , sign and timing of the underlying synaptic input . here , we investigate the synaptic mechanisms of cortical synchrony between excitatory neurons in layers 2/3 and 5 in behaving mice . measuring translaminar membrane potential ( vm ) synchrony and linking it to sensory processing and behavior require simultaneous vm recordings from different layers in awake animals . however , the vast majority of vm recordings of cortical neurons in behaving animals have been made from superficial layers ( bennett et al . , 2013 , poulet and petersen , 2008 , poulet et al . , 2012 , these studies have shown that internally generated , spontaneous network activity dominates the vm of cortical neurons across cortical regions and is correlated with the behavioral and arousal state . large - amplitude , slow fluctuations are highly correlated between neighboring layer 2/3 ( l2/3 ) neurons in resting animals but are abolished during movement , resulting in a desynchronized or active cortical state ( harris and thiele , 2011 , poulet and petersen , 2008 ) . the active state may result from arousal - related effects associated with movement and has been linked to a modulation in sensory responsiveness ( crochet and petersen , 2006 , otazu et al . , 2009 , pinto et al . , 2013 , , 2013 , reimer et al . , 2014 , schneider et al . , 2014 , , 2015 , zhou et al . , 2014 ) , adaptation ( castro - alamancos , 2004 ) , and even perception itself ( bennett et al . , 2013 , mcginley et al . , 2015 ) . few studies have examined the vm activity of deeper layer cortical neurons in behaving animals ( mcginley et al . , 2015 , schiemann et al . , 2015 ) . extracellular recordings , however , have shown higher spontaneous and sensory - evoked firing rates in deeper layer neurons ( de kock et al . , 2007 , oconnor et al . , 2010 ) and , intriguingly , that sensory - evoked and spontaneous spiking have different temporal structures across layers ( sakata and harris , 2009 ) . the rodent forepaw somatosensory system is a relevant and accessible model system to investigate cortical sensory processing during behavior . the forepaw has five digits ( figure 1a ) that can be used to grasp and manipulate objects as well as discriminate somatosensory stimuli ( milenkovic et al . , 2014 ) . we made whole - cell recordings from primary forepaw somatosensory cortex l2/3 and l5 excitatory neurons in awake mice to compare the synchrony and integration of external ( sensory ) and internal ( movement - evoked and spontaneous ) synaptic input . our data highlight layer - specific membrane properties that underlie differences in ap firing and show that translaminar vm synchrony is dependent both on the behavioral state and the source of synaptic input . to investigate the intrinsic membrane properties of l2/3 and l5 excitatory neurons , we made blind whole - cell patch clamp recordings targeted to the digit 3 cortical representation in primary somatosensory forepaw cortex ( s1 ) in awake mice . mice were head - restrained and had their right forepaw tethered to the platform ( figure 1b ) . the tips of the digits 2 and 4 overhung the edge of the platform while the tip of digit 3 was positioned on a flat , circular head of a combined movement sensor and force - feedback tactile stimulating arm . the stimulating arm head was held in contact with the glabrous skin of digit 3 throughout all recordings . whole - cell recordings from 17 l2/3 neurons were made at subpial depths between 121 and 384 m ( 245.30 17.91 m ) and from 28 l5 neurons between 538 and 823 m ( 649.43 14.28 m ) ( figure 1c ) . all neurons had evoked regular - spiking firing patterns during current injection , and a subset ( l2/3 = 4/17 and l5 = 15/28 neurons ) was confirmed by post hoc biocytin staining to be excitatory pyramidal neurons . we first examined the intrinsic membrane properties soon after break - in during quiet wakefulness . l5 neurons generated more aps in response to equivalent current injection amplitudes than l2/3 neurons ( figures 1d and 1e ) . the increased excitability may be due to the higher resting vm value of l5 neurons during quiet wakefulness ( l2/3mean = 56.92 1.21 mv , n = 12 cells versus l5mean = 50.70 0.65 mv , n = 19 cells ; p < 0.001 ) and their higher input resistance ( figure 1f ; l2/3i / r = 33.46 2.80 m , n = 11 cells versus l5 = 51.66 3.35 m , n = 18 cells ; p < 0.001 ) . interestingly , following the termination of a depolarizing current step , l5 neurons showed a larger - amplitude afterhyperpolarization than l2/3 neurons ( figure 1 g ; at 400 pa : l2/3ahp = 0.56 0.12 mv , n = 10 cells versus l5ahp = 2.42 0.17 mv , n = 22 cells ; p < 0.001 ) . overall , our data show distinct intrinsic membrane properties of forepaw s1 l5 and l2/3 neurons . we next compared the vm properties and dynamics of l2/3 and l5 neurons during forepaw behavior . spontaneous digit movements were monitored by the sensing arm and used to define periods of quiet wakefulness ( q ) and digit movement ( m ) ( see experimental procedures ) . q was characterized by large - amplitude , low - frequency vm fluctuations observed in all recordings from neurons in both layers ( figure 2a ) . slow fluctuations had a similar mean duration across layers ( l2/3 329.06 27.55 ms , n = 13 cells versus l5 266.94 11.15 ms , n = 23 cells ) but lower frequency in l2/3 ( l2/3 2.03 0.08 hz , n = 13 cells versus l5 2.29 0.04 hz , n = 23 cell ; p = 0.005 ) ( figure s1 ) . in both layers , m was accompanied by a reduction in the amplitude of low - frequency fluctuations , with fast fourier transform analysis highlighting a reduction in the power in the 1 to 5 hz range ( figures 2b and 2c ) , and a reduction in the sd of the vm ( figure 2d ) . mean ap firing rates were higher in l5 than l2/3 neurons during q ( figure 2e ; l2/3fr , q = 0.32 0.10 hz , n = 12 cells versus l5fr , q = 2.86 0.60 hz , n = 19 cells ; p < 0.001 ) . moreover , a significant increase in mean firing rates during m was observed in l5 neurons ( l5fr q = 2.86 0.60 hz versus m = 6.14 1.16 hz , n = 19 cells , p = 0.005 ) , but not l2/3 ( l2/3fr q = 0.32 0.10 hz versus m = 0.45 0.18 hz , n = 12 cells ; p = 0.851 ) . inter - spike intervals ( isis ) showed a skewed distribution in both layers , with 22% of l2/3 aps and 30% of l5 aps having an isi of < 25 ms ( figure s2 ) . we next analyzed ap bursting and observed similar burst durations across layers but overall more busts in l5 neurons . no differences were observed between q and m periods ( burst frequency l2/3bf , q = 0.02 0.004 hz , m = 0.03 0.01 hz , n = 13 cells versus l5bf , q = 0.14 0.05 hz , m = 0.25 0.12 hz , n = 23 cells , l2/3 versus l5 q , p = 0.019 ; figure s2 ) . to investigate what drove the laminar differences in mean ap rates , we next examined the ap threshold . ap threshold in l2/3 and l5 neurons varies dependent on the speed of pre - spike depolarization in vm , with faster depolarizing ramps evoking aps at lower threshold ( figure s3 ) . there was , however , no overall difference in ap threshold between l2/3 and l5 neurons in q or m periods ( figure 2f ) . measurement of the mean vm showed a depolarization during m in both l2/3 ( figure 2 g ; l2/3mean q = 56.92 1.21 mv versus m = 51.45 1.73 mv , n = 12 cells ; p < 0.001 ) and l5 neurons ( l5mean q = 50.70 0.65 mv versus m = 45.89 0.80 mv , 0.001 ) , with l5 neurons significantly more depolarized in both behavioral states ( q l2/3mean versus l5mean p < notably , plotting the mean spontaneous ap rates as a function of the difference between ap threshold and the mean value of the top 10% of the vm distribution ( max vm ) revealed an exponential decay ( figure 2h ) . l5 neurons were distributed on the falling slope and l2/3 on the tail of the slope ( figure 2h ) , suggesting that the simplest explanation for higher firing rates in l5 neurons is their more depolarized vm . most likely , this works in combination with the higher input resistance of l5 neurons ( figure 1 ) to push excitatory postsynaptic potentials over ap threshold and trigger more aps in l5 than l2/3 neurons in behaving mice . synchrony of cortical activity across layers is thought to be an important feature of cortical coding . to examine translaminar subthreshold and spiking synchrony , we next inserted two pipettes through neighboring craniotomies to target recording sites in the same vertical axis and made nine simultaneous , dual whole - cell recordings from l2/3 and l5 neurons ( figure 3a ) . we went on to measure vm synchrony at different timescales using cross - correlation , coherence analysis , and spike - triggered averaging . visual inspection of dual vm recordings and cross - correlation analysis shows that large - amplitude slow fluctuations in resting mice are highly correlated across layers ( figures 3a3e ) . as mice went from q to m , however , the correlation of the vm between l2/3 and l5 neurons was reduced in all pairs of cells ( figures 3d and 3e ) . unexpectedly , the peak time of the cross - correlation showed a significant time lag , indicating that l5 subthreshold activity preceded that in l2/3 by 8.04 1.40 ms in resting mice and by 4.35 1.85 ms during m ( figure 3f ) . coherence measurements suggested that the drop in correlation during m was due mostly to the reduction in low - frequency ( 15 hz ) coherence ( figures 3 g and 3h ) . thus , dual whole - cell recordings showed that subthreshold activity of cortical neurons in awake animals is under dynamic control , with cortical layers becoming more independent during active brain states . to quantify ap synchrony from dual recordings , we made spike - triggered peri - stimulus time histograms ( psths ) of spontaneous aps . the chance of observing an ap in a l5 neuron in a 10-ms window around a l2/3 ap is 6.1% during q and 5.6% during m , and the chance of observing an ap in a l2/3 neuron around a l5 ap is 1.0% during q and 0.4% during m. thus , while the chance of observing an ap is dependent upon the firing rate , ap firing appears asynchronous at fast timescales across cortical l2/3 and l5 in awake mice . , aps were triggered by large - amplitude and cell - specific depolarizing synaptic input ( figures 3i3k ) . at the time of the ap , a slow , small - amplitude depolarization was observed in the simultaneously recorded cell in resting mice , necessarily induced by the slow network fluctuations . a similar picture was present during movement : aps were triggered by large - amplitude , depolarizing inputs in the spiking cell that were absent in the simultaneously recorded neuron . these observations provide a synaptic basis for independent laminar spontaneous firing and suggest that spontaneous ap firing in l2/3 and l5 excitatory neurons is driven by sparsely connected excitatory networks . the time lag of the cross - correlation analysis and independent synaptic input during spiking suggested that there might be laminar differences in the fine temporal structure of spontaneous subthreshold fluctuations . we therefore first compared the onset timing of the slow depolarizing fluctuations in resting mice from dual l2/3 and l5 recordings ( figures 4a and 4b ) . we defined a slow depolarizing event ( sde ) as depolarizing epochs in q periods whose average vm level between onset and offset was > 60% of the vm range between the most hyperpolarized and depolarized values for at least 100 ms . plotting the duration and vm of isolated sdes across seven dual recordings as a heatmap ( figures 4b and s4 ) revealed a range of durations with a mean of 300 ms ( figure s1b for single and dual recordings , l2/3 = 329.06 27.55 ms , n = 13 cells , l5 = 266.94 11.15 ms , normally a sde in one neuron was accompanied by a sde in the simultaneously recorded neuron , but a minority of small - amplitude , short - duration sdes were observed in one layer only ( figure s5 ) , likely accounting for the slightly shorter durations of sdes in l5 . in those sdes with a measureable vm onset in both layers , plotting the trial - by - trial onset times ( figure 4c ; supplemental experimental procedures ) , the population averaged vm ( figure 4d ) and population spiking rates ( figure 4e ) revealed that l5 sdes started earlier than those in l2/3 ( figure 4f ; l5 leading l2/3 = 9.07 2.19 ms , n = 7 pairs , p = 0.031 ) . in contrast , the hyperpolarizing offset time was not significantly different across layers when sdes were triggered on l2/3 onset ( figures 4a and 4f ) . sdes thus have an earlier onset in higher - firing l5 neurons , supporting the proposal that l5 neurons are important drivers of supragranular slow network activity ( beltramo et al . , 2013 , sanchez - vives and mccormick , 2000 ) . is the laminar - specific synaptic input observed during ap firing ( figure 3 ) and spontaneous activity ( figure 4 ) a general feature of subthreshold processing in cortical neurons , or are there other sources of more synchronized synaptic input ? movement triggers an active , desynchronized cortical state , which can be driven by thalamic input ( poulet et al . to examine the fast dynamics of movement related synaptic input across layers , we next analyzed the vm dynamics at m onset ( detected by thresholding the first derivative of the digit movement signal ; supplemental experimental procedures ) . averaging the vm showed that m onset is accompanied by simultaneous depolarizing synaptic input to l2/3 and l5 neurons ( figures 5a and 5b ) . close inspection of the log - scale digit movement trace indicated that the depolarization in both layers was tightly coupled to the tiny initial movements of the digits and did not appear before movement . to quantify the timing of the depolarization , we performed cross - correlation analysis between the averaged digit movement and the averaged vm response at m onset . the peak of the cross - correlation showed no significant time lag ( figure 5b ) , suggesting an internal , non - sensory origin for synaptic input at movement onset . analysis of 2-s epochs of vm activity showed an overall reduction in the sd of the vm during extended m periods ( figure 2 ) , but how fast this occurs was unclear . we therefore quantified the variance of the l2/3 and l5 subthreshold activity around all recorded m onsets , including short - duration movements ( figures 5e and 5f ) . in all cells across both layers , we observed a rapid and robust reduction in variance within the first 200 ms of movement onset . thus , early movement - evoked input acts to reduce subthreshold variability simultaneously across cortical layers . the reduction in variance , however , does not result in synchronous ap firing . instead , l5 neurons showed higher firing rates after movement onset than l2/3 neurons ( figures 5c and 5d ) . thus , unlike the laminar - specific vm dynamics during sdes and spontaneous aps , movement onset triggers simultaneous vm depolarization across layers , which results in an increase in firing only in l5 neurons . thalamo - cortical axons are unequally distributed across the cortical layers , with some l5 neurons receiving direct thalamic input ( bureau et al . we therefore next examined whether there is laminar specificity in sensory processing across layers in behaving animals . we delivered brief ( 2 ms ) , light ( 10 mn ) tactile stimuli via the stimulating / sensing arm to digit 3 during single and dual whole - cell recordings . the sensing arm was in constant contact with the glabrous skin of digit 3 . while the first spike latency was similar for both behavioral conditions and layers ( figure s6b ) , the amplitude of the vm response was strongly dependent on the behavioral state ( figures 6a6d ) . in q periods , stimuli evoked a large - amplitude depolarizing response in both layers , whereas the amplitude of the response was reduced during m ( figures 6c and 6e ; l2/3amp q = 10.80 0.65 mv , m = 4.84 1.24 mv , n = 10 cells , p = 0.037 ; l5amp q = 6.19 0.69 mv , m = 2.41 0.60 mv , n = 20 cells , p = 0.001 ; q l2/3amp versus l5amp p < 0.001 , and m l2/3amp versus l5amp p = 0.005 ) . despite the difference in subthreshold response amplitude , tactile stimulation evoked few extra aps over the background rate in both layers in both behavioral conditions ( figures 6d , 6f , and s6a ; l2/3ap q = 1.59 0.62 hz , m = 1.95 1.36 hz , n = 10 cells , p = 0.375 ; l5ap q = 2.44 1.40 hz , m = 1.55 1.16 hz , n = 20 cells , p = 0.370 ; q l2/3ap versus l5ap p = 0.613 , m l2/3ap versus l5ap p = 0.523 ) . to examine why l5 neurons do not fire more aps in response to tactile stimulation despite having a more depolarized vm , we measured the tactile responses as a function of the vm just prior to stimulus onset . as neurons became more depolarized , the sensory response reduced in amplitude until eventually showing a hyperpolarizing response ( figure s7 ) . the vm level for the tactile - evoked reversal potential was similar during q and m periods and hyperpolarized relative to glutamate reversal potential ( figure 7 g , l2/3rev q = 46.67 1.60 mv , m = 47.09 1.59 mv , n = 10 cells , p = 0.492 ; l5rev q = 46.17 0.75 mv , m = 46.61 0.86 mv , n = 20 cells , p = 0.412 ; q l2/3rev versus l5rev p = 0.644 , m l2/3rev versus l5rev p = 0.775 ) . notably , a minority of cells with higher mean firing rates showed a smaller difference between ap threshold and the sensory reversal potential ( figure 6h ) . these data provide an explanation for why the evoked rates are similar across layers and suggest that sensory - evoked gaba - ergic inhibition plays a critical role in defining the sensory reversal potential in both layers ( crochet et al . , 2011 , we next examined correlations between the timing and amplitude of sensory - evoked synaptic responses across layers during dual recordings . while the onset latencies of the synaptic and spiking responses to tactile stimulation in l5 neurons were more broadly distributed than in l2/3 , there was no significant difference across layers ( figure 6i , l2/3psplat = 11.07 0.58 ms , n = 13 versus l5psplat = 11.34 0.76 ms , n = 21 , p = 0.972 ; figure s6b , l2/31staplat , q = 33.78 4.73 ms , n = 8 cells , m = 44.67 8.30 ms , n = 3 cells ; l51staplat , q = 39.52 4.72 ms , n = 19 cells , m = 31.10 5.90 ms , n = 9 cells ) . moreover , trial - by - trial analysis of the tactile - evoked postsynaptic potential ( psp ) showed that subthreshold response amplitudes were highly correlated between l2/3 and l5 neurons ( figures 6j and 6k ) . together , these data indicate that , in contrast to sdes and spontaneous aps , tactile stimulation , like movement onset , triggers synchronized subthreshold input across layers . following 75.2% of stimuli delivered in q periods , the mouse s forepaw remained stationary ( quiet - quiet [ qq ] trials ) . however , 24.8% of stimuli in q periods evoked short - latency ( < 100 ms ) forepaw digit movements ( figure 7 ) , termed quiet - move ( qm ) trials . we next compared sub- and supra - threshold responses in qq with qm trials ( figures 7a7c ) . tactile stimulation evoked a short - latency , large - amplitude subthreshold response in qq and qm trials in both layers ( figures 7a and 7b ) . notably , the amplitude of the subthreshold response was larger in qm than qq trials in both layers ( figure 7d ; l2/3 qq = 10.80 0.65 mv versus qm = 11.83 0.79 mv , n = 10 cells , p = 0.037 ; l5 qq = 6.19 0.69 mv versus qm = 8.16 0.78 mv , n = 20 cells , p = 0.002 ; and qq l2/3 versus l5 p < 0.001 , qm l2/3 versus l5 p = 0.005 ) . moreover , we observed that the vm prior to stimulus onset was significantly more hyperpolarized in qm trials than qq trials in l5 , but not in l2/3 neurons ( figures 7e ; l2/3 qq = 57.37 1.36 mv , qm = 58.19 1.58 mv , n = 10 cells , p = 0.232 ; l5 qq = 52.53 0.69 mv , qm = 54.24 0.61 mv , n = 20 cells , p = 0.009 ; and qq l2/3 versus l5 p = 0.006 , qm l2/3 versus l5 p = 0.033 ) . in qq trials , the peak response was then followed by a brief hyperpolarization and subsequent depolarization peaking at 350 ms ( figure 7b ) . this secondary depolarization was significantly larger in both layers during qm than qq trials ( figure 7f ) and evoked aps in l5 , but not l2/3 , neurons ( figures 7c and 7 g ; l2/3 qq = 0.18 0.13 hz , qm = 0.12 0.34 hz , n = 10 cells , p = 0.910 ; l5 qq = 0.01 0.24 hz , qm = 2.66 0.81 hz , n = 20 cells , p = 0.003 ; qq l2/3 versus l5 p = 0.613 , qm l2/3 versus l5 p = 0.011 ) . thus , movements evoked by a tactile input are linked to a more hyperpolarized pre - stimulus vm , a larger subthreshold early response , and a higher late increase in mean firing rate of l5 neurons . the coordinated activity of six layers of primary sensory cortical neurons underlies sensory perception . using whole - cell recordings in awake mice , we investigated synaptic mechanisms of translaminar synchronous activity . we show that laminar - specific differences in membrane properties drive distinct firing rates , that translaminar synchrony is dependent both on the behavioral state and the source of synaptic input ( spontaneous , sensory , and movement evoked ) , and that l5 neurons signal spontaneous and tactile - triggered movement . prior work has observed low firing rates of l2/3 excitatory neurons during synchronized states in resting and anesthetized animals ( barth and poulet , 2012 , jouhanneau et al . , 2014 ) and no change ( crochet and petersen , 2006 , polack et al . , 2013 , poulet and petersen , 2008 , zhou et al . , 2014 ) or a reduction ( bennett et al . , 2013 , we observed that l5 neurons have a higher mean spontaneous rate of aps ( 2.86 0.60 hz ) than l2/3 neurons ( 0.32 0.09 hz ) in resting mice , supporting previous reports in anesthetized and awake animals ( brecht et al . , 2003 , constantinople and bruno , 2011 , de kock et al . , 2007 , de kock and sakmann , 2009 , manns et al . , 2004 , oconnor et al . , 2010 , sakata and harris , 2009 ) , and , in contrast to l2/3 , a significant increase in mean firing rates during movement ( figure 2 ) . previous studies of deeper layers have observed both increases and decreases in firing rates of somatosensory ( curtis and kleinfeld , 2009 , de kock and sakmann , 2009 ) and motor ( carvell et al . , 1996 , schiemann et al . , 2015 , zagha et al . , 2015 ) our whole - cell recordings indicate that higher l5 rates were not the result of an intrinsic difference in ap threshold . moreover , ap triggered averaging showed that the depolarizing synaptic inputs driving aps had similar amplitudes and dynamics in l2/3 and l5 neurons ( figure 3 ) . instead , higher mean firing rates in l5 neurons appeared to result from a larger input resistance and a more depolarized vm , as observed in subsets of l5 pyramidal neurons in vitro ( lefort et al . , 2009 , mason and larkman , 1990 ) , which likely combine to push excitatory input over ap threshold more often than in l2/3 neurons . because most excitatory connections to pyramidal neurons are small in amplitude ( jouhanneau et al . , 2015 , lefort et al . , 2009 ) and the rise time of the depolarization prior to an ap is negatively correlated with ap threshold ( figure s3 ) ( azouz and gray , 2000 , poulet and petersen , 2008 ) , it is likely that coincident synaptic input is required to drive spontaneous ap firing in both layers . in resting mice , the absolute tactile - evoked firing rate was higher in l5 ( 5.03 1.74 hz ) than l2/3 ( 1.96 0.75 hz ) neurons , but this was superimposed on different background firing rates . in fact , a brief tactile stimulus added similarly few additional aps to the background rate in both layers ( evoked rate : l2/3 1.59 0.62 hz and l5 2.44 1.40 hz ) . our observation of low evoked rates across layers is similar to prior studies of s1 in anesthetized animals ( barth and poulet , 2012 , brecht et al . , 2003 , l5 pyramidal neurons ( de kock et al . , 2007 , sakata and harris , 2009 ) . we did not differentiate between subtypes of cortical excitatory l5 neurons , but we did observe a minority ( 3/20 ) of l5 neurons with sensory - evoked rates > 10 hz ( figure 6f ) . future work targeting whole - cell recordings to within - layer excitatory neuron subtypes in sensory cortex will be of great importance . cortical sensory responses are modulated by behavioral state in different sensory systems ( bennett et al . , 2013 , crochet and petersen , 2006 , niell and stryker , 2010 , otazu et al . , 2009 , schneider et al . , 2014 , zhou et al . , we report a reduction in subthreshold response amplitude in l2/3 neurons to brief tactile stimuli during movement . we go on to show that l5 neurons also have a reduced subthreshold response during movement ( figure 6 ) . despite this , the numbers of evoked aps remained the same in both layers during quiet and moving periods . in both layers , subthreshold responses were reduced in amplitude as the baseline vm values became more depolarized and exhibited a reversal potential more hyperpolarized than ap threshold . interestingly , in the cells with higher sensory - evoked firing rates , the sensory reversal potential was closer to threshold . together , this suggests that strong , local gabaergic inhibition plays a significant role in clamping the subthreshold sensory response below ap threshold and regulating ap firing during behavior in both l2/3 ( crochet et al . , 2011 ) and l5 neurons . synchronous activity in cortical networks is thought to be fundamental to sensory processing and perception . prior work has shown that spontaneous vm activity in l2/3 neurons during resting states is more correlated than in activated states in behaving or attentive animals ( okun et al . neurons showed large - amplitude , highly correlated fluctuations of the vm during resting periods and an active state with low sd , as well as a reduction in slow fluctuation amplitude and translaminar vm synchrony ( figures 2 and 3 ) . however , close inspection of the fine timing of subthreshold inputs evoked at different time points revealed differences in the timing of synaptic input across layers . spontaneous aps were driven by cell - specific , depolarizing inputs ( figure 3 ) . in contrast , movement- ( figure 5 ) and sensory - driven ( figure 6 ) synaptic input had a similar timing across layers . interestingly , slow subthreshold fluctuations in resting mice had an earlier onset in l5 , reminiscent of the earlier timing of upstate onsets in deeper layers observed in anesthetized and sleeping animals ( chauvette et al . , 2010 , sakata and harris , 2009 ) , cortical slices ( sanchez - vives and mccormick , 2000 ) , and extracellular recordings in awake rats ( sakata and harris , 2009 ) . thus , the type of input ( spontaneous , sensory , or movement evoked ) determines the timing of synaptic input across layers . one possibility is that they result from differences in the wiring supporting spontaneous , sensory , and motor events . in support of this suggestion , a recent anatomical study showed laminar differences in local and long - range cortico - cortical inputs to s1 excitatory neurons ( denardo et al . , 2015 ) . l2/3 neurons receive proportionally more input from distant l2/3 neurons than l5 neurons , which receive a greater proportion from distant l5 neurons . moreover , locally , l2/3 neurons receive a greater proportion of inhibitory inputs than l5 neurons . luczak et al . , 2007 ) and slow fluctuations in awake resting animals ( ferezou et al . , 2007 ) are thought to propagate as waves of activity across cortex supported by long - range cortico - cortical connections . thus , a later onset in l2/3 neurons could result from a combination of lower firing rates , increased inhibitory input , and laminar - specific cortico - cortical wiring . in contrast , because sensory thalamic neurons are driven both by sensory stimulation and movement ( poulet et al . , 2012 ) , we suggest that punctate thalamic input drives the synchronized synaptic input following tactile stimuli or movement onset . subthreshold sensory responses are correlated not only with the behavioral and cortical state but also with the behavioral outcome . both l2/3 and l5 neurons showed a larger - amplitude subthreshold sensory response in trials that lead to short - latency forepaw movements ( qm trials ) compared to trials with no movement before or after the stimulus ( qq trials ) . in l5 neurons , a difference was also observed in the pre - stimulus vm , with qm trials having a more hyperpolarized value than qq trials . this suggests that the link between subthreshold response amplitude and behavioral output is due to the prestimulus cortical state rather than delivery of different amplitude stimuli . our findings resemble recent vm recordings in auditory cortex during an auditory discrimination go / no - go task , where neurons from l4 and l5 showed more hyperpolarized vm value in hit trials than in false - positive trials in mice performing an auditory perception task ( mcginley et al . , 2015 ) . future experiments could address whether this is the result of a higher signal to noise ratio in a phase of low network activity resulting in an enhanced probability of signal transmission to downstream motor centers . during evoked movement trials , neurons in both layers showed a prominent late depolarization ( figure 7 ) , with l5 neurons also showing an increase in late spiking . because forepaw movements necessarily occurred soon after tactile stimulation in qm trials , it was difficult to assess whether the late activity is causally related to the perception of the stimulus ( sachidhanandam et al . , 2013 ) , an intrinsic part of the transformation of sensory input to motor output and/or the start of the active cortical state associated with attention or arousal . examining mice trained to delay sensory - triggered movements will help link late activity to perception and movement . we observed higher background firing rates in l5 neurons but no difference in the numbers of additional , sensory - evoked spikes across layers , suggesting that l2/3 and l5 have distinct sensory coding strategies ( sakata and harris , 2009 ) . the fine timing differences of synaptic input may be important in the processing of dynamic sensory stimuli and for changes of synaptic strength under spike - time - dependent plasticity rules . fast , laminar - specific manipulations of synchronized activity in trained mice are now required to define the causal role of translaminar synchrony in perception . all experiments were approved by the berlin animal ethics committee and carried out in accordance with european animal welfare law . head - restrained 6- to 9-week - old c57bl6j mice were paw - tethered , and digit movements were monitored by a force - feedback sensing and stimulating arm . the sensing arm ending was a 4.7-mm - diameter flat disk with one edge pressed up against the glabrous skin of digit 3 . blind whole - cell patch - clamp recordings were made from primary somatosensory forepaw cortex located by intrinsic optical imaging . all data were statistically analyzed using non - parametric tests , paired data with a wilcoxon signed rank test and unpaired data with a wilcoxon rank sum test .

summarythe synchronized activity of six layers of cortical neurons is critical for sensory perception and the control of voluntary behavior , but little is known about the synaptic mechanisms of cortical synchrony across layers in behaving animals . we made single and dual whole - cell recordings from the primary somatosensory forepaw cortex in awake mice and show that l2/3 and l5 excitatory neurons have layer - specific intrinsic properties and membrane potential dynamics that shape laminar - specific firing rates and subthreshold synchrony . first , while sensory and movement - evoked synaptic input was tightly correlated across layers , spontaneous action potentials and slow spontaneous subthreshold fluctuations had laminar - specific timing ; second , longer duration forepaw movement was associated with a decorrelation of subthreshold activity ; third , spontaneous and sensory - evoked forepaw movements were signaled more strongly by l5 than l2/3 neurons . together , our data suggest that the degree of translaminar synchrony is dependent upon the origin ( sensory , spontaneous , and movement ) of the synaptic input .

Introduction Results Discussion Experimental Procedures Author Contributions

gain- and loss - of - function experiments have shown laminar - specific effects on local cortical processing ( beltramo et al . the synchrony of action potential ( ap ) firing across cortical layers is thought to be a fundamental aspect of translaminar processing and is determined by the strength , sign and timing of the underlying synaptic input . here , we investigate the synaptic mechanisms of cortical synchrony between excitatory neurons in layers 2/3 and 5 in behaving mice . measuring translaminar membrane potential ( vm ) synchrony and linking it to sensory processing and behavior require simultaneous vm recordings from different layers in awake animals . however , the vast majority of vm recordings of cortical neurons in behaving animals have been made from superficial layers ( bennett et al . , 2012 , these studies have shown that internally generated , spontaneous network activity dominates the vm of cortical neurons across cortical regions and is correlated with the behavioral and arousal state . few studies have examined the vm activity of deeper layer cortical neurons in behaving animals ( mcginley et al . extracellular recordings , however , have shown higher spontaneous and sensory - evoked firing rates in deeper layer neurons ( de kock et al . , 2010 ) and , intriguingly , that sensory - evoked and spontaneous spiking have different temporal structures across layers ( sakata and harris , 2009 ) . we made whole - cell recordings from primary forepaw somatosensory cortex l2/3 and l5 excitatory neurons in awake mice to compare the synchrony and integration of external ( sensory ) and internal ( movement - evoked and spontaneous ) synaptic input . our data highlight layer - specific membrane properties that underlie differences in ap firing and show that translaminar vm synchrony is dependent both on the behavioral state and the source of synaptic input . to investigate the intrinsic membrane properties of l2/3 and l5 excitatory neurons , we made blind whole - cell patch clamp recordings targeted to the digit 3 cortical representation in primary somatosensory forepaw cortex ( s1 ) in awake mice . whole - cell recordings from 17 l2/3 neurons were made at subpial depths between 121 and 384 m ( 245.30 17.91 m ) and from 28 l5 neurons between 538 and 823 m ( 649.43 14.28 m ) ( figure 1c ) . l5 neurons generated more aps in response to equivalent current injection amplitudes than l2/3 neurons ( figures 1d and 1e ) . interestingly , following the termination of a depolarizing current step , l5 neurons showed a larger - amplitude afterhyperpolarization than l2/3 neurons ( figure 1 g ; at 400 pa : l2/3ahp = 0.56 0.12 mv , n = 10 cells versus l5ahp = 2.42 0.17 mv , n = 22 cells ; p < 0.001 ) . overall , our data show distinct intrinsic membrane properties of forepaw s1 l5 and l2/3 neurons . we next compared the vm properties and dynamics of l2/3 and l5 neurons during forepaw behavior . in both layers , m was accompanied by a reduction in the amplitude of low - frequency fluctuations , with fast fourier transform analysis highlighting a reduction in the power in the 1 to 5 hz range ( figures 2b and 2c ) , and a reduction in the sd of the vm ( figure 2d ) . mean ap firing rates were higher in l5 than l2/3 neurons during q ( figure 2e ; l2/3fr , q = 0.32 0.10 hz , n = 12 cells versus l5fr , q = 2.86 0.60 hz , n = 19 cells ; p < 0.001 ) . ap threshold in l2/3 and l5 neurons varies dependent on the speed of pre - spike depolarization in vm , with faster depolarizing ramps evoking aps at lower threshold ( figure s3 ) . measurement of the mean vm showed a depolarization during m in both l2/3 ( figure 2 g ; l2/3mean q = 56.92 1.21 mv versus m = 51.45 1.73 mv , n = 12 cells ; p < 0.001 ) and l5 neurons ( l5mean q = 50.70 0.65 mv versus m = 45.89 0.80 mv , 0.001 ) , with l5 neurons significantly more depolarized in both behavioral states ( q l2/3mean versus l5mean p < notably , plotting the mean spontaneous ap rates as a function of the difference between ap threshold and the mean value of the top 10% of the vm distribution ( max vm ) revealed an exponential decay ( figure 2h ) . l5 neurons were distributed on the falling slope and l2/3 on the tail of the slope ( figure 2h ) , suggesting that the simplest explanation for higher firing rates in l5 neurons is their more depolarized vm . most likely , this works in combination with the higher input resistance of l5 neurons ( figure 1 ) to push excitatory postsynaptic potentials over ap threshold and trigger more aps in l5 than l2/3 neurons in behaving mice . to examine translaminar subthreshold and spiking synchrony , we next inserted two pipettes through neighboring craniotomies to target recording sites in the same vertical axis and made nine simultaneous , dual whole - cell recordings from l2/3 and l5 neurons ( figure 3a ) . visual inspection of dual vm recordings and cross - correlation analysis shows that large - amplitude slow fluctuations in resting mice are highly correlated across layers ( figures 3a3e ) . as mice went from q to m , however , the correlation of the vm between l2/3 and l5 neurons was reduced in all pairs of cells ( figures 3d and 3e ) . unexpectedly , the peak time of the cross - correlation showed a significant time lag , indicating that l5 subthreshold activity preceded that in l2/3 by 8.04 1.40 ms in resting mice and by 4.35 1.85 ms during m ( figure 3f ) . thus , dual whole - cell recordings showed that subthreshold activity of cortical neurons in awake animals is under dynamic control , with cortical layers becoming more independent during active brain states . to quantify ap synchrony from dual recordings , we made spike - triggered peri - stimulus time histograms ( psths ) of spontaneous aps . the chance of observing an ap in a l5 neuron in a 10-ms window around a l2/3 ap is 6.1% during q and 5.6% during m , and the chance of observing an ap in a l2/3 neuron around a l5 ap is 1.0% during q and 0.4% during m. thus , while the chance of observing an ap is dependent upon the firing rate , ap firing appears asynchronous at fast timescales across cortical l2/3 and l5 in awake mice . , aps were triggered by large - amplitude and cell - specific depolarizing synaptic input ( figures 3i3k ) . these observations provide a synaptic basis for independent laminar spontaneous firing and suggest that spontaneous ap firing in l2/3 and l5 excitatory neurons is driven by sparsely connected excitatory networks . the time lag of the cross - correlation analysis and independent synaptic input during spiking suggested that there might be laminar differences in the fine temporal structure of spontaneous subthreshold fluctuations . we therefore first compared the onset timing of the slow depolarizing fluctuations in resting mice from dual l2/3 and l5 recordings ( figures 4a and 4b ) . plotting the duration and vm of isolated sdes across seven dual recordings as a heatmap ( figures 4b and s4 ) revealed a range of durations with a mean of 300 ms ( figure s1b for single and dual recordings , l2/3 = 329.06 27.55 ms , n = 13 cells , l5 = 266.94 11.15 ms , normally a sde in one neuron was accompanied by a sde in the simultaneously recorded neuron , but a minority of small - amplitude , short - duration sdes were observed in one layer only ( figure s5 ) , likely accounting for the slightly shorter durations of sdes in l5 . in those sdes with a measureable vm onset in both layers , plotting the trial - by - trial onset times ( figure 4c ; supplemental experimental procedures ) , the population averaged vm ( figure 4d ) and population spiking rates ( figure 4e ) revealed that l5 sdes started earlier than those in l2/3 ( figure 4f ; l5 leading l2/3 = 9.07 2.19 ms , n = 7 pairs , p = 0.031 ) . is the laminar - specific synaptic input observed during ap firing ( figure 3 ) and spontaneous activity ( figure 4 ) a general feature of subthreshold processing in cortical neurons , or are there other sources of more synchronized synaptic input ? to examine the fast dynamics of movement related synaptic input across layers , we next analyzed the vm dynamics at m onset ( detected by thresholding the first derivative of the digit movement signal ; supplemental experimental procedures ) . averaging the vm showed that m onset is accompanied by simultaneous depolarizing synaptic input to l2/3 and l5 neurons ( figures 5a and 5b ) . close inspection of the log - scale digit movement trace indicated that the depolarization in both layers was tightly coupled to the tiny initial movements of the digits and did not appear before movement . to quantify the timing of the depolarization , we performed cross - correlation analysis between the averaged digit movement and the averaged vm response at m onset . analysis of 2-s epochs of vm activity showed an overall reduction in the sd of the vm during extended m periods ( figure 2 ) , but how fast this occurs was unclear . we therefore quantified the variance of the l2/3 and l5 subthreshold activity around all recorded m onsets , including short - duration movements ( figures 5e and 5f ) . thus , early movement - evoked input acts to reduce subthreshold variability simultaneously across cortical layers . instead , l5 neurons showed higher firing rates after movement onset than l2/3 neurons ( figures 5c and 5d ) . thus , unlike the laminar - specific vm dynamics during sdes and spontaneous aps , movement onset triggers simultaneous vm depolarization across layers , which results in an increase in firing only in l5 neurons . we therefore next examined whether there is laminar specificity in sensory processing across layers in behaving animals . we delivered brief ( 2 ms ) , light ( 10 mn ) tactile stimuli via the stimulating / sensing arm to digit 3 during single and dual whole - cell recordings . in q periods , stimuli evoked a large - amplitude depolarizing response in both layers , whereas the amplitude of the response was reduced during m ( figures 6c and 6e ; l2/3amp q = 10.80 0.65 mv , m = 4.84 1.24 mv , n = 10 cells , p = 0.037 ; l5amp q = 6.19 0.69 mv , m = 2.41 0.60 mv , n = 20 cells , p = 0.001 ; q l2/3amp versus l5amp p < 0.001 , and m l2/3amp versus l5amp p = 0.005 ) . despite the difference in subthreshold response amplitude , tactile stimulation evoked few extra aps over the background rate in both layers in both behavioral conditions ( figures 6d , 6f , and s6a ; l2/3ap q = 1.59 0.62 hz , m = 1.95 1.36 hz , n = 10 cells , p = 0.375 ; l5ap q = 2.44 1.40 hz , m = 1.55 1.16 hz , n = 20 cells , p = 0.370 ; q l2/3ap versus l5ap p = 0.613 , m l2/3ap versus l5ap p = 0.523 ) . these data provide an explanation for why the evoked rates are similar across layers and suggest that sensory - evoked gaba - ergic inhibition plays a critical role in defining the sensory reversal potential in both layers ( crochet et al . , 2011 , we next examined correlations between the timing and amplitude of sensory - evoked synaptic responses across layers during dual recordings . while the onset latencies of the synaptic and spiking responses to tactile stimulation in l5 neurons were more broadly distributed than in l2/3 , there was no significant difference across layers ( figure 6i , l2/3psplat = 11.07 0.58 ms , n = 13 versus l5psplat = 11.34 0.76 ms , n = 21 , p = 0.972 ; figure s6b , l2/31staplat , q = 33.78 4.73 ms , n = 8 cells , m = 44.67 8.30 ms , n = 3 cells ; l51staplat , q = 39.52 4.72 ms , n = 19 cells , m = 31.10 5.90 ms , n = 9 cells ) . moreover , trial - by - trial analysis of the tactile - evoked postsynaptic potential ( psp ) showed that subthreshold response amplitudes were highly correlated between l2/3 and l5 neurons ( figures 6j and 6k ) . together , these data indicate that , in contrast to sdes and spontaneous aps , tactile stimulation , like movement onset , triggers synchronized subthreshold input across layers . moreover , we observed that the vm prior to stimulus onset was significantly more hyperpolarized in qm trials than qq trials in l5 , but not in l2/3 neurons ( figures 7e ; l2/3 qq = 57.37 1.36 mv , qm = 58.19 1.58 mv , n = 10 cells , p = 0.232 ; l5 qq = 52.53 0.69 mv , qm = 54.24 0.61 mv , n = 20 cells , p = 0.009 ; and qq l2/3 versus l5 p = 0.006 , qm l2/3 versus l5 p = 0.033 ) . the coordinated activity of six layers of primary sensory cortical neurons underlies sensory perception . using whole - cell recordings in awake mice , we investigated synaptic mechanisms of translaminar synchronous activity . we show that laminar - specific differences in membrane properties drive distinct firing rates , that translaminar synchrony is dependent both on the behavioral state and the source of synaptic input ( spontaneous , sensory , and movement evoked ) , and that l5 neurons signal spontaneous and tactile - triggered movement . prior work has observed low firing rates of l2/3 excitatory neurons during synchronized states in resting and anesthetized animals ( barth and poulet , 2012 , jouhanneau et al . , 2013 , we observed that l5 neurons have a higher mean spontaneous rate of aps ( 2.86 0.60 hz ) than l2/3 neurons ( 0.32 0.09 hz ) in resting mice , supporting previous reports in anesthetized and awake animals ( brecht et al . , 2015 ) our whole - cell recordings indicate that higher l5 rates were not the result of an intrinsic difference in ap threshold . moreover , ap triggered averaging showed that the depolarizing synaptic inputs driving aps had similar amplitudes and dynamics in l2/3 and l5 neurons ( figure 3 ) . , 2009 ) and the rise time of the depolarization prior to an ap is negatively correlated with ap threshold ( figure s3 ) ( azouz and gray , 2000 , poulet and petersen , 2008 ) , it is likely that coincident synaptic input is required to drive spontaneous ap firing in both layers . in resting mice , the absolute tactile - evoked firing rate was higher in l5 ( 5.03 1.74 hz ) than l2/3 ( 1.96 0.75 hz ) neurons , but this was superimposed on different background firing rates . we did not differentiate between subtypes of cortical excitatory l5 neurons , but we did observe a minority ( 3/20 ) of l5 neurons with sensory - evoked rates > 10 hz ( figure 6f ) . future work targeting whole - cell recordings to within - layer excitatory neuron subtypes in sensory cortex will be of great importance . interestingly , in the cells with higher sensory - evoked firing rates , the sensory reversal potential was closer to threshold . however , close inspection of the fine timing of subthreshold inputs evoked at different time points revealed differences in the timing of synaptic input across layers . in contrast , movement- ( figure 5 ) and sensory - driven ( figure 6 ) synaptic input had a similar timing across layers . interestingly , slow subthreshold fluctuations in resting mice had an earlier onset in l5 , reminiscent of the earlier timing of upstate onsets in deeper layers observed in anesthetized and sleeping animals ( chauvette et al . , 2010 , sakata and harris , 2009 ) , cortical slices ( sanchez - vives and mccormick , 2000 ) , and extracellular recordings in awake rats ( sakata and harris , 2009 ) . thus , the type of input ( spontaneous , sensory , or movement evoked ) determines the timing of synaptic input across layers . one possibility is that they result from differences in the wiring supporting spontaneous , sensory , and motor events . , 2007 ) and slow fluctuations in awake resting animals ( ferezou et al . thus , a later onset in l2/3 neurons could result from a combination of lower firing rates , increased inhibitory input , and laminar - specific cortico - cortical wiring . both l2/3 and l5 neurons showed a larger - amplitude subthreshold sensory response in trials that lead to short - latency forepaw movements ( qm trials ) compared to trials with no movement before or after the stimulus ( qq trials ) . because forepaw movements necessarily occurred soon after tactile stimulation in qm trials , it was difficult to assess whether the late activity is causally related to the perception of the stimulus ( sachidhanandam et al . examining mice trained to delay sensory - triggered movements will help link late activity to perception and movement . we observed higher background firing rates in l5 neurons but no difference in the numbers of additional , sensory - evoked spikes across layers , suggesting that l2/3 and l5 have distinct sensory coding strategies ( sakata and harris , 2009 ) . fast , laminar - specific manipulations of synchronized activity in trained mice are now required to define the causal role of translaminar synchrony in perception . blind whole - cell patch - clamp recordings were made from primary somatosensory forepaw cortex located by intrinsic optical imaging .

proteinaceous infectious particles , or prions , are self - perpetuating alternate conformations of proteins that are responsible for heritable non - mendelian traits in mammals , filamentous fungi , and yeast ( 15 ) . the yeast saccharomyces cerevisiae hosts many structurally and functionally unrelated proteins with prion properties ( 6 ) . [ psi ] is undoubtedly the best - known yeast prion and results from the conversion of the translation termination factor sup35p into fibrillar protein aggregates ( 6 ) . sup35p can populate structurally different heritable conformations that are at the origin of different [ psi ] strains , generally defined as weak or strong with respect to the severity of the nonsense suppression phenotypes they confer ( 6 ) . yeast prions are faithfully transmitted from mother to daughter cell during cell division or upon cytoplasmic mixing during mating . it is also possible to induce the prion state efficiently by introducing cytosolic fractions from prion - containing strains or prions assembled in vitro within prion - free yeast cells ( 7 , 8) . cells from the three domains of life , bacteria , archaea , and eukarya , secrete small membrane vesicles in the extracellular space ( 911 ) . these extracellular vesicles ( ev ) comprise exosomes ( ~30 to 100 nm ) , which originate from the fusion of multivesicular bodies with the plasma membrane , and microvesicles ( or ectosomes ) ( ~100 to 1,000 nm ) , which form directly from the plasma membrane ( 911 ) . ev have gained increased interest as their potential roles as vehicles for the intercellular transfer of nucleic acids , signaling molecules , and pathogenic factors have been uncovered over the past years ( 12 , 13 ) . ev also mediate clearance and possibly cell - to - cell transfer of protein aggregates associated with neurodegenerative diseases ( 14 ) . in yeasts and fungi , ev mediate the export of a wide range of proteins , lipids , rna , and polysaccharides ( 1519 ) . here we assess whether ev are vehicles for the export of [ psi ] prions in yeast . we purified ev from the extracellular medium of [ psi ] and strong or weak [ psi ] yeast cultures and found that they all contained sup35p . importantly , we demonstrate that sup35p within ev produced by strong and weak [ psi ] cells is in its aggregated infectious prion state . these findings are of importance , as they not only shed light on a new pathway for prion export in yeast but also provide a tool to investigate horizontal cell - to - cell transfer of genetic and epigenetic information in yeasts and possibly fungi . we first determined that maximal recovery of ev was achieved from the extracellular medium of yeast cultures grown to early stationary phase in rich yeast extract - peptone - dextrose ( ypd ) medium ( see text s1 in the supplemental material ) . we estimated that our purification procedure yielded ~2 10 ev per liter of extracellular medium under these conditions , which corresponds to a final ratio of ~200 ev per yeast cell ( calculation details can be found in text s1 ) . yeast ev were purified from a [ psi ] strain and from [ psi ] and [ psi ] strains , bearing strong and weak prion variants , respectively , by using the optimized protocol described in text s1 in the supplemental material ( see also fig . ev preparations from all three strains had similar electrophoretic protein profiles when examined by sds - page ( see fig . s1b ) and contained , as expected , membrane vesicles visible by electron microscopy ( see fig . the morphology of these vesicles was consistent from one preparation to another , most of them being round , ovoid , or cup - shaped ( see fig . the vesicle diameters spanned between ~20 and ~300 nm , with most ev in the ~50-to-100-nm range ( see fig . thus , harboring or not strong and weak [ psi ] prion strains affected neither the yeast cells abilities to produce and secrete ev nor the size and morphology of ev . we found significant amounts of sup35p in ev isolated from both [ psi ] and strong or weak [ psi ] cells ( fig . slight variations in the levels of sup35p detected in ev were observed in biological replicates , e.g. , independent preparations ( data not shown ) . ev isolated from [ psi ] cells contained in a consistent manner less sup35p than those isolated from [ psi ] cells ( see fig . cytosolic molecular chaperones of the hsp70 ( ssa1p ) , hsp110 ( sse1p ) , and hsp40 ( sis1p ) families were detected within ev preparations ( fig . these also contained the clathrin heavy - chain protein chc1p and the endoplasmic reticulum hsp70 family member kar2p , but they did not contain detectable amounts of proteasomes ( 20s ) or the ydj1p and hsp104 molecular chaperones ( fig . most of the proteins we found associated with ev were also identified in a proteomic study of yeast ev proteins ( 17 ) . the reasons why sup35p was not identified in that study are unclear , but this may have been due to the different yeast genetic backgrounds used and/or to the different growth conditions , particularly the use of sabouraud dextrose broth instead of ypd agar ( ypda ) medium and a growth temperature of 25c instead of 30c ( 17 ) . all of these parameters may have consequences on protein expression levels and ultimately on the composition of the ev proteome . furthermore , the yeast strains used by oliveira et al . were likely in the [ psi ] state and thus contained less sup35p than our [ psi ] cells ( for instance , see fig . alternatively , the identification of sup35p may have not withstood the stringent filtering criteria that were applied at each step of the mass spectrometry analysis ( 17 ) . immunogold labeling and electron microscopy revealed the presence of sup35p inside triton x-100-permeabilized ev ( fig . ev were not stained by the secondary gold - conjugated antibody in the absence of primary anti - sup35p antibodies , confirming that the immunogold labeling procedure was specific for sup35p ( see fig . most ( ~75% ) sup35p - positive vesicles had a diameter of ~30 to 100 nm , suggesting that they correspond to exosomes ( see fig . we demonstrated the ev intraluminal localization of sup35p and hsp70 by subjecting ev to proteinase k treatment . sup35p and hsp70 resisted cleavage in intact ev but not in the presence of triton x-100 ( fig . sup35p and hsp70 colocalized with ev proteins in sucrose gradients , and their flotation in the gradients was altered upon solubilization of ev with triton x-100 ( fig . furthermore , sup35p and hsp70 remained associated with ev in sucrose gradient separations under high ionic strength ( see fig . these data show that sup35p is packaged inside vesicles and secreted in the extracellular medium , both in [ psi ] and weak or strong [ psi ] cells . sup35p is localized inside ev produced by [ psi ] and [ psi ] cells . ( a ) the indicated strains were grown to early stationary phase in ypda medium . cell lysates and ev were then prepared and analyzed by sds - page and western blotting using antibodies against the indicated proteins . ( b ) ev were fixed with 2% paraformaldehyde , adsorbed onto electron microscopy grids , and permeabilized with 0.02% triton x-100 for 10 min . immunogold labeling of sup35p was then performed using primary polyclonal anti - sup35p antibody and secondary anti - rabbit 10-nm - gold - conjugated antibodies . electron microscopy grids were fixed with 1% glutaraldehyde for 5 min at room temperature and visualized by negative - stain electron microscopy . ( c ) purified ev were incubated with or without 2% triton x-100 for 30 min on ice , before the addition of 0.01 mg ml proteinase k. reaction mixtures were further incubated for 15 min on ice and stopped with the addition of 2 mm phenylmethylsulfonyl fluoride . an untreated control reaction mixture ( without triton x-100 and without proteinase k ) was run in parallel under identical conditions . reaction products were then analyzed by sds - page and western blotting using the indicated antibodies . ( d ) purified ev produced by [ psi ] cells were incubated with or without 1% triton x-100 for 30 min at 4c . the mixture was adjusted to 60% sucrose in 20 mm hepes - oh ( ph 7.5 ) and deposited in the bottom of an ultracentrifuge tube . equal volumes of 40% and 20% ( wt / wt ) sucrose solutions in 20 mm hepes - oh ( ph 7.5 ) were successively layered on top of the ev suspensions . following centrifugation for 17 h at 150,000 g and at 4c , 10 fractions were collected from the top of the gradients and analyzed by sds - page and western blotting using the indicated antibodies ( t stands for total and represents the input material ) . remarkably , sdd - age analysis revealed that sup35p in ev prepared from [ psi ] cells migrates as high - molecular - weight sds - resistant polymers , whereas only monomeric sup35p was detected in ev prepared from [ psi ] cells ( fig . 2a , right panel ) . as reported previously ( 20 ) , the size distribution of sds - resistant sup35p polymers visualized by sdd - age changed upon entry of cells into stationary phase ( fig . 2a ; compare log - phase and early - stationary - phase [ psi ] samples ) . sup35p sds - resistant polymers appeared slightly larger in [ psi ] than in [ psi ] cell lysates , regardless of the growth phase , as reported previously ( 21 , 22 ) . the apparent molecular weight of sup35p polymers within [ psi ] ev appeared much higher than that of sup35p polymers in lysates of the early - stationary - phase cells from which they originated ( fig . 2a ; compare early - stationary - phase and ev [ psi ] samples ) . the size distribution of sup35p polymers within [ psi ] ev was , however , comparable to that of sup35p polymers found within lysates prepared from exponentially growing cells ( fig . these observations suggest that most sup35p prion particles are packaged during the exponential phase of growth before being progressively released in the extracellular medium , possibly in relation to glucose consumption ( 23 ) . alternatively , conformational rearrangements of sup35p prion particles within ev could also account for the observed size differences between intracellular and intravesicular high - molecular - weight species . regardless , our data suggest that both soluble and aggregated sup35p molecular species are released from cells via export in ev . these results were confirmed by the finding that green fluorescent protein ( gfp)-tagged sup35p ( see text s1 in the supplemental material ) displayed a punctate pattern ( characteristic of aggregates ) and a diffuse dim pattern ( characteristic of soluble entities ) in ev purified from [ psi ] and [ psi ] cells , respectively , as visualized by fluorescence microscopy ( see fig . sup35p within [ psi ] ev is in its aggregated strain - specific prion state . ( a ) cell lysates ( 20 g ) from the indicated strains grown to log phase or early stationary phase and purified ev ( 20 g ) were analyzed by sdd - age followed by immunoblotting using anti - sup35p antibodies . the right panel corresponds to a longer exposure of the same membrane ( only ev lanes are shown ) . ( b ) ev and cell lysates were prepared as described for panel a. all samples ( ~1 to 10 g total protein ) were normalized by semiquantitative western blotting to contain equivalent amounts of sup35p and were then transformed into [ psi ] spheroplasts . random clones from each transformation reaction were then patched onto 1/4-ypd plates to assess their prion phenotype and to determine the [ psi ] conversion efficiency ( mean standard error ) . transformation with [ psi ] ev or cell lysates did not yield any [ psi ] clones , and the [ psi ] conversion frequency was then equal to 0 . ( c ) [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] or [ psi ] ev , and [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] remarkably , sup35p prion particles within ev prepared from [ psi ] cells were able to confer the prion state when transformed into [ psi ] cells ( see text s1 in the supplemental material for details ) ( fig . [ psi ] formation frequencies obtained upon transformation of [ psi ] spheroplasts with ev or cell lysates normalized to contain comparable amounts of aggregated sup35p were within similar ranges ( fig . indeed , ev originating from strong [ psi ] and weak [ psi ] cells induced the formation of strong and weak [ psi ] cells , respectively , in the protein transformation experiments ( fig . 2c ) . as expected , [ psi ] and [ psi ] cell lysates induced the formation of strong and weak [ psi ] clones , respectively , in control experiments ( see fig . , our study provides the first evidence for ev - mediated export of a prion in yeast ( fig . 1 ) . of particular importance is the finding that prion particles within ev fully retain their infectious and strain - specific prion state ( fig . 2 ) , meaning they could in principle mediate horizontal cell - to - cell propagation of yeast prions . we demonstrated using immunogold labeling and electron microscopy , proteolysis resistance assays , and flotation assays that both soluble and aggregated sup35p are within ev , not at their surface ( fig . sup35p was mostly found inside vesicles with a diameter of ~30 to 100 nm , e.g. , exosomes ( fig . was detected in larger vesicles ( ~100 to 180 nm in diameter ) that may , or may not , correspond to ectosomes directly originating from plasma membrane budding ( fig . 1b s3 ) ( 9 , 11 ) . given the limited differences in size and density , we were not able to obtain homogeneous preparations of each type of vesicle . in addition , specific differences in cargo content that may be helpful in distinguishing exosomes from ectosomes have yet to be established in yeast . therefore , other strategies will be required to ascertain the exosomal or ectosomal origin of sup35p - containing ev . important questions that also need to be addressed in future studies include how aggregated sup35p prion particles are addressed to vesicles and which cellular factors ( e.g. , molecular chaperones ) and/or cellular structures ( e.g. , cytoskeleton ) assist in this process . sup35p is a cytosolic protein that does not , to the extent of our knowledge , enter the secretory pathway . surprisingly though , both soluble and aggregated sup35p were found in ev ( fig . 2 ; see also fig . s6 in the supplemental material ) . a plausible explanation for this localization would be either that sup35p is secreted to the extracellular space or that it is directed toward autophagic destruction , or both ( 9 , 24 ) . the stochastic trapping of sup35p into prevesicular structures is unlikely to account for sup35p encapsulation within ev . indeed , only a subset of cytosolic molecular chaperones was found within ev ( fig . the sizes of sds - resistant sup35p assemblies within [ psi ] ev and cell lysates from log - phase [ psi ] cells were alike ( fig . these sup35p assemblies were , however , much larger than those found in lysates prepared from the early - stationary - phase [ psi ] cells harvested at the end of culturing ( fig . this observation suggests that the packaging of sup35p aggregates within vesicles occurs in actively dividing cells . entry into stationary phase upon glucose exhaustion may significantly slow vesicle formation and trafficking , but it may in turn trigger the release of preformed vesicles in the extracellular space ( 23 ) . in line with this notion , we were not able to isolate significant amounts of ev from cell cultures in the log phase of growth ( data not shown ) . although less likely , we can not rule out the alternate possibility that sup35p aggregates are remodeled within ev or that bigger aggregates are preferentially targeted for ev - mediated release . regardless , sup35p prion particles within ev fully retained their infectious potential ( fig . 2b ) and their ability to transmit their strain - specific conformations ( fig . 2c ) , indicating that they are unlikely to represent dead - end aggregation products . in light of our data , ev - mediated export of prions does not seem to constitute a prion clearance mechanism , as [ psi ] is faithfully maintained during growth . however , packaging and export of prions ( and possibly that of other aggregated or misfolded protein species ) in ev may constitute a way to reduce the burden on the cellular protein quality - control machineries and improve cell fitness . our data show that ev constitute a natural source of infectious sup35p particles , raising the intriguing possibility that ev could be new mediators of prion spreading among yeasts . our attempts to induce prion formation by incubating [ psi ] cells with purified ev or with ev - containing conditioned medium from [ psi ] cells were , as per now , inconclusive but did not yet rule out the existence of such a cell - to - cell transfer of yeast prions . uptake of exogenous vesicles by yeasts requires overcoming the cell wall physical barrier , which appears to be an unsurmountable obstacle for such big particles , at least under standard laboratory growth conditions ( 11 ) . the efficient internalization of ev trapped between the plasma membrane and the cell wall has been demonstrated to occur when the growth medium is replenished with glucose ( 23 ) . in contrast , neither the mechanism by which ev are able to cross the yeast cell wall to be released in the medium nor that of ev capture and uptake from the medium are known ( 11 ) . these processes may require particular and yet - to - be - identified growth conditions and/or environmental cues that may , for example , induce changes in the mechanical properties of the cell wall ( 25 ) . intercellular transfer of ev may also require direct cell - to - cell contacts , such as those occurring in colonies or biofilms . we can envision conditions in the wild where multiple individuals within a microbial community yeast and/or other microbes share a cell wall or analogous polysaccharide extracellular matrix that allows the exchange of information via the secretion and uptake of ev . ev are under intense scrutiny from the biomedical research community due to the key roles they may play in horizontal cell - to - cell communication and in the transfer of pathogenic and virulence factors , to their potential use as diagnostic markers in many cancers or neurodegenerative diseases , and to their use as vehicles for drug delivery . the findings we report here establish yeast prions as powerful tractable biological models to specifically investigate the molecular mechanisms of vesicle - mediated export of infectious protein assemblies and as a new tool to investigate the ev - mediated horizontal transfer of information in yeasts and fungi . we first determined that maximal recovery of ev was achieved from the extracellular medium of yeast cultures grown to early stationary phase in rich yeast extract - peptone - dextrose ( ypd ) medium ( see text s1 in the supplemental material ) . we estimated that our purification procedure yielded ~2 10 ev per liter of extracellular medium under these conditions , which corresponds to a final ratio of ~200 ev per yeast cell ( calculation details can be found in text s1 ) . yeast ev were purified from a [ psi ] strain and from [ psi ] and [ psi ] strains , bearing strong and weak prion variants , respectively , by using the optimized protocol described in text s1 in the supplemental material ( see also fig . ev preparations from all three strains had similar electrophoretic protein profiles when examined by sds - page ( see fig . s1b ) and contained , as expected , membrane vesicles visible by electron microscopy ( see fig . the morphology of these vesicles was consistent from one preparation to another , most of them being round , ovoid , or cup - shaped ( see fig . the vesicle diameters spanned between ~20 and ~300 nm , with most ev in the ~50-to-100-nm range ( see fig . thus , harboring or not strong and weak [ psi ] prion strains affected neither the yeast cells abilities to produce and secrete ev nor the size and morphology of ev . we found significant amounts of sup35p in ev isolated from both [ psi ] and strong or weak [ psi ] cells ( fig . slight variations in the levels of sup35p detected in ev were observed in biological replicates , e.g. , independent preparations ( data not shown ) . ev isolated from [ psi ] cells contained in a consistent manner less sup35p than those isolated from [ psi ] cells ( see fig . cytosolic molecular chaperones of the hsp70 ( ssa1p ) , hsp110 ( sse1p ) , and hsp40 ( sis1p ) families were detected within ev preparations ( fig . these also contained the clathrin heavy - chain protein chc1p and the endoplasmic reticulum hsp70 family member kar2p , but they did not contain detectable amounts of proteasomes ( 20s ) or the ydj1p and hsp104 molecular chaperones ( fig . most of the proteins we found associated with ev were also identified in a proteomic study of yeast ev proteins ( 17 ) . the reasons why sup35p was not identified in that study are unclear , but this may have been due to the different yeast genetic backgrounds used and/or to the different growth conditions , particularly the use of sabouraud dextrose broth instead of ypd agar ( ypda ) medium and a growth temperature of 25c instead of 30c ( 17 ) . all of these parameters may have consequences on protein expression levels and ultimately on the composition of the ev proteome . furthermore , the yeast strains used by oliveira et al . were likely in the [ psi ] state and thus contained less sup35p than our [ psi ] cells ( for instance , see fig . alternatively , the identification of sup35p may have not withstood the stringent filtering criteria that were applied at each step of the mass spectrometry analysis ( 17 ) . immunogold labeling and electron microscopy revealed the presence of sup35p inside triton x-100-permeabilized ev ( fig . ev were not stained by the secondary gold - conjugated antibody in the absence of primary anti - sup35p antibodies , confirming that the immunogold labeling procedure was specific for sup35p ( see fig . most ( ~75% ) sup35p - positive vesicles had a diameter of ~30 to 100 nm , suggesting that they correspond to exosomes ( see fig . we demonstrated the ev intraluminal localization of sup35p and hsp70 by subjecting ev to proteinase k treatment . sup35p and hsp70 resisted cleavage in intact ev but not in the presence of triton x-100 ( fig . sup35p and hsp70 colocalized with ev proteins in sucrose gradients , and their flotation in the gradients was altered upon solubilization of ev with triton x-100 ( fig . furthermore , sup35p and hsp70 remained associated with ev in sucrose gradient separations under high ionic strength ( see fig . these data show that sup35p is packaged inside vesicles and secreted in the extracellular medium , both in [ psi ] and weak or strong [ psi ] cells . sup35p is localized inside ev produced by [ psi ] and [ psi ] cells . ( a ) the indicated strains were grown to early stationary phase in ypda medium . cell lysates and ev were then prepared and analyzed by sds - page and western blotting using antibodies against the indicated proteins . ( b ) ev were fixed with 2% paraformaldehyde , adsorbed onto electron microscopy grids , and permeabilized with 0.02% triton x-100 for 10 min . immunogold labeling of sup35p was then performed using primary polyclonal anti - sup35p antibody and secondary anti - rabbit 10-nm - gold - conjugated antibodies . electron microscopy grids were fixed with 1% glutaraldehyde for 5 min at room temperature and visualized by negative - stain electron microscopy . ( c ) purified ev were incubated with or without 2% triton x-100 for 30 min on ice , before the addition of 0.01 mg ml proteinase k. reaction mixtures were further incubated for 15 min on ice and stopped with the addition of 2 mm phenylmethylsulfonyl fluoride . an untreated control reaction mixture ( without triton x-100 and without proteinase k ) was run in parallel under identical conditions . reaction products were then analyzed by sds - page and western blotting using the indicated antibodies . ( d ) purified ev produced by [ psi ] cells were incubated with or without 1% triton x-100 for 30 min at 4c . the mixture was adjusted to 60% sucrose in 20 mm hepes - oh ( ph 7.5 ) and deposited in the bottom of an ultracentrifuge tube . equal volumes of 40% and 20% ( wt / wt ) sucrose solutions in 20 mm hepes - oh ( ph 7.5 ) were successively layered on top of the ev suspensions . following centrifugation for 17 h at 150,000 g and at 4c , 10 fractions were collected from the top of the gradients and analyzed by sds - page and western blotting using the indicated antibodies ( t stands for total and represents the input material ) . remarkably , sdd - age analysis revealed that sup35p in ev prepared from [ psi ] cells migrates as high - molecular - weight sds - resistant polymers , whereas only monomeric sup35p was detected in ev prepared from [ psi ] cells ( fig . 2a , right panel ) . as reported previously ( 20 ) , the size distribution of sds - resistant sup35p polymers visualized by sdd - age changed upon entry of cells into stationary phase ( fig . 2a ; compare log - phase and early - stationary - phase [ psi ] samples ) . sup35p sds - resistant polymers appeared slightly larger in [ psi ] than in [ psi ] cell lysates , regardless of the growth phase , as reported previously ( 21 , 22 ) . the apparent molecular weight of sup35p polymers within [ psi ] ev appeared much higher than that of sup35p polymers in lysates of the early - stationary - phase cells from which they originated ( fig . 2a ; compare early - stationary - phase and ev [ psi ] samples ) . the size distribution of sup35p polymers within [ psi ] ev was , however , comparable to that of sup35p polymers found within lysates prepared from exponentially growing cells ( fig . these observations suggest that most sup35p prion particles are packaged during the exponential phase of growth before being progressively released in the extracellular medium , possibly in relation to glucose consumption ( 23 ) . alternatively , conformational rearrangements of sup35p prion particles within ev could also account for the observed size differences between intracellular and intravesicular high - molecular - weight species . regardless , our data suggest that both soluble and aggregated sup35p molecular species are released from cells via export in ev . these results were confirmed by the finding that green fluorescent protein ( gfp)-tagged sup35p ( see text s1 in the supplemental material ) displayed a punctate pattern ( characteristic of aggregates ) and a diffuse dim pattern ( characteristic of soluble entities ) in ev purified from [ psi ] and [ psi ] cells , respectively , as visualized by fluorescence microscopy ( see fig . sup35p within [ psi ] ev is in its aggregated strain - specific prion state . ( a ) cell lysates ( 20 g ) from the indicated strains grown to log phase or early stationary phase and purified ev ( 20 g ) were analyzed by sdd - age followed by immunoblotting using anti - sup35p antibodies . the right panel corresponds to a longer exposure of the same membrane ( only ev lanes are shown ) . ( b ) ev and cell lysates were prepared as described for panel a. all samples ( ~1 to 10 g total protein ) were normalized by semiquantitative western blotting to contain equivalent amounts of sup35p and were then transformed into [ psi ] spheroplasts . random clones from each transformation reaction were then patched onto 1/4-ypd plates to assess their prion phenotype and to determine the [ psi ] conversion efficiency ( mean standard error ) . transformation with [ psi ] ev or cell lysates did not yield any [ psi ] clones , and the [ psi ] conversion frequency was then equal to 0 . ( c ) [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] or [ psi ] ev , and [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] ev , were streaked onto 1/4-ypd plates to assess their prion phenotype . remarkably , sup35p prion particles within ev prepared from [ psi ] cells were able to confer the prion state when transformed into [ psi ] cells ( see text s1 in the supplemental material for details ) ( fig . [ psi ] formation frequencies obtained upon transformation of [ psi ] spheroplasts with ev or cell lysates normalized to contain comparable amounts of aggregated sup35p were within similar ranges ( fig . importantly , sup35p prion particles within ev also retained their strain - specific characteristics . indeed , ev originating from strong [ psi ] and weak [ psi ] cells induced the formation of strong and weak [ psi ] cells , respectively , in the protein transformation experiments ( fig . 2c ) . as expected , [ psi ] and [ psi ] cell lysates induced the formation of strong and weak [ psi ] clones , respectively , in control experiments ( see fig . , our study provides the first evidence for ev - mediated export of a prion in yeast ( fig . 1 ) . of particular importance is the finding that prion particles within ev fully retain their infectious and strain - specific prion state ( fig . 2 ) , meaning they could in principle mediate horizontal cell - to - cell propagation of yeast prions . we demonstrated using immunogold labeling and electron microscopy , proteolysis resistance assays , and flotation assays that both soluble and aggregated sup35p are within ev , not at their surface ( fig . sup35p was mostly found inside vesicles with a diameter of ~30 to 100 nm , e.g. , exosomes ( fig . was detected in larger vesicles ( ~100 to 180 nm in diameter ) that may , or may not , correspond to ectosomes directly originating from plasma membrane budding ( fig . s3 ) ( 9 , 11 ) . given the limited differences in size and density , we were not able to obtain homogeneous preparations of each type of vesicle . in addition , specific differences in cargo content that may be helpful in distinguishing exosomes from ectosomes have yet to be established in yeast . therefore , other strategies will be required to ascertain the exosomal or ectosomal origin of sup35p - containing ev . important questions that also need to be addressed in future studies include how aggregated sup35p prion particles are addressed to vesicles and which cellular factors ( e.g. , molecular chaperones ) and/or cellular structures ( e.g. , cytoskeleton ) assist in this process . sup35p is a cytosolic protein that does not , to the extent of our knowledge , enter the secretory pathway . surprisingly though , both soluble and aggregated sup35p were found in ev ( fig . 2 ; see also fig . s6 in the supplemental material ) . a plausible explanation for this localization would be either that sup35p is secreted to the extracellular space or that it is directed toward autophagic destruction , or both ( 9 , 24 ) . the stochastic trapping of sup35p into prevesicular structures is unlikely to account for sup35p encapsulation within ev . indeed , only a subset of cytosolic molecular chaperones was found within ev ( fig . the sizes of sds - resistant sup35p assemblies within [ psi ] ev and cell lysates from log - phase [ psi ] cells were alike ( fig . these sup35p assemblies were , however , much larger than those found in lysates prepared from the early - stationary - phase [ psi ] cells harvested at the end of culturing ( fig . this observation suggests that the packaging of sup35p aggregates within vesicles occurs in actively dividing cells . entry into stationary phase upon glucose exhaustion may significantly slow vesicle formation and trafficking , but it may in turn trigger the release of preformed vesicles in the extracellular space ( 23 ) . in line with this notion , we were not able to isolate significant amounts of ev from cell cultures in the log phase of growth ( data not shown ) . although less likely , we can not rule out the alternate possibility that sup35p aggregates are remodeled within ev or that bigger aggregates are preferentially targeted for ev - mediated release . regardless , sup35p prion particles within ev fully retained their infectious potential ( fig . 2b ) and their ability to transmit their strain - specific conformations ( fig . 2c ) , indicating that they are unlikely to represent dead - end aggregation products . in light of our data , ev - mediated export of prions does not seem to constitute a prion clearance mechanism , as [ psi ] is faithfully maintained during growth . however , packaging and export of prions ( and possibly that of other aggregated or misfolded protein species ) in ev may constitute a way to reduce the burden on the cellular protein quality - control machineries and improve cell fitness . our data show that ev constitute a natural source of infectious sup35p particles , raising the intriguing possibility that ev could be new mediators of prion spreading among yeasts . our attempts to induce prion formation by incubating [ psi ] cells with purified ev or with ev - containing conditioned medium from [ psi ] cells were , as per now , inconclusive but did not yet rule out the existence of such a cell - to - cell transfer of yeast prions . uptake of exogenous vesicles by yeasts requires overcoming the cell wall physical barrier , which appears to be an unsurmountable obstacle for such big particles , at least under standard laboratory growth conditions ( 11 ) . the efficient internalization of ev trapped between the plasma membrane and the cell wall has been demonstrated to occur when the growth medium is replenished with glucose ( 23 ) . in contrast , neither the mechanism by which ev are able to cross the yeast cell wall to be released in the medium nor that of ev capture and uptake from the medium are known ( 11 ) . these processes may require particular and yet - to - be - identified growth conditions and/or environmental cues that may , for example , induce changes in the mechanical properties of the cell wall ( 25 ) . intercellular transfer of ev may also require direct cell - to - cell contacts , such as those occurring in colonies or biofilms . we can envision conditions in the wild where multiple individuals within a microbial community yeast and/or other microbes share a cell wall or analogous polysaccharide extracellular matrix that allows the exchange of information via the secretion and uptake of ev . ev are under intense scrutiny from the biomedical research community due to the key roles they may play in horizontal cell - to - cell communication and in the transfer of pathogenic and virulence factors , to their potential use as diagnostic markers in many cancers or neurodegenerative diseases , and to their use as vehicles for drug delivery . the findings we report here establish yeast prions as powerful tractable biological models to specifically investigate the molecular mechanisms of vesicle - mediated export of infectious protein assemblies and as a new tool to investigate the ev - mediated horizontal transfer of information in yeasts and fungi . download text s1 , pdf file , 0.1 mb characterization of ev purified from [ psi ] , [ psi ] , and [ psi ] yeast cultures . ( a ) prion phenotypes of the strains used in this study were assessed on 1/4-ypd plates . ( b ) purified ev from the indicated strains were analyzed by sds - page and silver staining . ( d ) the size distribution of the indicated ev preparations was determined by analyzing electron micrographs , such as those shown in panel c , by using the imagej software . the number ( n ) of vesicles measured in each analysis is indicated ( three independent ev preparations were analyzed and combined for each strain ) . download figure s1 , pdf file , 0.2 mb slight variations in the levels of sup35p were observed among different ev preparations . purified ev ( ~3 g ) from [ psi ] or [ psi ] strains were analyzed by sds - page and western blotting by using anti - sup35p antibodies ( upper panel ) . the membrane was stained with amido black ( lower panel ) to reveal total ev proteins . download figure s2 , pdf file , 0.1 mb ev are not stained by gold - conjugated secondary antibodies in the absence of primary anti - sup35p antibody . ev were fixed with 2% paraformaldehyde , adsorbed onto electron microscopy grids , and permeabilized with 0.02% triton x-100 for 10 min . electron microscopy grids were incubated with anti - rabbit gold - conjugated antibodies , fixed with 1% glutaraldehyde , and then visualized by negative - stain electron microscopy . download figure s3 , pdf file , 0.8 mb size distribution of immunogold - labeled sup35p - positive vesicles . immunogold labeling of sup35p and negative - stain electron microscopy of ev isolated from the indicated strains were performed as described for fig . the size distribution of immunogold - labeled sup35p - positive vesicles was determined using the imagej software , and and results are presented as a box plot , showing the median ( middle line within each box ) , 25th ( lower boundaries of boxes ) and 75th ( upper boundaries of boxes ) percentiles , the minimal and maximal values ( whiskers ) , and outliers ( dots ) . the number of immunogold - labeled sup35p - positive vesicles analyzed for each ev type is indicated at the bottom of the plot . download figure s4 , pdf file , 0.1 mb sup35p and hsp70 colocalize with [ psi ] ev in discontinuous sucrose gradients under low- or high - salt conditions . purified ev from the [ psi ] strain were adjusted to 1.75 m sucrose in phosphate - buffered saline ( pbs ; low salt condition ) supplemented or not with 400 mm nacl ( high - salt condition ) and deposited at the bottom of ultracentrifugation tubes . equal volumes of 1.17 m and 0.58 m sucrose solutions in pbs ( low - salt condition ) supplemented or not with 400 mm nacl ( high - salt condition ) were layered on top of the ev suspensions . following centrifugation for 17 h at 150,000 g at 4c , eight fractions were collected from the tops of the gradients and analyzed by sds - page and coomassie blue staining ( upper panel ) or western blotting ( middle and lower panels ) , using the indicated antibodies ( t stands for total and represents the input material ) . ( b ) the fractions highlighted by colored dashed - line boxes from the sucrose gradients described for panel a were concentrated by centrifugation for 1 h at 100,000 g at 4c , washed in pbs , and then analyzed by immunogold labeling of sup35p and by negative - stain electron microscopy . download figure s5 , pdf file , 0.3 mb ev isolated from a [ psi ] sup35-gfp strain display a punctate fluorescence pattern . ( a ) negative - stained electron micrographs of ev preparations from the indicated strains . ( b ) ev preparations from the indicated strains were directly observed under a fluorescence microscope . download figure s6 , pdf file , 0.2 mb cell lysates from strong and weak [ psi ] strains induce strong and weak [ psi ] clones , respectively , in protein transformation assays . representative [ psi ] and [ psi ] clones obtained by transforming [ psi ] spheroplasts with the indicated cell lysates ( as described for fig .

abstractthe yeast saccharomyces cerevisiae harbors several prions that constitute powerful models to investigate the mechanisms of epigenetic structural inheritance . [ psi+ ] is undoubtedly the best - known yeast prion and results from the conversion of the translation termination factor sup35p into self - perpetuating protein aggregates . structurally different conformers of sup35p aggregates can lead to [ psi+ ] strains with weak or strong prion phenotypes . yeast prions are faithfully transmitted from mother to daughter cells during cell division , upon cytoplasmic mixing during mating , or when sup35p fibrils made in test tubes are introduced into spheroplasts . virtually all living cells in the three domains of life , bacteria , archaea , and eukarya , secrete small membrane vesicles in the extracellular space . these extracellular vesicles ( ev ) have gained increasing interest as vehicles for the intercellular transfer of signaling molecules , nucleic acids , and pathogenic factors , as well as prion - like protein aggregates associated with neurodegenerative diseases . to begin to explore the question of whether ev could represent a natural mean for yeast prion transmission from cell to cell , we purified these extracellular vesicles and assessed whether they contained sup35p . here , we show that sup35p is secreted within ev released in the extracellular medium of yeast cultures . we demonstrate that sup35p within ev isolated from strong and weak [ psi+ ] cells is in an infectious prion conformation . among the possible implications of our work is the possibility of previously unsuspected ev - mediated horizontal cell - to - cell transfer of fungal prions .

OBSERVATION Purification and characterization of yeast EV. Sup35p is localized inside EV originating from [ Sup35p within [ Packaging of soluble and aggregated Sup35p in EV. EV as vehicles for horizontal cell-to-cell transfer of yeast prions? Conclusions. SUPPLEMENTAL MATERIAL

proteinaceous infectious particles , or prions , are self - perpetuating alternate conformations of proteins that are responsible for heritable non - mendelian traits in mammals , filamentous fungi , and yeast ( 15 ) . the yeast saccharomyces cerevisiae hosts many structurally and functionally unrelated proteins with prion properties ( 6 ) . [ psi ] is undoubtedly the best - known yeast prion and results from the conversion of the translation termination factor sup35p into fibrillar protein aggregates ( 6 ) . sup35p can populate structurally different heritable conformations that are at the origin of different [ psi ] strains , generally defined as weak or strong with respect to the severity of the nonsense suppression phenotypes they confer ( 6 ) . yeast prions are faithfully transmitted from mother to daughter cell during cell division or upon cytoplasmic mixing during mating . cells from the three domains of life , bacteria , archaea , and eukarya , secrete small membrane vesicles in the extracellular space ( 911 ) . these extracellular vesicles ( ev ) comprise exosomes ( ~30 to 100 nm ) , which originate from the fusion of multivesicular bodies with the plasma membrane , and microvesicles ( or ectosomes ) ( ~100 to 1,000 nm ) , which form directly from the plasma membrane ( 911 ) . ev have gained increased interest as their potential roles as vehicles for the intercellular transfer of nucleic acids , signaling molecules , and pathogenic factors have been uncovered over the past years ( 12 , 13 ) . ev also mediate clearance and possibly cell - to - cell transfer of protein aggregates associated with neurodegenerative diseases ( 14 ) . here we assess whether ev are vehicles for the export of [ psi ] prions in yeast . we purified ev from the extracellular medium of [ psi ] and strong or weak [ psi ] yeast cultures and found that they all contained sup35p . importantly , we demonstrate that sup35p within ev produced by strong and weak [ psi ] cells is in its aggregated infectious prion state . these findings are of importance , as they not only shed light on a new pathway for prion export in yeast but also provide a tool to investigate horizontal cell - to - cell transfer of genetic and epigenetic information in yeasts and possibly fungi . we first determined that maximal recovery of ev was achieved from the extracellular medium of yeast cultures grown to early stationary phase in rich yeast extract - peptone - dextrose ( ypd ) medium ( see text s1 in the supplemental material ) . yeast ev were purified from a [ psi ] strain and from [ psi ] and [ psi ] strains , bearing strong and weak prion variants , respectively , by using the optimized protocol described in text s1 in the supplemental material ( see also fig . s1b ) and contained , as expected , membrane vesicles visible by electron microscopy ( see fig . thus , harboring or not strong and weak [ psi ] prion strains affected neither the yeast cells abilities to produce and secrete ev nor the size and morphology of ev . we found significant amounts of sup35p in ev isolated from both [ psi ] and strong or weak [ psi ] cells ( fig . slight variations in the levels of sup35p detected in ev were observed in biological replicates , e.g. ev isolated from [ psi ] cells contained in a consistent manner less sup35p than those isolated from [ psi ] cells ( see fig . cytosolic molecular chaperones of the hsp70 ( ssa1p ) , hsp110 ( sse1p ) , and hsp40 ( sis1p ) families were detected within ev preparations ( fig . most of the proteins we found associated with ev were also identified in a proteomic study of yeast ev proteins ( 17 ) . sup35p and hsp70 colocalized with ev proteins in sucrose gradients , and their flotation in the gradients was altered upon solubilization of ev with triton x-100 ( fig . these data show that sup35p is packaged inside vesicles and secreted in the extracellular medium , both in [ psi ] and weak or strong [ psi ] cells . sup35p is localized inside ev produced by [ psi ] and [ psi ] cells . following centrifugation for 17 h at 150,000 g and at 4c , 10 fractions were collected from the top of the gradients and analyzed by sds - page and western blotting using the indicated antibodies ( t stands for total and represents the input material ) . sup35p sds - resistant polymers appeared slightly larger in [ psi ] than in [ psi ] cell lysates , regardless of the growth phase , as reported previously ( 21 , 22 ) . these observations suggest that most sup35p prion particles are packaged during the exponential phase of growth before being progressively released in the extracellular medium , possibly in relation to glucose consumption ( 23 ) . alternatively , conformational rearrangements of sup35p prion particles within ev could also account for the observed size differences between intracellular and intravesicular high - molecular - weight species . these results were confirmed by the finding that green fluorescent protein ( gfp)-tagged sup35p ( see text s1 in the supplemental material ) displayed a punctate pattern ( characteristic of aggregates ) and a diffuse dim pattern ( characteristic of soluble entities ) in ev purified from [ psi ] and [ psi ] cells , respectively , as visualized by fluorescence microscopy ( see fig . sup35p within [ psi ] ev is in its aggregated strain - specific prion state . ( c ) [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] or [ psi ] ev , and [ psi ] clones , obtained by transforming [ psi ] spheroplasts with [ psi ] remarkably , sup35p prion particles within ev prepared from [ psi ] cells were able to confer the prion state when transformed into [ psi ] cells ( see text s1 in the supplemental material for details ) ( fig . indeed , ev originating from strong [ psi ] and weak [ psi ] cells induced the formation of strong and weak [ psi ] cells , respectively , in the protein transformation experiments ( fig . as expected , [ psi ] and [ psi ] cell lysates induced the formation of strong and weak [ psi ] clones , respectively , in control experiments ( see fig . , our study provides the first evidence for ev - mediated export of a prion in yeast ( fig . of particular importance is the finding that prion particles within ev fully retain their infectious and strain - specific prion state ( fig . 2 ) , meaning they could in principle mediate horizontal cell - to - cell propagation of yeast prions . we demonstrated using immunogold labeling and electron microscopy , proteolysis resistance assays , and flotation assays that both soluble and aggregated sup35p are within ev , not at their surface ( fig . was detected in larger vesicles ( ~100 to 180 nm in diameter ) that may , or may not , correspond to ectosomes directly originating from plasma membrane budding ( fig . sup35p is a cytosolic protein that does not , to the extent of our knowledge , enter the secretory pathway . a plausible explanation for this localization would be either that sup35p is secreted to the extracellular space or that it is directed toward autophagic destruction , or both ( 9 , 24 ) . the stochastic trapping of sup35p into prevesicular structures is unlikely to account for sup35p encapsulation within ev . these sup35p assemblies were , however , much larger than those found in lysates prepared from the early - stationary - phase [ psi ] cells harvested at the end of culturing ( fig . this observation suggests that the packaging of sup35p aggregates within vesicles occurs in actively dividing cells . entry into stationary phase upon glucose exhaustion may significantly slow vesicle formation and trafficking , but it may in turn trigger the release of preformed vesicles in the extracellular space ( 23 ) . in line with this notion , we were not able to isolate significant amounts of ev from cell cultures in the log phase of growth ( data not shown ) . although less likely , we can not rule out the alternate possibility that sup35p aggregates are remodeled within ev or that bigger aggregates are preferentially targeted for ev - mediated release . in light of our data , ev - mediated export of prions does not seem to constitute a prion clearance mechanism , as [ psi ] is faithfully maintained during growth . our data show that ev constitute a natural source of infectious sup35p particles , raising the intriguing possibility that ev could be new mediators of prion spreading among yeasts . our attempts to induce prion formation by incubating [ psi ] cells with purified ev or with ev - containing conditioned medium from [ psi ] cells were , as per now , inconclusive but did not yet rule out the existence of such a cell - to - cell transfer of yeast prions . in contrast , neither the mechanism by which ev are able to cross the yeast cell wall to be released in the medium nor that of ev capture and uptake from the medium are known ( 11 ) . these processes may require particular and yet - to - be - identified growth conditions and/or environmental cues that may , for example , induce changes in the mechanical properties of the cell wall ( 25 ) . intercellular transfer of ev may also require direct cell - to - cell contacts , such as those occurring in colonies or biofilms . ev are under intense scrutiny from the biomedical research community due to the key roles they may play in horizontal cell - to - cell communication and in the transfer of pathogenic and virulence factors , to their potential use as diagnostic markers in many cancers or neurodegenerative diseases , and to their use as vehicles for drug delivery . the findings we report here establish yeast prions as powerful tractable biological models to specifically investigate the molecular mechanisms of vesicle - mediated export of infectious protein assemblies and as a new tool to investigate the ev - mediated horizontal transfer of information in yeasts and fungi . we first determined that maximal recovery of ev was achieved from the extracellular medium of yeast cultures grown to early stationary phase in rich yeast extract - peptone - dextrose ( ypd ) medium ( see text s1 in the supplemental material ) . yeast ev were purified from a [ psi ] strain and from [ psi ] and [ psi ] strains , bearing strong and weak prion variants , respectively , by using the optimized protocol described in text s1 in the supplemental material ( see also fig . s1b ) and contained , as expected , membrane vesicles visible by electron microscopy ( see fig . thus , harboring or not strong and weak [ psi ] prion strains affected neither the yeast cells abilities to produce and secrete ev nor the size and morphology of ev . we found significant amounts of sup35p in ev isolated from both [ psi ] and strong or weak [ psi ] cells ( fig . slight variations in the levels of sup35p detected in ev were observed in biological replicates , e.g. ev isolated from [ psi ] cells contained in a consistent manner less sup35p than those isolated from [ psi ] cells ( see fig . cytosolic molecular chaperones of the hsp70 ( ssa1p ) , hsp110 ( sse1p ) , and hsp40 ( sis1p ) families were detected within ev preparations ( fig . most of the proteins we found associated with ev were also identified in a proteomic study of yeast ev proteins ( 17 ) . were likely in the [ psi ] state and thus contained less sup35p than our [ psi ] cells ( for instance , see fig . sup35p and hsp70 colocalized with ev proteins in sucrose gradients , and their flotation in the gradients was altered upon solubilization of ev with triton x-100 ( fig . these data show that sup35p is packaged inside vesicles and secreted in the extracellular medium , both in [ psi ] and weak or strong [ psi ] cells . sup35p is localized inside ev produced by [ psi ] and [ psi ] cells . following centrifugation for 17 h at 150,000 g and at 4c , 10 fractions were collected from the top of the gradients and analyzed by sds - page and western blotting using the indicated antibodies ( t stands for total and represents the input material ) . sup35p sds - resistant polymers appeared slightly larger in [ psi ] than in [ psi ] cell lysates , regardless of the growth phase , as reported previously ( 21 , 22 ) . the apparent molecular weight of sup35p polymers within [ psi ] ev appeared much higher than that of sup35p polymers in lysates of the early - stationary - phase cells from which they originated ( fig . these observations suggest that most sup35p prion particles are packaged during the exponential phase of growth before being progressively released in the extracellular medium , possibly in relation to glucose consumption ( 23 ) . alternatively , conformational rearrangements of sup35p prion particles within ev could also account for the observed size differences between intracellular and intravesicular high - molecular - weight species . these results were confirmed by the finding that green fluorescent protein ( gfp)-tagged sup35p ( see text s1 in the supplemental material ) displayed a punctate pattern ( characteristic of aggregates ) and a diffuse dim pattern ( characteristic of soluble entities ) in ev purified from [ psi ] and [ psi ] cells , respectively , as visualized by fluorescence microscopy ( see fig . remarkably , sup35p prion particles within ev prepared from [ psi ] cells were able to confer the prion state when transformed into [ psi ] cells ( see text s1 in the supplemental material for details ) ( fig . indeed , ev originating from strong [ psi ] and weak [ psi ] cells induced the formation of strong and weak [ psi ] cells , respectively , in the protein transformation experiments ( fig . as expected , [ psi ] and [ psi ] cell lysates induced the formation of strong and weak [ psi ] clones , respectively , in control experiments ( see fig . of particular importance is the finding that prion particles within ev fully retain their infectious and strain - specific prion state ( fig . 2 ) , meaning they could in principle mediate horizontal cell - to - cell propagation of yeast prions . we demonstrated using immunogold labeling and electron microscopy , proteolysis resistance assays , and flotation assays that both soluble and aggregated sup35p are within ev , not at their surface ( fig . was detected in larger vesicles ( ~100 to 180 nm in diameter ) that may , or may not , correspond to ectosomes directly originating from plasma membrane budding ( fig . sup35p is a cytosolic protein that does not , to the extent of our knowledge , enter the secretory pathway . a plausible explanation for this localization would be either that sup35p is secreted to the extracellular space or that it is directed toward autophagic destruction , or both ( 9 , 24 ) . the stochastic trapping of sup35p into prevesicular structures is unlikely to account for sup35p encapsulation within ev . these sup35p assemblies were , however , much larger than those found in lysates prepared from the early - stationary - phase [ psi ] cells harvested at the end of culturing ( fig . this observation suggests that the packaging of sup35p aggregates within vesicles occurs in actively dividing cells . entry into stationary phase upon glucose exhaustion may significantly slow vesicle formation and trafficking , but it may in turn trigger the release of preformed vesicles in the extracellular space ( 23 ) . in line with this notion , we were not able to isolate significant amounts of ev from cell cultures in the log phase of growth ( data not shown ) . although less likely , we can not rule out the alternate possibility that sup35p aggregates are remodeled within ev or that bigger aggregates are preferentially targeted for ev - mediated release . in light of our data , ev - mediated export of prions does not seem to constitute a prion clearance mechanism , as [ psi ] is faithfully maintained during growth . our data show that ev constitute a natural source of infectious sup35p particles , raising the intriguing possibility that ev could be new mediators of prion spreading among yeasts . our attempts to induce prion formation by incubating [ psi ] cells with purified ev or with ev - containing conditioned medium from [ psi ] cells were , as per now , inconclusive but did not yet rule out the existence of such a cell - to - cell transfer of yeast prions . in contrast , neither the mechanism by which ev are able to cross the yeast cell wall to be released in the medium nor that of ev capture and uptake from the medium are known ( 11 ) . these processes may require particular and yet - to - be - identified growth conditions and/or environmental cues that may , for example , induce changes in the mechanical properties of the cell wall ( 25 ) . intercellular transfer of ev may also require direct cell - to - cell contacts , such as those occurring in colonies or biofilms . ev are under intense scrutiny from the biomedical research community due to the key roles they may play in horizontal cell - to - cell communication and in the transfer of pathogenic and virulence factors , to their potential use as diagnostic markers in many cancers or neurodegenerative diseases , and to their use as vehicles for drug delivery . the findings we report here establish yeast prions as powerful tractable biological models to specifically investigate the molecular mechanisms of vesicle - mediated export of infectious protein assemblies and as a new tool to investigate the ev - mediated horizontal transfer of information in yeasts and fungi . download text s1 , pdf file , 0.1 mb characterization of ev purified from [ psi ] , [ psi ] , and [ psi ] yeast cultures . download figure s1 , pdf file , 0.2 mb slight variations in the levels of sup35p were observed among different ev preparations . immunogold labeling of sup35p and negative - stain electron microscopy of ev isolated from the indicated strains were performed as described for fig . the size distribution of immunogold - labeled sup35p - positive vesicles was determined using the imagej software , and and results are presented as a box plot , showing the median ( middle line within each box ) , 25th ( lower boundaries of boxes ) and 75th ( upper boundaries of boxes ) percentiles , the minimal and maximal values ( whiskers ) , and outliers ( dots ) . following centrifugation for 17 h at 150,000 g at 4c , eight fractions were collected from the tops of the gradients and analyzed by sds - page and coomassie blue staining ( upper panel ) or western blotting ( middle and lower panels ) , using the indicated antibodies ( t stands for total and represents the input material ) . ( b ) the fractions highlighted by colored dashed - line boxes from the sucrose gradients described for panel a were concentrated by centrifugation for 1 h at 100,000 g at 4c , washed in pbs , and then analyzed by immunogold labeling of sup35p and by negative - stain electron microscopy . download figure s6 , pdf file , 0.2 mb cell lysates from strong and weak [ psi ] strains induce strong and weak [ psi ] clones , respectively , in protein transformation assays .

molecular simulations have made significant contributions in the prediction of chemical processes and thermodynamic properties with applications ranging from protein folding to enhanced oil recovery . the interactions between atoms , which induce motion in classical molecular dynamics ( md ) simulations or translation in monte carlo ( mc ) simulations , are calculated using force fields . in general , force fields consist of simplified pairwise potential energy functions with associated atom - type dependent parameters . various force fields have been developed differing in genericity , coarsening , accuracy , fitted properties , and the used set of molecules within the optimization . we mention three well - known force fields : amber , compass , and opls . the amber force field is designed for simulation of proteins and nucleic acids . amber was originally developed as a united - atom force field ; however , a more accurate all - atom representation was published 2 years later . an extended version , including parameters optimized for organic molecules , is known as the general amber force field ( gaff ) . within both amber and gaff , the van der waals and hydrogen bonding parameters are obtained from crystal structures and lattice energies . the atomic partial charges , required for electrostatic interactions , are derived using ab initio quantum mechanics . the all - atom compass and compass ii force fields are developed for simulations of organic molecules , inorganic small molecules , and polymers . the van der waals parameters are obtained by fitting enthalpy of vaporizations and densities , calculated using md simulations , to experimental data . the atomic partial charges are derived using ab initio quantum mechanics and empirically adjusted to take hydrogen bonding effects into account . the opls force field ( published in a united - atom version and an all - atom version ) and improved opls3 force field are created for liquid simulations containing organic molecules and proteins . the van der waals parameters are , comparable to compass , optimized using experimental liquid properties , mainly enthalpy of vaporizations and densities . the atomic partial charges are derived using quantum calculations as well as using experimental condensed - phase properties . a reliable md or mc simulation fully depends on the quality of the force field . the accuracy of various force fields is extensively tested by van der spoel et al . the opls - aa force field outperforms gaff in the prediction of experimental enthalpy of vaporization with a measured root - mean - square error ( rmse ) of 6.5 kj / mol versus 10.6 kj / mol . for the calculation of hydration free energies and solvation free energies using nonpolarizable force fields , obtained a minimum average unsigned error ( aue ) of 1.03 kcal / mol (= 4.3 kj / mol ) , mobley et al . obtained a minimum rmse of 1.00 kcal / mol (= 4.2 kj / mol ) , sherman et al obtained for opls_2005 an aue of 1.10 kcal / mol (= 4.6 kj / mol ) , and van der spoel obtained for gaff a rmse of 3.7 kj / mol . for an accurate calculation of mixing free energies , the error should be significantly lower . the difference in free energy between an ideal binary mixture and a phase separated system is maximum 1.7 kj / mol at 298 k. an extensive accuracy test of mixing free energies calculated using md or mc is , to our knowledge , not published yet . jedlovszky et al . did perform mixing free energy calculations of several important binary mixtures using various force fields . although the maximum absolute error was always lower than 1 kbt (= 2.5 kj / mol at 298 k ) , the weakness of force fields was exposed . a mixture of acetone and water turned out to be immiscible at 298 k for all considered combinations of force fields with the exception of the combination of the acetone model developed by pereyra , asar , and carignano with the tip5p - e water model . also none of the force field combinations were able to predict a positive entropy of mixing at a dmso mole fraction below 0.8 for a mixture of dmso and water . a disadvantage of md and mc simulations is the extensive computational time which is often required to obtain precise results . a less time - consuming approach to obtain mixing free energies is the use of methods based on the quasi - chemical approximation of guggenheim . quasi - chemical models represent condensed molecular mixtures as a collection of independent interacting pairs of sites or molecules . the probability to obtain an interacting pair is derived from the theory of chemical reactions . the quasi - chemical approximation is basically an improvement of the regular solution model which assumes a random distribution of interacting pairs of sites or molecules . the two best known quasi - chemical methods are cosmo - rs and unifac . the cosmo - rs model represents liquid mixtures as a collection of interacting charged surface panels , whereas the unifac model represents liquid mixtures as a collection of interacting functional groups . we recently published a quasi - chemical method based on force fields : the pair configuration to molecular activity coefficient ( pac - mac ) model . the pac - mac model represents molecular mixtures as a collection of interacting molecular pairs . activity coefficients and related mixing free energies are calculated using a large set of sampled molecular pair configurations . the rmse of pac - mac in the calculation of mixing free energies using the opls - aa force field turned out to be 0.153 kbt ( or 0.43 kj / mol ) , and the calculation time is only a fraction of the required simulation time of corresponding md or mc simulations . since only two - body interactions are calculated within pac - mac , in order to reduce calculation time , we expect the accuracy of md and mc , which involve n - body interactions , to be higher . we perform an accuracy test of the opls - aa force field based on mixing free energies calculated using thermodynamic integration ( ti ) of md simulations for an extensive set of equimolar binary mixtures and compare the results with the pac - mac model . we improve the accuracy of the pac - mac method by optimizing force field parameters and pac - mac specific parameters . we check whether or not the optimized force field parameters induce accuracy improvement of the mixing free energies calculated using ti of md simulations . first , a large data set of mixing free energies is useful for comparison between quasi - chemical thermodynamics and force field simulations and could be a new tool for further analysis of force field performance . second , pac - mac as a force field based quasi - chemical method performs , after optimization , comparable to existing quasi - chemical thermodynamics . the performance is achieved without the need for quantum calculations ( cosmo - rs ) or group assignments ( unifac ) but relies on well - established force field parameters . the amount of additional empirical parameters is very little in comparison with other quasi - chemical methods , especially in comparison with unifac which contains hundreds of parameters optimized using experimental miscibility data . subsequently , ti of md simulations on the same data set indicates that mixtures of most chemical classes are modeled quite well by the opls - aa force field , even though such experimental data was not used in the original force field parameter optimization . we indicate that a very modest modification of water charge assignment , by reduction of 6.4% in partial atomic charges , is already enough to improve the results on average by 0.3 kbt . finally , pac - mac could potentially serve as a fast proxy method for md simulations , with a correlation coefficient of 0.82 between both methods . such correlation could already be sufficient for the use of pac - mac as a fast indicator for new intermolecular force field parameters in case of missing interactions . full replacement of md , for force field parameter improvement , is a tantalizing prospect but not achieved fully here . we briefly consider quantitatively to what extent correlations need to be improved further to achieve such goal in future research . , we first explain the theory of thermodynamic integration for the calculation of free energies of mixing and then briefly summarize the pac - mac method . subsequently , within results and discussion , we first test the accuracy of the opls - aa force field in the prediction of mixing free energies using ti of md simulations . then , we optimize a set of force field parameters using the pac - mac method . finally , we calculate the predictive capacity of the obtained opls - pm force field using ti . all experimental and calculated free energies of mixing for 1053 binary mixtures are presented in the supporting information . in this section , we discuss the approach to calculate excess mixing free energies using ti of md simulations . also the formulation of the pac - mac method is briefly explained . for more information concerning the pac - mac method we calculate the molar excess gibbs free energy of mixing for an equimolar binary mixture of molecules a and b , so xa = xb = 0.5 . the excess gibbs free energy of mixing is given by1 in which the excess helmholtz free energy of mixing f and the excess molar volume vme are given by23 vm , a , vm , b , and vm , ab in eq 3 represent the molar volumes of respectively pure component a , pure component b , and a mixture of a and b with equal mole fractions . the molar volumes are calculated using md simulations in the npt ensemble with a barostat set to a virtual pressure p of 1 atm . f in eq 2 represents the excess helmholtz free energy caused by volume expansion or compression of an ideal gas mixture:4the other free energy terms in eq 2 are calculated using thermodynamic integration by switching on intermolecular interactions using a coupling parameter 5 in which ... indicates an ensemble average at a given value for , and ui( ) represents the total potential energy in a simulated frame of system i { a , b , ab}. the decoupled state , in which the molecules behave as an ideal gas , refers to = 0 , and the coupled state , in which the molecules fully interact , refers to = 1 . to avoid singularities , caused by the repulsive r term in the lennard - jones potential , we choose a scaling of and for respectively the van der waals and electrostatic interaction between two atoms i and j separated by a distance rij(32)67where ij and ij in eq 6 are respectively the energy and distance parameter of the lennard - jones potential between atom i and atom j , and qi in eq 7 is the partial charge of atom i. summation of uijvdw ( rij, ) and uijelec ( rij, ) over all combinations of atoms i and j in a simulated frame , using a cutoff radius of 12.5 , results in the total van der waals and electrostatic energy:89the integrand of eq 5 can be split in a van der waals and electrostatic part because other potential energy terms ( bond , angle , and torsion energies ) are not a function of 10 in which uivdw() and uielec() represent an ensemble average of the total inter- and intramolecular van der waals and electrostatic energies at a given value for . the ensemble average potential energies are evaluated using md simulations in the nvt ensemble at 11 evenly spaced values for : 0.0 , 0.1 , ... , 0.9 , and 1.0 . the integral of eq 5 is obtained using a not - a - knot spline fitted through uivdw() and uielec(). furthermore , the block averaging approach is used to predict the standard deviation of the calculated ensemble average potential energies and subsequently the standard deviation of the calculated mixing free energy . the force field parameters , used to calculate interactions between the atoms , are taken from the opls - aa force field . our protocol to obtain the excess mixing free energy of an equimolar binary mixture of molecules a and b is summarized below : 1 . a cubic box , with an edge length of 30 , is filled with randomly oriented molecule a to an amount wherein the density is equal to the experimental density of a. if the total amount of molecules in the box is less than 120 , then the edge length is increased to an extent at which 120 molecules are required for a density equal to the experimental density . the potential energy of the box is minimized using the quick - min method for a maximum of 10000 steps . an andersen thermostat is used to control the simulation temperature at the desired temperature . a group - based cutoff is used to truncate long - range van der waals and electrostatic interactions . we are aware that the use of a particle mesh ewald ( pme ) , to better incorporate long - range electrostatics , is more common in md simulations . however , a pme would increase the total calculation time by over a factor of 3 . moreover , the use of neutral subunits within the molecules in opls - aa is well suited for a group - based cutoff . we expect the added value of a pme to be negligible in comparison with a group - based cutoff of 12.5 , in accordance with the results obtained by piana et al . the equilibration of the system is continued for 25 ps in the npt ensemble . the simulation pressure is set to 1.0 atm , controlled by the andersen barostat . a production simulation is performed for 50 ps in the npt ensemble to calculate the average box volume . the ensemble average box volume is used to calculate the molar volume vm , a in eq 3 and eq 4 . the box volume is rescaled and fixed for the upcoming md simulations to the ensemble average box volume calculated in step 5 . the system is equilibrated for 25 ps in the nvt ensemble using a coupling parameter of = 1.0 for the van der waals and electrostatic potential given in eq 6 and eq 7 respectively . a production simulation is performed for 25 ps in the nvt ensemble using a coupling parameter of = 1.0 to calculate uavdw() and uaelec() in eq 10 . steps 7 and 8 are repeated for other coupling parameters { 0.9 , 0.8 , ... , 0.1}. 10 . the free energy difference fa between a fully interacting condensed system and an ideal gas system is calculated using eq 5 . 11 . steps 110 are repeated for a box filled with only molecule b and for a box filled with an equal amount of molecules a and b. 12 . the excess molar gibbs and helmholtz free energy of mixing are calculated using respectively eq 1 and eq 2 . the theoretical basis of pac - mac has been extensively explained in previous papers . the pac - mac method consists of three consecutive steps briefly explained below : surface generation , pair sampling , and free energy minimization . first , the surface of a molecule is defined by the outer surface of spheres around the atomic nuclei in the molecule . the radius of these spheres is given by11 in which kk is the lennard - jones distance parameter of atom k , and s is an atom - type independent multiplication factor with an optimized value of 0.62 . subsequently , the generated molecular surface of a molecule i is divided in li surface panels with an area of about 0.5 each . second , a sampling of pair configurations is performed between all possible combinations of two molecules in the mixture . a pair configuration represents a possible composition of two nearest neighboring molecules in a condensed phase ; therefore , the sampling is performed within the first coordination shell of the molecules . the total amount of sampled pair configurations between two molecules is m = 5 10 , by default . of each pair configuration i [ 1 .. m ] between molecules i and j , we track two properties for the last step of the pac - mac method . the first property is the intermolecular energy wiij calculated using the opls - aa force field , and the second property is a depiction of the surface panels j which are covered ( oiij = 1 ) and uncovered ( oiij = 0 ) by the neighboring molecule . examples of sampled pair configurations with occupied surface panels ( black colored ) and intermolecular energy for ethanol ethanol ( 1 ) , ethanol acetonitrile ( 2 ) , acetonitrile ethanol ( 3 ) , and acetonitrile acetonitrile ( 4 ) . in pac - mac , the expression for the free energy of mixing is based on the quasi - chemical approximation of guggenheim and is given by12the 5 contributing free energy terms in eq 12 are given by the entropy of mixing of an ideal solution ( f ) , a modified staverman ( f ) , the combinatorial expression according to the quasi - chemical approximation ( f ) , the entropic contribution due to inhomogeneities in the occupation of the molecular surface ( f ) , and the total intermolecular energy ( f ) . the expressions of the free energy terms in eq 12 are , for a binary mixture containing molecules a and b , given by1314151617 in which xi , i , and yi are respectively the mole , volume , and coordination fraction of component i { a , b } in the mixture , z represents the average coordination number , xiij is the fraction of pair configuration i between molecules i and j in the mixture , aji represents the surface area of surface panel j in component i , ai is the average occupied area in an interaction on molecule i , xj , ivac represents the unoccupied area fraction of surface panel j on molecule i , and x is the total unoccupied surface fraction of a molecule . only the latter variable , x , included in eq 16 , is an empirically obtained general parameter equal to 0.6 . the expression for the free energy , eq 12 , is minimized subject to the constraint stating the occupation fraction of each surface panel to be equal to the probability that a panel is covered by neighboring molecules:18finally , various thermodynamic miscibility properties can be obtained from the minimized expression for the free energy . examples of these properties are activity coefficients , flory huggins -interaction parameters , and vapor liquid equilibrium phase diagrams . we refer to the supporting information of a previously published paper for more details regarding the derivation of the activity coefficients . furthermore , an analytical expression is derived for the calculation of the standard deviation of the obtained mixing free energy . assuming the set of sampled pair configurations i [ 1 .. m ] between molecules i { a , b } and j { a , b } to be uncorrelated , then the following relation holds for the variance of the mixing free energy:19 the excess gibbs free energy of mixing , calculated using ti and eq 1 , is compared with experimental data from the nist database . for a binary mixture , containing molecules a and b , the experimental excess free energy of mixing is calculated using20with xiliq and i being respectively the mole fraction and activity coefficient of component i in the liquid phase . the activity coefficient of component i is calculated using the extended raoult s law assuming gaseous ideality21 in which xigas represents the mole fraction of component i in the vapor phase , and p(xiliq ) is the vapor pressure of the binary mixture at mole fraction xiliq taken from experimental vapor the used subset of the nist database , containing 1092 experimental excess free energies of mixing of equimolar binary mixtures , is presented in a previously published article . mixing free energies are not calculated with ti for 39 binary mixtures because intramolecular force field parameters are missing ( 32 binary mixtures ) or the experimental vapor pressure ( > 5 atm ) is far above the simulation pressure of 1 atm resulting in gas formation within the md simulation ( 7 binary mixtures ) . although the use of a higher simulation pressure would be justified in the latter case , because the pressure volume work term pv is often negligible in condensed phase systems , we decided to be consistent in the used pressure of 1 atm and remove the 7 mixtures from the data set . the combinations of molecules in the remaining set of 1053 binary mixtures are shown in figure 2 . the data set contains 159 different molecules which are clustered in 9 categories . the category molecules containing oxygen ( excluding alcohols ) contains 11 ketones , 4 aldehydes , 16 ethers , and 11 esters . the category molecules containing nitrogen contains 8 amines , 3 nitriles , and 3 nitrogen - containing aromatic compounds . dimethyl disulfide , 2-ethoxyethanol , and hexafluoroisopropanol are three examples of the 19 molecules with different or multiple functional groups . a comparison of the calculated excess gibbs free energy of mixing , by ti performed on md simulations using the opls - aa force field , with experimental data for 1053 binary mixtures is shown in figure 3 . the error bar attached to each point indicates the predicted standard deviation of the calculation , obtained using block averaging . the root mean squared error ( rmse ) is calculated using22 in which n is the amount of data points ( n = 1053 ) , and h is the index of a data point . note that mixtures with an obtained g above 0.69 kbt are metastable since this is the upper limit for binary mixtures to initiate phase separation . phase separation is not observed in the performed md simulations because of the relatively large surface tension that has to be overcome due to the limited size of the box . scatterplot of the excess gibbs free energy of mixing : ti using the opls - aa force field versus experimental data for 1053 binary mixtures . figure 3 clearly shows correlation between the free energy obtained using ti and experimental data . the obtained correlation coefficient of 0.79 is lower than the correlation coefficient for the prediction of densities ( = 0.99 ) and enthalpies of vaporization ( = 0.94 ) using opls - aa . however , this is expected for two reasons . first , the opls - aa force field parameters are optimized using experimental data of mainly enthalpies of vaporization and densities and not using experimental free energies of mixing . as a consequence , greater predictability second , the range of values for the excess free energy of mixing is much smaller than the range of values for the enthalpy of vaporization . the calculated mixing free energies vary between 0.5 kbt and 1.0 kbt , whereas the calculated enthalpies of vaporization are up to 40 kbt . therefore , a small absolute error can cause a big relative error . on the other hand , because of the smaller range of values , the observed rmse of 0.132 kbt ( or 0.37 kj / mol ) for the prediction of excess mixing free energies is much smaller than the rmse of 6.5 kj / mol for the prediction of enthalpies of vaporization . we find it remarkable that opls - aa gives such a small error , given that the data set was not included in the original parametrization . the accuracy of the opls - aa force field is even higher than indicated by the observed error , since the standard deviation of the ti calculation itself affects the observed rmse . two types of calculation errors contribute to the observed deviations from experiments : the error introduced by wrong values of the force field parameters and the standard deviation of the calculation itself . the average observed error is defined by rmseobs , the average effective error induced by the force field is defined by rmseeff , and the average predicted standard deviation of the ti calculations is defined by rmsestd . first , the standard deviations of the ti calculations are assumed to be independent of the errors induced by the force field . second , the standard deviations of the ti calculations and the errors induced by the force field are assumed to be the only factors affecting the observed errors . finally , the standard deviations of the ti calculations are assumed to be unbiased . if the three proposed assumptions are fulfilled , then the following relation holds between rmseobs , rmseeff , and rmsestd:23the root mean squared standard deviation of the ti calculations equals 0.064 kbt resulting in an effective rmse for the opls - aa force field of 0.115 kbt if infinitely long md simulations are performed . as shown in figure 3 , the opls - aa force field , as calculated by ti , performs quite well over all categories , except for binary mixtures containing water . the mixing free energies of binary mixtures containing water are , in general , overestimated by molecular simulations . the bias is caused by the used flexible tip3p water model for which the force field parameters are optimized using the enthalpy of vaporization and density of pure liquid water , so experimental miscibility data is not taken into account . show a similar systematic overestimation of hydration free energies calculated using the amber force field and a spc / e water model . the largest overestimation is obtained for hexafluoroisopropanol water at 298.15 k ( g experimental : 0.026 kbt , g ti : 0.850 kbt ) . the representation of the strongly electron withdrawing trifluoromethyl groups by nonpolarizable partial charges of the opls - aa force field might also affect the results in this case . tetrahydrofuran at 298.144 k ( g experimental : 0.586 kbt , g ti : 0.009 kbt ) . in accordance with the obtained overestimation for hexafluoroisopropanolwater , this result might have been affected by a weak representation of trifluoromethyl groups within the opls - aa force field as well . in accordance with the results of jedlovszky , we also observe acetone to be incorrectly immiscible with water at equal mole fraction . the observed mixing free energy is a positive 0.263 0.083 kj / mol at a temperature of 308 k. the helmholtz free energy of mixing for an equal molar acetone also this value is in accordance with the results of simulations performed by jedlovszky et al . , even though they fixed the internal coordinates of the atoms within the molecules ; they calculated a helmholtz free energy of mixing of 0.97 kj / mol using the comparable opls - ua force field . the pac - mac method predicts mixing free energies assuming incompressible fluids , so the excess molar volume vme equals 0 . as a consequence , there is no distinction between gibbs and helmholtz free energy of mixing in pac - mac : f = g. the pv term is usually negligible in condensed phase systems . mean squared value of pvme equals 4.4 10kbt ( or 0.13 j / mol ) . in a previous publication , the total observed rmse of pac - mac is proven to be 0.153 kbt , with a correlation coefficient of 0.695 , for the calculation of excess free energies in comparison with experimental data.figure 4 shows comparable results for the subset of 1053 binary mixtures . of note is that figure 4 is almost the same as figure 9 from our previously published paper . we included figure 4 for easier comparison with the information from the md simulations . first , the used database of binary mixtures for figure 4 is a subset of the used database for figure 9 in ref ( 30 ) . second , each data point in figure 4 contains an error bar , indicating the predicted standard deviation of the calculation . scatterplot of the excess gibbs free energy of mixing : pac - mac using the opls - aa force field versus experimental data for 1053 binary mixtures . error bars indicate the predicted standard deviation of the calculations . it is shown in figure 4 that the standard deviation is usually within the size of a dot . the values calculated using pac - mac are precise : the root mean squared standard deviation equals 0.014 kbt . therefore , the standard deviation only slightly reduces the effective rmse to 0.150 kbt , calculated using eq 23 , for an infinite amount of sampled pair configurations . the effective rmse of pac - mac is 30% higher than the effective rmse of ti caused by several assumptions made within the model . the three assumptions with the highest impact are neglecting multiple body interactions , neglecting interactions beyond the first coordination shell , and using an expression for the free energy which is not exact . md simulations , which contain no additional assumptions besides the force field , are therefore always more accurate than pac - mac using the same force field . the benefit of the assumptions made within pac - mac is a great reduction of calculation time : a g calculation using pac - mac takes 10 min on a single core ( intel xeon e5 - 2620 ) , whereas the same calculation using ti takes 5 days . so the calculation speed is increased with a factor 700 at the expense of a 14% reduction in accuracy . in principle , the reduction of calculation time is even much greater . a g calculation using ti requires on average the calculation of 1.0 10 molecular pair interactions , whereas a g calculation using pac - mac requires the calculation of only 2.0 10 molecular pair interactions . the calculation of the molecular pair interactions takes about 20% of the total calculation time of pac - mac . if we assume that the calculation time of a molecular pair interaction in pac - mac is equal to the calculation time of a molecular pair interaction in md , then the calculation of mixing free energies can potentially be 1.0 10 times faster using pac - mac than using ti . furthermore , in accordance with ti and shown in figure 4 , also pac - mac in general overestimates mixing free energies of binary mixtures containing water . water at 298.15k ( g experimental : 0.026 kbt , g pac - mac : 0.712 kbt ) . correlation in the predicted mixing free energy is expected since both methods use the same opls - aa force field . a proof of correlation between the results obtained by ti and pac - mac is given in figure 5 . scatterplot of the excess gibbs free energy of mixing : ti versus pac - mac using the opls - aa force field for 1053 binary mixtures . figure 5 shows a correlation coefficient of 0.815 between ti and pac - mac for the calculation of mixing free energies . indicating that the overestimation of g by pac - mac , shown in figure 4 , is , in accordance with ti , also caused by the tip3p water force field and not by other assumptions made within the model . the high correlation between ti and pac - mac offers opportunities to optimize force field parameters , required in md simulation , using pac - mac as a quick auxiliary method . an accurate predictive capacity is essential for a good auxiliary method . our protocol to optimize force field parameters consists of three consecutive steps : 1 . the correlation between ti and pac - mac is increased in order to improve pac - mac as the predictive auxiliary method . force field parameters are optimized by minimizing the rmse of pac - mac in comparison with experimental data . the accuracy of the optimized force field parameters in the prediction of mixing free energies is tested using ti . the correlation between ti and pac - mac is further increased by optimizing the x parameter in eq 16 and by introducing 16 atom - type dependent skseg instead of a single general s parameter equal to 0.62 in eq 11 . the different atom - types k are taken from the dreiding force field and are expressed in daylight smarts notation . the skseg and x parameters are optimized by minimizing the rmse of pac - mac compared to ti , in the calculation of excess free energies of mixing , using the gauss an upper and lower limit for skseg is set to 0.8 and 0.4 , respectively , to avoid unphysical values . an overview of the optimized skseg and x parameters is given in table 1 . the column # in dataset contains the amount of binary mixtures in which the parameter is present . figure 6 shows a reduction of the rmse and an increase in the correlation coefficient to respectively 0.109 kbt and 0.861 by using the optimized parameters shown in table 1 . scatterplot of the excess gibbs free energy of mixing : ti versus pac - mac using the opls - aa force field and optimized skseg and x parameters for 1053 binary mixtures . subsequently , the accuracy of the pac - mac model is increased by optimizing force field parameters given the skseg and x parameters presented in table 1 . the tuned parameters contain 16 lennard - jones kk - values and 16 lennard - jones kk - values of the dreiding atom - types k presented in table 1 . note that the atom - type [ h][!#7;!#8;!#9 ] refers to a hydrogen atom unable to form hydrogen bonds . hydrogen atoms attached to oxygen or nitrogen keep lennard - jones parameters = 0.0 kcal / mol and = 0.0 , in accordance with the opls - aa force field . also atomic partial charges of the 18 most observed opls - aa charge groups in the used data set are optimized by scaling all partial charges of the atoms within the neutral charge group with a factor tkcg . other charge groups a comparable optimization is performed by jin et al . to fit hydration free energies using 40 atom - type dependent dispersion term scaling factors . all 50 parameters are optimized by minimizing the rmse of pac - mac compared to experimental data , in the calculation of excess free energies of mixing , using the gauss we comply with the rule of thumb that the number of data points should be at least 5 times bigger than the number of parameters . moreover , the optimized parameters may deviate a maximum of 20% from the initial parameters to avoid unphysical values . an overview of the optimized lennard - jones kk- and kk - parameters is given in table 2 . the atom - types k are written in daylight smarts notation . the column # in dataset contains the amount of binary mixtures in which the atom - type is present . an overview of the 18 most observed opls - aa charge groups in the used data set and corresponding optimized scaling factors tkcg is given in table 3 . the column # in dataset contains the amount of binary mixtures in which the opls - aa charge group is present . the obtained results for pac - mac using the 50 optimized force field parameters , shown in table 2 and table 3 and from now on referred to as the opls - pm force field , are presented in figure 7 . scatterplot of the excess gibbs free energy of mixing : pac - mac using force field parameters shown in table 2 and table 3 versus experimental data for 1053 binary mixtures . error bars indicate the predicted standard deviation of the calculations . as shown in figure 7 , the total rmse is reduced from 0.151 kbt to 0.099 kbt by the opls - pm force field . binary mixtures containing water show , with the exception of a single data point , a big improvement ; the positive bias obtained in figure 4 is not visible anymore . the impact is remarkable since the force field of water is only moderately modified ( the atomic charges are reduced by 6.4% ) indicating that small changes can have big effects . the calculated mixing free energies of the 71 binary mixtures containing water are on average reduced by 0.30 kbt using the opls - pm force field . internal calculations showed that the change in van der waals interactions and electrostatic interactions contributed respectively 31% and 69% to the average reduction of g. so the reduced atomic charges of the water molecule have the biggest impact on the change in calculated mixing free energies of the binary mixtures containing water . in accordance with figure 3 and figure 4 , water at 298.15 k ( g experimental : 0.026 kbt , g pac - mac : 0.935 kbt ) . hexafluoroisopropanol occurs only once in the used data set , and the atomic charges of the cf3 groups remain unchanged since they are not present in the 18 most observed opls - aa charge groups ; so the error is mainly influenced by the water force field . the calculated g increases from 0.712 kbt to 0.935 kbt as a sacrifice to reduce the error of the other 70 binary mixtures containing water . the adapted force field parameters have an influence on the properties calculated using md simulations . internal md calculations show that the calculated density of water at 298.15 k is reduced from 1065.6 1.7 kg / m for the opls - pm force field ( experimental : 997.06 0.01 the calculated enthalpy of vaporization of water at 298.15 k is reduced from 49.05 0.04 kj / mol for the flexible tip3p model to 35.94 0.02 kj / mol for the opls - pm force field ( experimental : 43.90 0.04 kj / mol ) . it is tested whether or not the opls - pm force field is more accurate than the opls - aa force field in the calculation of g using ti . we use the obtained densities from the performed md simulations with the opls - aa force field for the md simulations with the opls - pm force field , because the opls - aa force field is optimized using experimental densities whereas the opls - pm force field is not . so only steps 712 are repeated of the protocol described in simulation setup . a comparison of the calculated excess gibbs free energies of mixing , using force field parameters shown in table 2 and table 3 , with experimental data for 1053 binary mixtures is shown in figure 8 . scatterplot of the excess gibbs free energy of mixing : ti using force field parameters shown in table 2 and table 3 versus experimental data for 1053 binary mixtures . the opls - pm force field ( figure 8) shows comparable results as the opls - aa force field ( figure 3 ) . the rmse is slightly reduced by opls - pm ( respectively 0.128 kbt versus 0.132 kbt ) ; however , the correlation coefficient is also slightly reduced ( respectively 0.769 versus 0.791 ) . a rmse of 0.128 kbt is expected since the obtained rmse of 0.109 kbt for ti in comparison with pac - mac , shown in figure 6 , is the limiting accuracy ( assuming pac - mac to predict experimental data perfectly ) . the following relation holds between the obtained rmses , assuming pac - mac and ti to be unbiased24 in which equals 0.497 and represents the correlation coefficient between the errors of pac - mac and ti in comparison with experimental data . ti represents the deviation between pac - mac and ti , which is increased from 0.109 kbt to 0.117 kbt after the optimization of 50 force field parameters . furthermore , rmsepm exp represents the deviation between pac - mac and experimental data , which is equal to 0.099 kbt and is shown in figure 7 . finally , rmseti exp represents the deviation between ti and experimental data . 0.099 kbt and 0.117 kbt for respectively rmsepm exp and rmsepm ti . solving eq 24 results in a value for rmseti exp equal to 0.129 kbt , supporting the obtained rmse in figure 8 . the accuracy of g calculations with ti is hardly changed using the parameters shown in table 2 and table 3 . the correlation between pac - mac and ti has to be increased in order to use pac - mac as a force field optimization engine . on the other hand , using pac - mac , it is possible to obtain force field parameters with a comparable accuracy as the opls - aa force field for the calculation of g , without multiple iterations of time - consuming md simulations . so , if intermolecular opls - aa force field parameters are missing in a molecule , then pac - mac can be used as the auxiliary method to quickly estimate the missing force field parameters with comparable accuracy as the opls - aa force field . the 71 binary mixtures containing water are heavily affected by the adapted force field : the opls - aa force field mainly overestimates the mixing free energy , whereas the opls - pm force field mainly underestimates the mixing free energy ( with the exception of the earlier discussed mixture hexafluoroisopropanol again confirms the statement that small changes to the force field parameters can have big effects in the outcome . figure 9 illustrates two requirements to further increase the accuracy of the force field for the prediction of mixing free energies using pac - mac as the correlated auxiliary method . first , a higher correlation between pac - mac and ti will result in a reduction of rmseti exp . the correlation can be increased by using a physical approach or by using a statistical approach . in the physical approach , the pac - mac method is brought closer to ti by reducing disparities between both methods , for example by taking three - body interactions or long - range interactions into account . in the statistical approach , the correlation is increased by increasing the number of fitting parameters shown in table 1 . perfect correlation will be obtained if the amount of fitting parameters is at least equal to the amount of experimental data points , for example by using binary mixture dependent s and x parameters . this statistical approach is used by van westen et al . to optimize force field parameters using the pc - saft equation of state as the correlated auxiliary function . second , a higher accuracy of the pac - mac method will result in a reduction of rmseti exp . again , a physical or statistical approach can be used . in the physical approach , the pac - mac method is modified to get closer to reality not only by taking three - body interactions or long - range interactions into account but also by improving potential energy functions . in the statistical approach , the correlation is increased by increasing the number of tunable force field parameters shown in table 2 and table 3 . one can make a quantitative estimate whether such a goal is achievable . equation 24 can be used to calculate the required accuracy of pac - mac calculations to achieve a desired reduction in error . for example , suppose that pac - mac could be improved to the same level as the best unifac methods ( i.e. , a rmse of about 0.07 kbt compared with experiment ) . perhaps such an improvement is not unrealistic , given prospects of including better packing models and potentially three - body interactions . in such cases , rmsepm ti could potentially be reduced to 0.08 kbt and rmsepm exp to 0.07 kbt , and then it follows that rmseti exp would be as low as 0.09 kbt . this is a tantalizing improvement of 30% in comparison with the current opls - aa force field . in this paper , excess free energies of mixing are calculated , by thermodynamic integration of md simulations using the opls - aa force field , for an extensive and diverse set of 1053 binary mixtures . the obtained results are compared with g obtained from experimental data and the pac - mac method . correlation with experimental data is obtained , and the observed rmse of 0.132 kbt ( and an effective rmse , for infinitely long md simulations , of 0.115 kbt ) is much smaller than a rmse of 2.6 kbt observed for the enthalpy of vaporization or a minimum rmse of 1.5 kbt observed for the free energy of solvation . furthermore , the flexible tip3p water force field is proven to overestimate mixing free energies . the error of the pac - mac method is higher than the error of ti since pac - mac contains , besides the chosen force field , several other assumptions in comparison with md simulations . however , an observed effective rmse of 0.150 kbt is only 30% above ti indicating that the assumptions made within the method are plausible . the benefit of the small loss in accuracy is a potential 1.0 10 times faster calculation , making pac - mac a quick alternative for ti in the calculation of mixing free energies using classical force fields . the calculation speed of pac - mac allows us to increase its accuracy by optimizing force field parameters based on experimental miscibility data instead of enthalpies of vaporization or densities . the rmse is reduced to 0.099 kbt using the optimized opls - pm force field . especially the prediction of mixing free energies of mixtures containing water is greatly increased by the adapted water force field : in contrast to the tip3p force field , a positive bias is not observed . the opls - pm force field , optimized without multiple iterations of time - consuming md simulations , also shows comparable accuracy as the opls - aa force field in the prediction of mixing free energies using ti of md simulations . by increasing the correlation between pac - mac and ti or by increasing the accuracy of pac - mac , it should be possible to obtain even better force field parameters using pac - mac as an auxiliary function .

we have calculated the excess free energy of mixing of 1053 binary mixtures with the opls - aa force field using two different methods : thermodynamic integration ( ti ) of molecular dynamics simulations and the pair configuration to molecular activity coefficient ( pac - mac ) method . pac - mac is a force field based quasi - chemical method for predicting miscibility properties of various binary mixtures . the ti calculations yield a root mean squared error ( rmse ) compared to experimental data of 0.132 kbt ( 0.37 kj / mol ) . pac - mac shows a rmse of 0.151 kbt with a calculation speed being potentially 1.0 104 times greater than ti . opls - aa force field parameters are optimized using pac - mac based on vapor liquid equilibrium data , instead of enthalpies of vaporization or densities . the rmse of pac - mac is reduced to 0.099 kbt by optimizing 50 force field parameters . the resulting opls - pm force field has a comparable accuracy as the opls - aa force field in the calculation of mixing free energies using ti .

Introduction Theoretical Basis Results and Discussion Conclusion

the opls - aa force field outperforms gaff in the prediction of experimental enthalpy of vaporization with a measured root - mean - square error ( rmse ) of 6.5 kj / mol versus 10.6 kj / mol . for the calculation of hydration free energies and solvation free energies using nonpolarizable force fields , obtained a minimum average unsigned error ( aue ) of 1.03 kcal / mol (= 4.3 kj / mol ) , mobley et al . obtained a minimum rmse of 1.00 kcal / mol (= 4.2 kj / mol ) , sherman et al obtained for opls_2005 an aue of 1.10 kcal / mol (= 4.6 kj / mol ) , and van der spoel obtained for gaff a rmse of 3.7 kj / mol . the difference in free energy between an ideal binary mixture and a phase separated system is maximum 1.7 kj / mol at 298 k. an extensive accuracy test of mixing free energies calculated using md or mc is , to our knowledge , not published yet . a less time - consuming approach to obtain mixing free energies is the use of methods based on the quasi - chemical approximation of guggenheim . we recently published a quasi - chemical method based on force fields : the pair configuration to molecular activity coefficient ( pac - mac ) model . the rmse of pac - mac in the calculation of mixing free energies using the opls - aa force field turned out to be 0.153 kbt ( or 0.43 kj / mol ) , and the calculation time is only a fraction of the required simulation time of corresponding md or mc simulations . we perform an accuracy test of the opls - aa force field based on mixing free energies calculated using thermodynamic integration ( ti ) of md simulations for an extensive set of equimolar binary mixtures and compare the results with the pac - mac model . we improve the accuracy of the pac - mac method by optimizing force field parameters and pac - mac specific parameters . first , a large data set of mixing free energies is useful for comparison between quasi - chemical thermodynamics and force field simulations and could be a new tool for further analysis of force field performance . second , pac - mac as a force field based quasi - chemical method performs , after optimization , comparable to existing quasi - chemical thermodynamics . subsequently , ti of md simulations on the same data set indicates that mixtures of most chemical classes are modeled quite well by the opls - aa force field , even though such experimental data was not used in the original force field parameter optimization . , we first explain the theory of thermodynamic integration for the calculation of free energies of mixing and then briefly summarize the pac - mac method . subsequently , within results and discussion , we first test the accuracy of the opls - aa force field in the prediction of mixing free energies using ti of md simulations . finally , we calculate the predictive capacity of the obtained opls - pm force field using ti . for more information concerning the pac - mac method we calculate the molar excess gibbs free energy of mixing for an equimolar binary mixture of molecules a and b , so xa = xb = 0.5 . the excess gibbs free energy of mixing is given by1 in which the excess helmholtz free energy of mixing f and the excess molar volume vme are given by23 vm , a , vm , b , and vm , ab in eq 3 represent the molar volumes of respectively pure component a , pure component b , and a mixture of a and b with equal mole fractions . the force field parameters , used to calculate interactions between the atoms , are taken from the opls - aa force field . the first property is the intermolecular energy wiij calculated using the opls - aa force field , and the second property is a depiction of the surface panels j which are covered ( oiij = 1 ) and uncovered ( oiij = 0 ) by the neighboring molecule . in pac - mac , the expression for the free energy of mixing is based on the quasi - chemical approximation of guggenheim and is given by12the 5 contributing free energy terms in eq 12 are given by the entropy of mixing of an ideal solution ( f ) , a modified staverman ( f ) , the combinatorial expression according to the quasi - chemical approximation ( f ) , the entropic contribution due to inhomogeneities in the occupation of the molecular surface ( f ) , and the total intermolecular energy ( f ) . assuming the set of sampled pair configurations i [ 1 .. m ] between molecules i { a , b } and j { a , b } to be uncorrelated , then the following relation holds for the variance of the mixing free energy:19 the excess gibbs free energy of mixing , calculated using ti and eq 1 , is compared with experimental data from the nist database . for a binary mixture , containing molecules a and b , the experimental excess free energy of mixing is calculated using20with xiliq and i being respectively the mole fraction and activity coefficient of component i in the liquid phase . the activity coefficient of component i is calculated using the extended raoult s law assuming gaseous ideality21 in which xigas represents the mole fraction of component i in the vapor phase , and p(xiliq ) is the vapor pressure of the binary mixture at mole fraction xiliq taken from experimental vapor the used subset of the nist database , containing 1092 experimental excess free energies of mixing of equimolar binary mixtures , is presented in a previously published article . mixing free energies are not calculated with ti for 39 binary mixtures because intramolecular force field parameters are missing ( 32 binary mixtures ) or the experimental vapor pressure ( > 5 atm ) is far above the simulation pressure of 1 atm resulting in gas formation within the md simulation ( 7 binary mixtures ) . a comparison of the calculated excess gibbs free energy of mixing , by ti performed on md simulations using the opls - aa force field , with experimental data for 1053 binary mixtures is shown in figure 3 . the root mean squared error ( rmse ) is calculated using22 in which n is the amount of data points ( n = 1053 ) , and h is the index of a data point . scatterplot of the excess gibbs free energy of mixing : ti using the opls - aa force field versus experimental data for 1053 binary mixtures . first , the opls - aa force field parameters are optimized using experimental data of mainly enthalpies of vaporization and densities and not using experimental free energies of mixing . as a consequence , greater predictability second , the range of values for the excess free energy of mixing is much smaller than the range of values for the enthalpy of vaporization . on the other hand , because of the smaller range of values , the observed rmse of 0.132 kbt ( or 0.37 kj / mol ) for the prediction of excess mixing free energies is much smaller than the rmse of 6.5 kj / mol for the prediction of enthalpies of vaporization . the accuracy of the opls - aa force field is even higher than indicated by the observed error , since the standard deviation of the ti calculation itself affects the observed rmse . if the three proposed assumptions are fulfilled , then the following relation holds between rmseobs , rmseeff , and rmsestd:23the root mean squared standard deviation of the ti calculations equals 0.064 kbt resulting in an effective rmse for the opls - aa force field of 0.115 kbt if infinitely long md simulations are performed . as shown in figure 3 , the opls - aa force field , as calculated by ti , performs quite well over all categories , except for binary mixtures containing water . the bias is caused by the used flexible tip3p water model for which the force field parameters are optimized using the enthalpy of vaporization and density of pure liquid water , so experimental miscibility data is not taken into account . in accordance with the obtained overestimation for hexafluoroisopropanolwater , this result might have been affected by a weak representation of trifluoromethyl groups within the opls - aa force field as well . the observed mixing free energy is a positive 0.263 0.083 kj / mol at a temperature of 308 k. the helmholtz free energy of mixing for an equal molar acetone also this value is in accordance with the results of simulations performed by jedlovszky et al . , even though they fixed the internal coordinates of the atoms within the molecules ; they calculated a helmholtz free energy of mixing of 0.97 kj / mol using the comparable opls - ua force field . in a previous publication , the total observed rmse of pac - mac is proven to be 0.153 kbt , with a correlation coefficient of 0.695 , for the calculation of excess free energies in comparison with experimental data.figure 4 shows comparable results for the subset of 1053 binary mixtures . scatterplot of the excess gibbs free energy of mixing : pac - mac using the opls - aa force field versus experimental data for 1053 binary mixtures . the effective rmse of pac - mac is 30% higher than the effective rmse of ti caused by several assumptions made within the model . the benefit of the assumptions made within pac - mac is a great reduction of calculation time : a g calculation using pac - mac takes 10 min on a single core ( intel xeon e5 - 2620 ) , whereas the same calculation using ti takes 5 days . a g calculation using ti requires on average the calculation of 1.0 10 molecular pair interactions , whereas a g calculation using pac - mac requires the calculation of only 2.0 10 molecular pair interactions . the calculation of the molecular pair interactions takes about 20% of the total calculation time of pac - mac . if we assume that the calculation time of a molecular pair interaction in pac - mac is equal to the calculation time of a molecular pair interaction in md , then the calculation of mixing free energies can potentially be 1.0 10 times faster using pac - mac than using ti . correlation in the predicted mixing free energy is expected since both methods use the same opls - aa force field . scatterplot of the excess gibbs free energy of mixing : ti versus pac - mac using the opls - aa force field for 1053 binary mixtures . figure 5 shows a correlation coefficient of 0.815 between ti and pac - mac for the calculation of mixing free energies . force field parameters are optimized by minimizing the rmse of pac - mac in comparison with experimental data . the accuracy of the optimized force field parameters in the prediction of mixing free energies is tested using ti . the correlation between ti and pac - mac is further increased by optimizing the x parameter in eq 16 and by introducing 16 atom - type dependent skseg instead of a single general s parameter equal to 0.62 in eq 11 . the skseg and x parameters are optimized by minimizing the rmse of pac - mac compared to ti , in the calculation of excess free energies of mixing , using the gauss an upper and lower limit for skseg is set to 0.8 and 0.4 , respectively , to avoid unphysical values . scatterplot of the excess gibbs free energy of mixing : ti versus pac - mac using the opls - aa force field and optimized skseg and x parameters for 1053 binary mixtures . hydrogen atoms attached to oxygen or nitrogen keep lennard - jones parameters = 0.0 kcal / mol and = 0.0 , in accordance with the opls - aa force field . all 50 parameters are optimized by minimizing the rmse of pac - mac compared to experimental data , in the calculation of excess free energies of mixing , using the gauss we comply with the rule of thumb that the number of data points should be at least 5 times bigger than the number of parameters . the obtained results for pac - mac using the 50 optimized force field parameters , shown in table 2 and table 3 and from now on referred to as the opls - pm force field , are presented in figure 7 . scatterplot of the excess gibbs free energy of mixing : pac - mac using force field parameters shown in table 2 and table 3 versus experimental data for 1053 binary mixtures . as shown in figure 7 , the total rmse is reduced from 0.151 kbt to 0.099 kbt by the opls - pm force field . the calculated mixing free energies of the 71 binary mixtures containing water are on average reduced by 0.30 kbt using the opls - pm force field . internal md calculations show that the calculated density of water at 298.15 k is reduced from 1065.6 1.7 kg / m for the opls - pm force field ( experimental : 997.06 0.01 the calculated enthalpy of vaporization of water at 298.15 k is reduced from 49.05 0.04 kj / mol for the flexible tip3p model to 35.94 0.02 kj / mol for the opls - pm force field ( experimental : 43.90 0.04 kj / mol ) . it is tested whether or not the opls - pm force field is more accurate than the opls - aa force field in the calculation of g using ti . we use the obtained densities from the performed md simulations with the opls - aa force field for the md simulations with the opls - pm force field , because the opls - aa force field is optimized using experimental densities whereas the opls - pm force field is not . a comparison of the calculated excess gibbs free energies of mixing , using force field parameters shown in table 2 and table 3 , with experimental data for 1053 binary mixtures is shown in figure 8 . scatterplot of the excess gibbs free energy of mixing : ti using force field parameters shown in table 2 and table 3 versus experimental data for 1053 binary mixtures . the opls - pm force field ( figure 8) shows comparable results as the opls - aa force field ( figure 3 ) . a rmse of 0.128 kbt is expected since the obtained rmse of 0.109 kbt for ti in comparison with pac - mac , shown in figure 6 , is the limiting accuracy ( assuming pac - mac to predict experimental data perfectly ) . ti represents the deviation between pac - mac and ti , which is increased from 0.109 kbt to 0.117 kbt after the optimization of 50 force field parameters . furthermore , rmsepm exp represents the deviation between pac - mac and experimental data , which is equal to 0.099 kbt and is shown in figure 7 . on the other hand , using pac - mac , it is possible to obtain force field parameters with a comparable accuracy as the opls - aa force field for the calculation of g , without multiple iterations of time - consuming md simulations . so , if intermolecular opls - aa force field parameters are missing in a molecule , then pac - mac can be used as the auxiliary method to quickly estimate the missing force field parameters with comparable accuracy as the opls - aa force field . the 71 binary mixtures containing water are heavily affected by the adapted force field : the opls - aa force field mainly overestimates the mixing free energy , whereas the opls - pm force field mainly underestimates the mixing free energy ( with the exception of the earlier discussed mixture hexafluoroisopropanol again confirms the statement that small changes to the force field parameters can have big effects in the outcome . figure 9 illustrates two requirements to further increase the accuracy of the force field for the prediction of mixing free energies using pac - mac as the correlated auxiliary method . this is a tantalizing improvement of 30% in comparison with the current opls - aa force field . in this paper , excess free energies of mixing are calculated , by thermodynamic integration of md simulations using the opls - aa force field , for an extensive and diverse set of 1053 binary mixtures . correlation with experimental data is obtained , and the observed rmse of 0.132 kbt ( and an effective rmse , for infinitely long md simulations , of 0.115 kbt ) is much smaller than a rmse of 2.6 kbt observed for the enthalpy of vaporization or a minimum rmse of 1.5 kbt observed for the free energy of solvation . the benefit of the small loss in accuracy is a potential 1.0 10 times faster calculation , making pac - mac a quick alternative for ti in the calculation of mixing free energies using classical force fields . the calculation speed of pac - mac allows us to increase its accuracy by optimizing force field parameters based on experimental miscibility data instead of enthalpies of vaporization or densities . the rmse is reduced to 0.099 kbt using the optimized opls - pm force field . the opls - pm force field , optimized without multiple iterations of time - consuming md simulations , also shows comparable accuracy as the opls - aa force field in the prediction of mixing free energies using ti of md simulations . by increasing the correlation between pac - mac and ti or by increasing the accuracy of pac - mac , it should be possible to obtain even better force field parameters using pac - mac as an auxiliary function .

the online version of this article ( doi:10.1007/s00280 - 013 - 2209 - 7 ) contains supplementary material , which is available to authorized users . an increasing number of nanomedicines , including liposomes , polymer conjugates , block copolymer micelles , nanoparticles and other complex hybrid technologies , are being developed as anticancer therapeutics , imaging agents and theranostics ( reviewed in [ 14 ] ) . several products are already in routine clinical use ( e.g. doxil , abraxane ) with a growing number of technologies including polymer therapeutics in clinical development [ 2 , 3 ] . intravenously ( i.v . ) administered long - circulating nanosized constructs have long been known to exhibit passive tumour targeting due to the enhanced permeability and retention ( epr ) effect [ 5 , 6 ] . this phenomenon has been demonstrated in many in vivo tumour models and the features of polymer conjugates [ 7 , 8 ] , nanoparticles and block copolymer micelles governing extravasation , as well as tumour characteristics regulating the efficiency of the process have been discussed . liposomal and polymer conjugate - based gamma camera imaging probes have also demonstrated the epr effect in some tumours in patients [ 1113 ] . a diverse array of tumour pathophysiological features regulate the efficiency of epr - mediated tumour targeting including heterogeneity of intratumoural blood flow , angiogenic vascular permeability , tumour microenvironment including extracellular matrix features , interstitial pressure and lymphatic drainage , and thus , no single biomarker adequately predicts tumour targeting and/or antitumour activity . as a functional epr effect is the primary factor governing nanomedicine tumour access ( fig . 1 ) , it is surprising that tumour models used to evaluate pharmacokinetics ( pk ) and/or antitumour activity have been rarely pre - calibrated for this characteristic this makes comparison of results , both between experiments and between laboratories , difficult and prevents effective benchmarking of emerging technologies against those for which there is now a considerable clinical database . moreover , although many constructs have been designed for intratumoural activation , particularly intralysosomal drug release ( using low ph or lysosomal enzymes such as cathepsin b ) ( fig . 1 ) , few tumour models are calibrated with respect to these activation mechanisms . therefore , the aim of this study was to quantify systematically the early - phase epr - mediated tumour targeting using a panel of murine and human xenograft tumours . the albumin - binding dye evans blue , widely used as a physiological marker of vascular permeability , and n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymer gflg dox ( fce28068 ) ( fig . 1 ) , a conjugate that has undergone phase i / ii clinical trials [ 11 , 12 ] , were selected as probes of the epr effect in tumour models . in certain experiments , doxorubicin ( dox ) alone was used as a reference control . to establish the comparability of evans blue and fce28068 as epr probes b16f10 model previously used widely to document the pk and antitumour activity of polymer conjugates and a s.c . l1210 model known to be sensitive to dox . fce28068 was then used to quantify the effect of tumour size on early - phase ( 1 h ) tumour accumulation in a panel of murine and human xenograft tumours ( table 1 ) and to study the effect of tumour size on passive targeting . in addition , as cathepsin b mediates dox liberation from fce28068 , and this enzyme is also responsible for activation of polyglutamic acid anticancer conjugates currently in clinical trials ( e.g. paclitaxel poliglumex ( ppx ) ) , the extent of dox released at 1 h was also quantified . it should be noted that the 1 h time point was selected for this study as it minimises additional kinetic effects introduced by differences in blood clearance rate , probe- or tumour model - dependent tumour efflux rates and/or inter - tumoural differences in the degradation rate of biodegradable probes , e.g. proteolysis of albumin over time [ 8 , 17].fig . 1structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activationtable 1summary of the tumour models usedcodetype of tumourmousemurine modelsl1210lymphocytic leukaemiadba2b16f10melanomac57 blkmac 15aadenocarcinomanmrimac 26adenocarcinomanmrimeta 7lung tumourbalb / chuman xenograftsrxf 1220renal cell carcinomanu / nurxf 486renal cell carcinomanu / nupaxf 546pancreatic carcinomanu / numexf carcinomanu / nucor l23human non - small cell lung carcinomanu / nusk - n - shneuroblastomascidimr 32neuroblastomascidsk - n - dzneuroblastomascid and for others [ 17 , 29 ] structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activation summary of the tumour models used and for others [ 17 , 29 ] hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy . meta-7 and cor l23 cells were from european collection of cell cultures ( ecacc ) , wiltshire , uk . c57 black , dba2 , balb - c and nu / nu mice were supplied by bantin and kingman ltd . , uk . all animal experiments were performed in accordance with the united kingdom co - ordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( 1998 ) and with uk home office guidelines . models ( see table 1 and [ 14 , 15 , 17 ] for full details ) . generally , cells were implanted into the posterior right flank of mice , and the studies were initiated when tumour reached 25289 mm ( product of two diagonal width ) . evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . via the tail vein . fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model . after 1 h , all animals were humanely killed , and tumour was carefully removed , washed with pbs , weighed and snap - frozen in liquid nitrogen before extraction and analysis . it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c . l1210 tumour model was conducted prior to the 2nd edition of ukcccr guidelines , thus accounting the use of tumours in excess of 2.5 g. evans blue was extracted from the tumour samples using a method adapted from harada et al . the homogenate ( 1,900 l ) was mixed with aqueous sodium sulphate solution ( 0.5 % w / v , 3 ml ) and acetone ( 7 ml ) in polypropylene tubes and then incubated at room temperature ( 20 c ) overnight before centrifugation ( 1,000g , 30 min , 20 c ) to precipitate the tissue . the concentration of evans blue in the supernatant was quantified spectrophotometrically at 590 nm using a standard curve prepared by spiking foetal bovine serum ( fbs ) samples with known concentrations of evans blue . administration of free dox , drug in tumour samples was quantified using a previously described hplc method [ 20 , 21 ] . throughout the extraction procedure , all samples were maintained at 4 c and kept in the dark ( wrapped in foil ) . briefly , tumour samples were thawed , homogenates were prepared in pbs , and samples ( 900 l ) were placed in polypropylene tubes . a daunomycin ( dnm ) standard ( donated by rhne - poulenc , france ) ( 100 ng , 100 l ) in pbs was added as an internal standard , followed by ammonium formate buffer ( 1 m ; ph 8.5 ; 100 l ) and chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) . after thorough vortex - mixing ( 3 10 s ; within 30 min ) , the tubes were centrifuged ( 1,000g ; 30 min , 10 c ) . the remaining organic phase was evaporated to dryness using a sample concentrator under constant nitrogen gas ( n2 ) flow over 20 min . the samples obtained were re - dissolved in hplc grade methanol ( 100 l ) by vortex - mixing ( 30 s ) and centrifuged ( 1,000g , 10 min , 10 c ) to remove any remaining biological materials . samples ( 100 l ) were then analysed by hplc using a c18 bondapak column eluted with propan-2-ol in water ( 29 % v / v ) adjusted to ph 3.2 with orthophosphoric acid with a fluorimetric detector ( excitation 480 nm , emission 560 nm ) . data were collected and analysed using a powerchrom integrator and software programme ( powerchrom v 2.0.7 ) . as hpma copolymer dox does not extract directly into the organic solvent , free dox present in the tumour samples could be extracted and quantified as described above [ 20 , 21 ] . dox , a previously described acid hydrolysis method [ 20 , 21 ] was used to first liberate the dox aglycone from the conjugate as follows . tumours were homogenised in pbs ( 2,000 l ) . then to duplicate samples ( 900 l ) , dnm was added as an internal standard ( 500 ng dnm ; 100 l in pbs ) followed by hcl ( 2 m , 1 ml ) before heating at 80 c for 20 min . samples were neutralised by the addition of naoh ( 2 m ; 1 ml ) , ammonium formate buffer ( 1 m ; ph 8.5 ; 1.5 ml ) and then chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) were added , and the samples were extracted . hplc analysis was conducted exactly as described above for free dox . to ensure quantitation , hpma copolymer data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) . statistical significance was calculated using student s t test for comparison of the mean of two small samples . statistical differences of at least p < 0.05 were considered statistically significant . to allow comparison between small and large tumours , the three smallest and three largest tumours available were grouped according to the classifiers for small and large described in the figure legends where tumour size as a variable is examined . hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy . meta-7 and cor l23 cells were from european collection of cell cultures ( ecacc ) , wiltshire , uk . c57 black , dba2 , balb - c and nu / nu mice were supplied by bantin and kingman ltd . , uk . all animal experiments were performed in accordance with the united kingdom co - ordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( 1998 ) and with uk home office guidelines . models ( see table 1 and [ 14 , 15 , 17 ] for full details ) . generally , cells were implanted into the posterior right flank of mice , and the studies were initiated when tumour reached 25289 mm ( product of two diagonal width ) . evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . via the tail vein . fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model . after 1 h , all animals were humanely killed , and tumour was carefully removed , washed with pbs , weighed and snap - frozen in liquid nitrogen before extraction and analysis . it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c . l1210 tumour model was conducted prior to the 2nd edition of ukcccr guidelines , thus accounting the use of tumours in excess of 2.5 g. evans blue was extracted from the tumour samples using a method adapted from harada et al . the homogenate ( 1,900 l ) was mixed with aqueous sodium sulphate solution ( 0.5 % w / v , 3 ml ) and acetone ( 7 ml ) in polypropylene tubes and then incubated at room temperature ( 20 c ) overnight before centrifugation ( 1,000g , 30 min , 20 c ) to precipitate the tissue . the concentration of evans blue in the supernatant was quantified spectrophotometrically at 590 nm using a standard curve prepared by spiking foetal bovine serum ( fbs ) samples with known concentrations of evans blue . administration of free dox , drug in tumour samples was quantified using a previously described hplc method [ 20 , 21 ] . throughout the extraction procedure , all samples were maintained at 4 c and kept in the dark ( wrapped in foil ) . briefly , tumour samples were thawed , homogenates were prepared in pbs , and samples ( 900 l ) were placed in polypropylene tubes . a daunomycin ( dnm ) standard ( donated by rhne - poulenc , france ) ( 100 ng , 100 l ) in pbs was added as an internal standard , followed by ammonium formate buffer ( 1 m ; ph 8.5 ; 100 l ) and chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) . after thorough vortex - mixing ( 3 10 s ; within 30 min ) , the tubes were centrifuged ( 1,000g ; 30 min , 10 c ) . the remaining organic phase was evaporated to dryness using a sample concentrator under constant nitrogen gas ( n2 ) flow over 20 min . the samples obtained were re - dissolved in hplc grade methanol ( 100 l ) by vortex - mixing ( 30 s ) and centrifuged ( 1,000g , 10 min , 10 c ) to remove any remaining biological materials . samples ( 100 l ) were then analysed by hplc using a c18 bondapak column eluted with propan-2-ol in water ( 29 % v / v ) adjusted to ph 3.2 with orthophosphoric acid with a fluorimetric detector ( excitation 480 nm , emission 560 nm ) . data were collected and analysed using a powerchrom integrator and software programme ( powerchrom v 2.0.7 ) . for each experiment , a dox standard curve was prepared in parallel . as hpma copolymer dox does not extract directly into the organic solvent , free dox present in the tumour samples could be extracted and quantified as described above [ 20 , 21 ] . to quantify hpma copolymer dox , a previously described acid hydrolysis method [ 20 , 21 ] was used to first liberate the dox aglycone from the conjugate as follows . tumours were homogenised in pbs ( 2,000 l ) . then to duplicate samples ( 900 l ) , dnm was added as an internal standard ( 500 ng dnm ; 100 l in pbs ) followed by hcl ( 2 m , 1 ml ) before heating at 80 c for 20 min . samples were neutralised by the addition of naoh ( 2 m ; 1 ml ) , ammonium formate buffer ( 1 m ; ph 8.5 ; 1.5 ml ) and then chloroform / propan-2-ol ( 4:1 v / v , 5 ml ) were added , and the samples were extracted . hplc analysis was conducted exactly as described above for free dox . to ensure quantitation , hpma copolymer data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) . statistical significance was calculated using student s t test for comparison of the mean of two small samples . statistical differences of at least p < 0.05 were considered statistically significant . to allow comparison between small and large tumours , the three smallest and three largest tumours available were grouped according to the classifiers for small and large described in the figure legends where tumour size as a variable is examined . to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v . b16f10 or l1210 tumours are shown in figs . 2 and 3 . when the tumour levels were expressed as % dose for evans blue ( fig . 2a , c ) , tumour levels increased in an approximately linear fashion with increasing tumour size up to a maximum of 46 % dose / tumour for the b16f10 model and ~15 % dose / tumour for the l1210 model ( fig . 3a ) were quantitatively similar across the b16f10 tumour size range , as expected for agents requiring permeable blood vessels for distribution . administration of free drug were consistently much lower ( < 0.8 % dose per tumour ; fig . 3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig . 3b ) levels expressed as % dose / g tumour decreased as tumour size increased ( figs . values fell to 0.43.7 % dose / g in the largest tumours ( > 600 mg ; fig . 3b ) . the observed tumour size dependence of evans blue accumulation while still evident was less marked in the l1210 model ( fig . figure 4 summarises data with respect to size of b16f10 tumours in relation to accumulated systemically administered evans blue , fce28028 and dox.fig . a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour . a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour . 4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . . not significantly different levels of evans blue detected in s.c . b16f10 and l1210 tumours at 1 h after i.v . administration . a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour . each point represents a single tumour sample levels of fce28068 and free dox detected in s.c . a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour . each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . statistical significance . not significantly different in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5 ) , the amount of conjugate in the tumour did not show statistically significant changes as tumour size increased . while the mac tumours accumulated fce28068 to a similar degree as the b16f10 model , approximately two to three lower conjugate levels were seen in the meta-7 lung model ( fig . 5d).fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours . the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig . 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively 0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours . the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig . 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively . statistical significance * * p < 0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines . the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 1 ) . although paxf 546 showed a similar trend , it should be noted that the three smallest and the three largest tumours of paxf 546 pancreatic carcinoma tumours did not give statistically significance different values . in contrast , the rxf 1220 , cor l23 , imr 32 , sk - n - sh and sk - n - dz tumours ( supplemental fig . ( only two sk - n - dz tumours were available , but the data are included for completeness ) . comparison of summary mean values obtained for fce28068 accumulation in all the tumour xenografts ( fig . 6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) . early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig . 6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) . tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p < 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. conjugated drug + free ) is shown in fig . only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours . notably , the extent of dox release in both mac tumours was very low ( supplemental fig . 3 ) . overall , there were a > 200-fold difference in the free dox levels seen at 1 h in the murine models studied and a 30-fold variation in the xenograft models . the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig . statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly . panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) . tumour size classification as per figs . 4 and 6 comparison of the dox released from fce28068 in the different tumour models . statistical significance : * * p < 0.02 ; * * * p < panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) . statistical significance : * * p < 0.02 ; ns not significantly different . to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v . b16f10 or l1210 tumours are shown in figs . 2 and 3 . when the tumour levels were expressed as % dose for evans blue ( fig . 2a , c ) , tumour levels increased in an approximately linear fashion with increasing tumour size up to a maximum of 46 % dose / tumour for the b16f10 model and ~15 % dose / tumour for the l1210 model ( fig . 3a ) were quantitatively similar across the b16f10 tumour size range , as expected for agents requiring permeable blood vessels for distribution . administration of free drug were consistently much lower ( < 0.8 % dose per tumour ; fig . 3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig . 3b ) levels expressed as % dose / g tumour decreased as tumour size increased ( figs . values fell to 0.43.7 % dose / g in the largest tumours ( > 600 mg ; fig . 3b ) . the observed tumour size dependence of evans blue accumulation while still evident was less marked in the l1210 model ( fig . figure 4 summarises data with respect to size of b16f10 tumours in relation to accumulated systemically administered evans blue , fce28028 and dox.fig . a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour . a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour . 4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . . not significantly different levels of evans blue detected in s.c . b16f10 and l1210 tumours at 1 h after i.v . administration . a , c tumour levels expressed as % dose per tumour and b , d tumour levels expressed as % dose / g / tumour . each point represents a single tumour sample levels of fce28068 and free dox detected in s.c . a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour . each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . statistical significance in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5 ) , the amount of conjugate in the tumour did not show statistically significant changes as tumour size increased . while the mac tumours accumulated fce28068 to a similar degree as the b16f10 model , approximately two to three lower conjugate levels were seen in the meta-7 lung model ( fig . 5d).fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . administration . the data in a c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours . the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig . 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively . statistical significance * * p < 0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . administration . the data in a c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours . the distribution of fce 28608 for the b16f10 model is shown for comparison . in fig . 5 small b16f10 , mac 15a mac 26 and meta-7 tumours were < 100 , < 470 , < 480 and < 150 mg , respectively . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively . 0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines . the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 1 ) . although paxf 546 showed a similar trend , it should be noted that the three smallest and the three largest tumours of paxf 546 pancreatic carcinoma tumours did not give statistically significance different values . in contrast , the rxf 1220 , cor l23 , imr 32 , sk - n - sh and sk - n - dz tumours ( supplemental fig . 2 ) displayed size - independent accumulation of fce28068 . ( only two sk - n - dz tumours were available , but the data are included for completeness ) . comparison of summary mean values obtained for fce28068 accumulation in all the tumour xenografts ( fig . 6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) . early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig . 6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p < 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) . tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . statistical significance * p < 0.05 * * * p < 0.01 , ns not significantly different . for the full data set , see supplementary figs . 1 and 2 after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. conjugated drug + free ) is shown in fig . only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours . notably , the extent of dox release in both mac tumours was very low ( supplemental fig . 3 ) . overall , there were a > 200-fold difference in the free dox levels seen at 1 h in the murine models studied and a 30-fold variation in the xenograft models . the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig . panel ( a ) murine tumour models ( mean se , n = 3 ) . statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly . panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) . statistical significance : * * p < 0.02 ; ns not significantly different . tumour size classification as per figs . 4 and 6 comparison of the dox released from fce28068 in the different tumour models . statistical significance : * * p < 0.02 ; * * * p < 0.01 ; ns not significantly . panel ( b ) human xenograft models ( mean se , n = 3 ; except sk - n - sh , n = 5 and sk - n - dz , n = 2 ) . statistical significance : * * p < 0.02 ; ns not significantly different . in vitro and in vivo pre - clinical models used to screen novel low molecular weight anticancer agents and biologics have evolved significantly to better mimic the clinical disease setting ( reviewed in [ 22 , 23 ] ) . while these advances give hope of lead compounds with an increased probability of a successful clinical outcome , many of the in vitro and in vivo methods / models used today are not optimal for nanomedicine evaluation given their very different cellular and whole - body pharmacokinetics compared to low molecular weight agents ( discussed in ) . administration , low molecular weight drugs distribute rapidly throughout the body with little or no tumour selectivity ( evidenced here for dox in fig . typically < 0.1 % of the administered drug dose is recoverable in the circulation , usually in the form of metabolites and/or protein - bound drug . in contrast , nanomedicines ( including liposomes , polymer conjugates and nanoparticles ) are retained within the bloodstream due to the tight vascular endothelial barrier present in most organs and limitation of cellular uptake to the endocytic pathways ( discussed in ) . in the absence of receptor - mediated targeting , nanomedicine blood clearance is largely governed by the rate of reticuloendothelial system ( res ) uptake and/or renal elimination . 1b ) , and a low epr effect is typically seen when a nanocarrier is cleared rapidly by professional phagocytes . however , it widely recognised that thereafter vasculature complexity ( e.g. different classes of angiogenic vessels , vessel disorganisation and heterogeneity of tumour perfusion ) and intratumoural biological barriers ( e.g. high interstitial pressure , the extracellular matrix , coupled with the presence or absence of lymphatic drainage , etc . [ 2 , 10 , 26 ] ) play an important role in passive targeting ultimately achieved in any particular tumour mass . in the 1950s and 1960s , unknowingly , these imaging agents were already capitalising on the epr effect for tumour selectivity , as do all chemotherapeutic and phototherapy agents that bind to plasma proteins . using an evans blue albumin complex , matsumura and maeda visualised and quantified tumour - specific passive accumulation in a sarcoma 180 model ( 32 % dose / g tumour at 48 h ) . in the experiments reported here , evans blue and fce28068 accumulation by l1210 and b1f10 ( figs . 2 , 3 ) tumours was in the range ~122 % dose / g at 1 h. there was good correlation between evans blue and the nanomedicine fce 28068 across the tumour size range . the data are in agreement with the previous reports describing evans blue , hpma copolymer fractions ( molecular weight from 4.5 to 800 kda ) , pamam dendrimer - dox and liposomal doxorubicin tumour accumulation . rapid passive targeting gave similar tumour values ( % dose / g ) irrespective of polymer molecular weight or construct architecture [ 2831 ] . a significant observation in these experiments is a 12-fold variation in functional epr , with many models displaying tumour size - dependent accumulation . the higher tumour localisation was noticeably in the smaller tumours , and this maybe a reflection of greater angiogenic activity in smaller tumours , reduced or absent tumour blood supply in hypoxic regions of larger necrotic tumours , an increase in tumour interstitial fluid pressure as the tumour grows , or a combination of all of these factors . kb human head and neck squamous cell xenografts also displayed tumour levels inversely correlated with tumour size : 15.1 10.8 % dose / g in tumours of < 0.1 g and 3.0 1.3 % dose / g in tumours of > 1 g . in this regard , the current results of experiments raise the possibility of a novel development strategy for nanomedicines , focusing on systemic delivery of such constructs in the adjuvant setting or with small - volume whatever the mechanism , there is a growing body of evidence that nanosized vectors show greatest targeting to the smaller tumours and this could provide an important opportunity to localise to those micrometastases so difficult to diagnose and treat effectively . use of in - dtpa - labelled pegylated liposomes to image 17 patients with locally advanced cancers gave positive tumour images in 15/17 patients and tumour levels in the range of ~0.53.5 % dose at 72 h. highest liposome localisation was observed in patients with head and neck cancers ( ~0.033 % injected dose / g tumour ) ( also noted for fce2068 in phase i clinical studies ) and lower levels in breast cancers ( ~0.005 % injected dose / g ) . the breast tumours were larger than the lung tumours , supporting a relationship between small tumour site and improved localisation , but the relationship was not unequivocal as it was noted that some tumours of similar size but different histological type displayed different levels of liposomal uptake . using tc - dtpa - labelled pegylated liposomes that localised to lung and head and neck cancers , others have suggested a correlation between tumour targeting and microvessel density as defined by anti - cd31 staining . although tumour vessel density ( vegf expression ) does correlate with tumour vascular permeability ( evans blue ) in some human tumour xenografts , other studies have indicated that a more complex multifactorial mechanism underpins epr - mediated targeting and efficacy , e.g. inflammatory status . at this time , it is difficult to predict tumour localisation / antitumour activity using a single biomarker . overexpression of lysosomal thiol - dependent proteases in many human tumours has been well documented , and these enzymes play an important role in tumour progression ( e.g. metastasis and angiogenesis ) . a growing number of polymer conjugates are being designed for lysosomotropic delivery [ 34 , 35 ] and cathepsin b - mediated drug release [ 1 , 2 ] . the wide variation in early - phase dox release from fc28068 ( ~200-fold ) observed here illustrates the need to characterise tumour models around the basis for delivery to tumours and response of tumours to nanomedicines as part of evolving an optimal clinical strategy for their development . for example , retrospective analysis of clinical data from ppx phase iii trials in advanced lung cancer patients showed improved survival in female but not in male patients , and it has been postulated that patient oestradiol level might play a pivotal role as oestrogens are known to induce cathepsin b activity . b16f10 tumours [ 15 , 37 ] indicated an ~30 min time lag before dox release began , supporting the hypothesis of drug release following endocytic internalisation . however , it should be noted that other factors may play a role in controlling the rate of drug release : ( i ) rate of conjugate diffusion through the tumour interstitium , ( ii ) the rate of endocytic internalisation ( endocytic gateways and intracellular trafficking pathways are often dysregulated in cancer ) and ( iii ) exposure to cathepsin b in the extracellular milieu . this complexity underlines the importance of functional tumour calibration in terms of drug release and also the future role of the real - time tumour imaging techniques that are emerging for quantitation of proteolysis per se and polymer degradation . the wide variation in passive targeting ( % dose / g ) and dox liberation observed here ( 1 h ) highlights the need to calibrate in vivo tumour models in respect of these parameters before they are used to define pharmacokinetics and/or antitumour activity of nanomedicines . this would provide more clinically relevant models for optimisation of lead candidates and benchmarking performance against anticancer nanomedicines already in routine clinical use . evans blue is a useful tool for routine evaluation of passive targeting , and the 1 h time point enables comparison of different nanocarriers minimising complications arising due to different blood clearance rate , tumour efflux and intratumoural degradation of the probe over time . these observations also emphasise the potential to select patients for early clinical trial involving nanomedicine therapy that are most likely to respond , i.e. by use of clinical imaging to verify functional epr , and monitoring of tumour biopsy samples for biomarkers relevant to activating conditions , e.g. in this case cathepsin b. fig . 1 fce28068 levels in mexf 276 , paxf 546 , maxf 449 and rxf 486 tumours at 1 h after i.v . administration . the data in panels ( a ) - ( d ) ( ppt 165 kb ) fig . 2 fce28068 levels in rxf 1220 , imr 32 , cor l23 , sk - n - sh and sk - n - dz tumours at 1 h after i.v . administration . the data in panels ( a ) - ( e ) show individual tumours . ( ppt 178 kb ) fig . 3 summary of tumour uptake of fce28068 and the rate of dox release in the mac tumours ( mean se , n = 11 ) .

purposeintravenously ( i.v . ) administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention ( epr ) effect , but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release . here the effect of tumour type and tumour size on epr - mediated tumour localisation and cathepsin b - mediated drug release was studied.methodsevans blue ( 10 mg / kg ) and an n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymer doxorubicin ( dox ) conjugate ( fce28068 ) ( 5 mg / kg dox - equiv ) were used as probes and tumour levels ( and dox release ) measured at 1 h after i.v . administration in a panel of murine and human xenograft tumours.resultsevans blue and fce28068 displayed similar tumour levels in the range of 218 % dose / g at 1 h for b16f10 and l1210 . approximately half of the tumour models evaluated exhibited tumour size - dependent accumulation of fce28068 ; smaller tumours had the highest accumulation . administration of free dox ( 5 mg / kg ) produced tumour levels of < 2.5 % dose / g independent of tumour size . whereas the degree of epr - mediated targeting showed ~12-fold difference across the tumour models evaluated , dox release from fce28068 at 1 h displayed ~200-fold variation.conclusionsmarked heterogeneity was seen in terms of epr effect and dox release rate , underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre - clinical and clinical development.electronic supplementary materialthe online version of this article ( doi:10.1007/s00280 - 013 - 2209 - 7 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Materials and cells Tumour models and administration of probes Quantitation of Evans Blue in tumour samples Quantitation of free Dox in tumour samples Quantitation of total HPMA copolymerDox and released free Dox in tumour samples Data expression Results Accumulation of Evans Blue, FCE28068 and free Dox in s.c. B16F10 and L1210 tumours Comparison of FCE28068 levels in murine and human xenograft tumours Dox released from FCE28068 in murine and human xenograft tumours Discussion Conclusions Electronic supplementary material

the online version of this article ( doi:10.1007/s00280 - 013 - 2209 - 7 ) contains supplementary material , which is available to authorized users . administered long - circulating nanosized constructs have long been known to exhibit passive tumour targeting due to the enhanced permeability and retention ( epr ) effect [ 5 , 6 ] . a diverse array of tumour pathophysiological features regulate the efficiency of epr - mediated tumour targeting including heterogeneity of intratumoural blood flow , angiogenic vascular permeability , tumour microenvironment including extracellular matrix features , interstitial pressure and lymphatic drainage , and thus , no single biomarker adequately predicts tumour targeting and/or antitumour activity . 1 ) , few tumour models are calibrated with respect to these activation mechanisms . therefore , the aim of this study was to quantify systematically the early - phase epr - mediated tumour targeting using a panel of murine and human xenograft tumours . the albumin - binding dye evans blue , widely used as a physiological marker of vascular permeability , and n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymer gflg dox ( fce28068 ) ( fig . fce28068 was then used to quantify the effect of tumour size on early - phase ( 1 h ) tumour accumulation in a panel of murine and human xenograft tumours ( table 1 ) and to study the effect of tumour size on passive targeting . 1structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activationtable 1summary of the tumour models usedcodetype of tumourmousemurine modelsl1210lymphocytic leukaemiadba2b16f10melanomac57 blkmac 15aadenocarcinomanmrimac 26adenocarcinomanmrimeta 7lung tumourbalb / chuman xenograftsrxf 1220renal cell carcinomanu / nurxf 486renal cell carcinomanu / nupaxf 546pancreatic carcinomanu / numexf carcinomanu / nucor l23human non - small cell lung carcinomanu / nusk - n - shneuroblastomascidimr 32neuroblastomascidsk - n - dzneuroblastomascid and for others [ 17 , 29 ] structure of a fce28068 , evans blue and a schematic representation of pendant dox molecules conjugated to a nanomedicine and b a schematic diagram showing the key steps in tumour targeting and lysosomotropic activation summary of the tumour models used and for others [ 17 , 29 ] hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy . evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model . it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c . to ensure quantitation , hpma copolymer data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) . hpma copolymer gflg dox ( fce28068 ; also known as pk1 ) ( mw ~ 30,000 g / mol ; mw / mn = 1.3 ; total dox 68 wt% ; free dox < 1 % in respect of total ) and dox were a gift from pharmacia and upjohn , italy . evans blue ( 2 mg / ml in 0.9 % nacl ; 10 mg / kg ) , fce28068 or free dox ( 1 mg / ml in pbs ; 5 mg / kg dox - equiv ) was administered i.v . fce28068 was given the higher dose of 40 mg / kg dox - equiv to nmri mice bearing the murine adenocarcinomas mac 15a and mac 26 to enable comparison with efficacy experiments in this model . it should be noted that most of this study was conducted according to the 2nd edition of the ukcccr guidelines for the welfare of animals in experimental neoplasia , which states that the maximum tumour burden should not exceed 10 % of the host animal s normal body weight or 2.5 g. the initial study involving measurement of evans blue levels in the s.c . to ensure quantitation , hpma copolymer data describing tumour accumulation of evans blue or hpma copolymer dox were expressed as either ( i ) the % administered dose per tumour ( for the conjugate the value is given as % dox - equiv dose administered ) or ( ii ) the administered dose % dose / g tumour ) . to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v . when the tumour levels were expressed as % dose for evans blue ( fig . 3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig . a , c tumour levels expressed as % dose per tumour , and b , d tumour levels expressed as % dose / g / tumour . 4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . b16f10 and l1210 tumours at 1 h after i.v . each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . not significantly different in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v c individual tumour samples and d a comparison of the levels after measured in the three smallest and three largest tumours . large b16f10 , mac 15a , mac 26 and meta-7 tumours were > 400 , > 900 , > 1,000 and > 300 mg , respectively 0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . statistical significance * * p < 0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines . the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) . early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig . 6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . 1 and 2 after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours . the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig . to assess evidence for the operation of the epr effect , intratumour levels of evans blue , fce28028 and dox detected at 1 h following i.v . 3c ) , and expression in terms of % dose / g tumour tissue showed dox localisation to be constant across all tumour sizes studied ( in the range 1.12.5 % dose / g tumour ; fig . 3b ) levels expressed as % dose / g tumour decreased as tumour size increased ( figs . 4comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . b16f10 and l1210 tumours at 1 h after i.v . each point represents a single tumour comparison of the levels of fce28068 , evans blue and free dox detected in small ( < 100 mg ) and large ( > 400 mg ) b16f10 tumours ( mean se ) . statistical significance in contrast to the results obtained in the b16f10 models , when fce28068 was administered to mice bearing mac 15a ( 8.212.6 % dose / g ) , mac 26 adenocarcinoma ( 6.910.8 % dose / g ) or meta-7 lung ( 3.54.7 % dose / g ) tumours ( fig . 5fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . statistical significance * * p < 0.02 ; ns not significantly different fce28068 levels in mac 15a , mac 26 and meta-7 tumours at 1 h after i.v . 0.02 ; ns not significantly different we therefore examined a series of additional models , as a tumour size - dependent nanomedicine delivery has been incompletely explored , and the existence of this phenomenon has important implications for the design of pre - clinical and clinical studies with nanomedicines . the mexf 276 , maxf 449 and rxf 486 human xenograft models displayed a tumour size - dependent pattern of fce28068 accumulation ( supplemental fig . 6 ) revealed the highest values in the non - small cell lung cancer cor l23 ( 4.712.2 % dose / g ) and the lowest values in the larger ( 0.20.4 g ) maxf 449 tumours ( 1.0 0.1 % dose / g ) . early - phase tumour levels of fce28068 across all the xenograft tumours ( and sizes ) examined displayed ~12-fold variation in magnitude.fig . 6comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , tumour model size cut - offs ( mg small , mg large ) are as follows : mexf276 ( < 50 , > 200 ) ; maxf449 ( < 50 , > 200 ) , paxf 546 ( < 100 , > 500 ) , rxf 486 ( < 100 , > 600 ) , rxf 1220 ( < 100 , > 400 ) , corl 23 ( < 100 , > 400 ) , imr32 ( < 120 , > 220 ) . 1 and 2 comparison of the levels of fce28068 detected in small and large human xenograft tumours at 1 h ( mean se , n = 3 ) . 1 and 2 after administration of fce28068 , the free dox detected in all tumour samples at 1 h expressed as percentage of total dox ( i.e. only the mac 15a , mac 26 murine tumours and the cor l23 xenograft showed tumour size dependency in terms of dox liberation at 1 h. this is interesting as none of these tumour models displayed tumour size - dependent fce28208 levels at 1 h. dox release was greater in the smaller tumours , and the difference is particularly striking for the smaller ( < 100 mg ) cor l23 tumours . the fastest dox release was observed in b16f10 ( 22.9 1.2 % at 1 h ) and the smaller in cor l23 tumours ( 30.1 5.3 % ) with the slowest release rate observed in the larger mac 26 tumours ( 0.06 0.01 % ) and the sk - n - sh neuroblastoma ( 1.0 0.1 % ) .fig . in vitro and in vivo pre - clinical models used to screen novel low molecular weight anticancer agents and biologics have evolved significantly to better mimic the clinical disease setting ( reviewed in [ 22 , 23 ] ) . 2 , 3 ) tumours was in the range ~122 % dose / g at 1 h. there was good correlation between evans blue and the nanomedicine fce 28068 across the tumour size range . the higher tumour localisation was noticeably in the smaller tumours , and this maybe a reflection of greater angiogenic activity in smaller tumours , reduced or absent tumour blood supply in hypoxic regions of larger necrotic tumours , an increase in tumour interstitial fluid pressure as the tumour grows , or a combination of all of these factors . kb human head and neck squamous cell xenografts also displayed tumour levels inversely correlated with tumour size : 15.1 10.8 % dose / g in tumours of < 0.1 g and 3.0 1.3 % dose / g in tumours of > 1 g . use of in - dtpa - labelled pegylated liposomes to image 17 patients with locally advanced cancers gave positive tumour images in 15/17 patients and tumour levels in the range of ~0.53.5 % dose at 72 h. highest liposome localisation was observed in patients with head and neck cancers ( ~0.033 % injected dose / g tumour ) ( also noted for fce2068 in phase i clinical studies ) and lower levels in breast cancers ( ~0.005 % injected dose / g ) . a growing number of polymer conjugates are being designed for lysosomotropic delivery [ 34 , 35 ] and cathepsin b - mediated drug release [ 1 , 2 ] . the wide variation in early - phase dox release from fc28068 ( ~200-fold ) observed here illustrates the need to characterise tumour models around the basis for delivery to tumours and response of tumours to nanomedicines as part of evolving an optimal clinical strategy for their development . however , it should be noted that other factors may play a role in controlling the rate of drug release : ( i ) rate of conjugate diffusion through the tumour interstitium , ( ii ) the rate of endocytic internalisation ( endocytic gateways and intracellular trafficking pathways are often dysregulated in cancer ) and ( iii ) exposure to cathepsin b in the extracellular milieu . the wide variation in passive targeting ( % dose / g ) and dox liberation observed here ( 1 h ) highlights the need to calibrate in vivo tumour models in respect of these parameters before they are used to define pharmacokinetics and/or antitumour activity of nanomedicines . 1 fce28068 levels in mexf 276 , paxf 546 , maxf 449 and rxf 486 tumours at 1 h after i.v . 2 fce28068 levels in rxf 1220 , imr 32 , cor l23 , sk - n - sh and sk - n - dz tumours at 1 h after i.v . 3 summary of tumour uptake of fce28068 and the rate of dox release in the mac tumours ( mean se , n = 11 ) .

rhodiola rosea ( r. rosea ) is an herb that grows in mountainous regions of north america , europe , and asia . it has been used in traditional folk medicine for centuries as a treatment for fatigue and mood disorders . r. rosea has been extensively studied by scientists in the former soviet union and has predominantly been found to result in favourable effects on exercise performance . however , this prior work has been lacking in experimental control . some recent studies have supported the results of earlier work by identifying antioxidant and anti - inflammatory properties of r. rosea [ 3 , 4 ] . another work has suggested ingestion of r. rosea appears to be effective , either acutely or with daily supplementation , for reducing perceived fatigue and improving cognition [ 5 , 7 ] , as well as reducing markers of physiological and psychological stress [ 7 , 8 ] . although r. rosea ingestion has been identified as a means to reduce physical fatigue , its efficacy during exercise is unclear . animal based research has shown increased swim time to exhaustion in rats [ 3 , 9 ] but the impact of r. rosea ingestion on exercise performance in humans is equivocal . some studies have shown no effect of r. rosea ingestion on exercise performance [ 4 , 1012 ] whilst others have supported its use [ 13 , 14 ] . the array of protocols and supplementation doses used in prior studies has also limited comparisons between studies and provides one potential explanation for the discrepancy in their findings . for example , de bock et al . reported that an acute dose of r. rosea increased time to exhaustion during an incremental cycle ergometer protocol . conversely , in the same study , 4 weeks of r. rosea supplementation did not significantly improve cycling performance . this was the first study to show an acute impact of r. rosea ingestion on human exercise performance . more recent research by noreen et al . examined the efficacy of a 3 mgkg body mass dose of r. rosea on 6-mile cycle time trial performance in 18 active women . they reported that r. rosea significantly decreased submaximal exercise heart rate , reduced rpe , and improved time trial performance time . noreen et al . subsequently suggested that acute r. rosea ingestion might be employed by active individuals while exercising to complete a set distance in the shortest time . such data are interesting ; as if r. rosea does positively impact on exercise , it may be safe and legal way to enhance exercise performance in athletes and the exercise experience in recreational gym users . abidov et al . demonstrated that an extract of r. rosea significantly prolonged the duration of exhaustive swimming in rats and stimulated atp synthesis in muscle during exercise . these findings led perfumi and mattioli to speculate that , in addition to enhancing the catecholaminergic system , the ability of r. rosea to increase performance involves an improvement in cellular energy metabolism . however , some studies have suggested that r. rosea acts to acutely increase endogenous opioid production or receptor sensitivity [ 2 , 13 ] which subsequently impacts on brain dopamine and serotonin and cardiac activity and attenuates the perception of effort at a given workload . however , as only two studies have examined acute r. rosea ingestion on exercise performance to date , further data is needed on this topic . moreover , both de bock et al . and noreen et al . employed performance based protocols to examine the effect of r. rosea ingestion . while these are valuable in understanding the effect of a nutritional supplement on performance responses , different protocols may be better placed to evidence any effect of r. rosea on exercise performance . for example , using steady state submaximal exercise in the presence of r. rosea and placebo would allow for power output to remain constant across a given period . this then allows for better like - for - like comparison of data between the active supplement and placebo trials . moreover , when employing protocols where intensity is modified throughout ( as is the case with both time trials and time to exhaustion ) understanding any impact of the exercise protocol of substance ingested becomes more difficult because even small increases in exercise intensity have been shown to markedly change feelings of pleasure during exercise . furthermore , given the postulation that r. rosea acts through increased opioid production , it is perhaps surprising that few studies have examined its impact on affect . noreen et al . reported no effect of r. rosea ingestion on mood state , as assessed using the poms questionnaire . however , changes in mood state with exercise have been reported following ingestion of a range of substances including caffeine [ 18 , 19 ] and carbohydrate . hedonic theory postulates that hedonic responses ( i.e. , pleasure versus displeasure ) following a behaviour influence decisions regarding whether or not to repeat that behaviour . as a consequence if nutritional manipulation enhances the pleasure response to exercise , according to hedonic theory , it may mean individuals are more likely to exercise . this is important as different exercise intensities and individual fitness will create variability in the affective response to exercise [ 2224 ] and the effect of nutritional manipulation on these responses is not well known . changes in mood and feelings of pleasure may be one reason why individuals do not participate in regular exercise and physical activity and favourable affective states benefit exercise performance . has also identified that affective and perceptual responses to exercise differ depending on adiposity status . assuming that the affective response to exercise is the same in recreational exercises compared to high level performers may therefore lead to erroneous conclusions regarding the exercise - affect relationship . thus , while the extant literature examining the effect of r. rosea ingestion has focused on performance measures , the current study seeks to develop this work by employing a model that can be applied to recreational exercisers and is more applicable to the exercising public in general . the aim of this study was to examine the effect of acute r. rosea ingestion on substrate utilisation , mood state , rpe , perceptions of arousal , and pleasure / displeasure in a population of recreationally active men . it was hypothesized that ingestion of r. rosea would positively influence perceived exertion during exercise as well as enhancing mood and perceptions of arousal and pleasure / displeasure before and after cycling exercise . testing these hypotheses will address gaps in knowledge on the effect of r. rosea by examining their effects on an active but not performance oriented group , more typical of regularly active individuals in the general population . the study will also combine physiological and psychological measures to address gaps in the literature base by assessing substrate utilisation , heart rate , rpe , mood state , and perceptions of arousal and pleasure / displeasure to provide a multidisciplinary investigation of the effect of r. rosea during exercise . this study employed a within - participants double - blind cross - over design whereby participants visited the laboratory on 3 occasions at the same time of day in a well - rested and well - hydrated state . all participants were asked to refrain from vigorous exercise and maintain normal dietary patterns in the 48 hours prior to testing and were asked not to consume caffeine for 24 hours before testing . during the first visit participants completed a familiarization session with the measures to be used in the subsequent experimental trials and completed an incremental exercise test to assess vo2max. inclusion criteria included being male and habitually engaged in recreational physical activity of more than 3 but less than 10 hours per week and not including formal competitive sports performance . participants were excluded if they had a musculoskeletal or cardiovascular contraindication to exercise , were taking any medication that could impact mood / affect , engaged in less than 3 or more than 10 hours physical activity per week , or were engaged in competitive sports activity as part of their habitual physical activity . the incremental exercise test was performed on a mechanically braked cycle ergometer ( monark exercise ab , sweden ) to assess vo2max following previously published guidelines . initially , participants were fitted to the ergometer and performed a 5 min warm - up at 25 watts . the initial work rate for the test was 80 watts with the work rate increasing by 40 watts every 3 minutes . this protocol , using 3-minute stages rather than a ramp protocol , was employed based on prior research suggesting that this form of protocol provides more reliable physiological measures , particular for the population characteristics examined in this study ( i.e. , active but not cycling trained specifically ) . participants were asked to maintain their pedal cadence at 70 rpm and were given visual feedback from the monark control box in order to do this . expired air was collected via the douglas bag technique during the final minute of each incremental exercise stage . samples were analyzed for oxygen and carbon dioxide content ( servomex , crowborough , england ) and expired air volume ( harvard dry gas meter , harvard apparatus , kent , england ) with values for oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) subsequently calculated . heart rate ( polar electro , kempele , finland ) and rating of perceived exertion ( rpe ) , using the borg 620 rpe scale , were recorded during the final 15 seconds of each workload . participants were judged to have reached vo2max if they presented at least 3 of the following : ( a ) a respiratory exchange ratio of greater than 1.1 , ( b ) a heart rate during the last stage of testing that was 10 beats of age predicted maximum heart rate , ( c ) an rpe of 18 or greater , ( d ) a plateau in vo2 with an increase in workload , and ( e ) volitional fatigue . . of participants ' baseline vo2max values was 50.5 6.6 mlkgmin ( range : 38.858.4 mlkgmin ) . descriptive information for the participants is presented in table 1 . on completion of the vo2max testing and following a period of at least 72 hours participants completed two 30-minute submaximal cycling trials at a workload of 70% vo2max in a fasted state . self - report of dietary intake was employed to assess and control dietary intake in the 24 hours prior to exercise trials . this was used to ensure that the same / similar per testing meals were consumed prior to experimental trials and that caffeine and alcohol had not been consumed in the 24 hours prior to testing . the duration and intensity of exercise were chosen as it sits within the american college of sports medicine guidelines for the prescription of exercise for health benefit . on a molecular basis , the main active ingredients of r. rosea appear to be tyrosol and its glucoside known as salidroside and following oral ingestion of 100 mgkg salidroside , the half - life of salidroside appears to be 1.32 0.22 h . therefore , conditions were randomised , separated by 4872 hours , and consisted of a r. rosea condition where 3 mgkg body mass of r. rosea ( indigo herbs , glastonbury , uk ) placed in a coloured , opaque gelatin capsule or a placebo ( 3 mgkg body mass of maltodextrin , ( myprotein , northwich , uk ) ) was ingested with 250 ml water . the capsules were indistinguishable between r. rosea and placebo conditions and were ingested 60 min before exercise on an empty stomach . the amount of total maltodextrin ingested was approximately 170 mg and thus highly unlikely to have had any impact on exercise performance or metabolism . the dose of r. rosea used was based on the previous work of de bock et al . and noreen et al . who observed improvements in endurance performance following acute ingestion of r. rosea . during each of the cycling trials , heart rate was monitored ( polar rs400 , polar electro oy , kempele , finland ) continuously and assessed every 10 minutes . participants were also asked to provide ratings of perceived exertion ( rpe ) using the borg 620 scale . prior to commencing the experimental trials the memory - anchoring approach was employed whereby participants were asked to remember a time when they were at rest ( attributed to rpe of 6 ) and a time when they had reached a level of exertion that was maximal attributed to rpe of 20 . the rpe scale was presented to students for 1 minute prior to their rating of perceived exertion at each time point and was not visible other than this . prior to any substance ingestion , 60 min after ingestion ( at the onset of each exercise bout ) and immediately on completion of each exercise bout , participants also completed the feeling scale ( fs ) as a measure of pleasure and displeasure . this is an 11-item single - item scale ranging from + 5 ( very good ) to 5 ( very bad ) that is used to quantify pleasure / displeasure . the felt arousal scale ( fas ) was also used as a measure of arousal . this is a six - item scale ranging from 1 ( low arousal ) to 6 ( high arousal ) . mood state was also assessed using the fatigue and vigour subscales of the brunel mood state inventory ( brums ) . this is a well - established , reliable , and valid measure of mood state that has been previously employed to assess the mood state response to various exercise modes [ 18 , 35 , 36 ] . the fatigue and vigor subscales were chosen in particular as prior research [ 1 , 7 ] has suggested r. rosea may particularly influence feelings of fatigue and/or vigor . participants were introduced to these scales during their first visit to the laboratory ( prior to establishment of vo2max ) . standardised instructions for completing the fs and fas were read to participants at the beginning of each trial . prior to cycling ( but whilst seated on the exercise bike ) at 14 min and 29 min during each exercise bout expired air samples were collected using the douglas bag technique to estimate fat and carbohydrate ( cho ) oxidation rates . the collection time was 60 s and samples were analysed for oxygen and carbon dioxide content ( servomex , crowborough , england ) that was calibrated at two points before each assessment using gases of known concentrations ( 15.12% o2 , 5.1% co2 ) in accordance with manufacturers guidelines and expired air volume ( harvard dry gas meter , harvard apparatus , kent , england ) with values for oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) subsequently calculated . energy expenditure ( kcalmin ) and the oxidation rates for carbohydrate and fat ( gmin ) were then calculated according to the weir and frayn equations , respectively . a series of 2 ( substance ingested ) 4 ( time point ) ways repeated measures analysis of variance was used to examine any differences in heart rate and rpe . a series of 2 ( substance ingested ) x 3 ( time point , before ingestion , after ingestion but before exercise , and after exercise ) ways repeated measures analysis of variance was used to examine any differences in mood state and perceptions of arousal and pleasure / displeasure and substrate utilisation . where any significant differences were discovered , bonferroni pairwise multiple comparisons were used to determine where the differences lay . partial was used as a measure of effect size , statistical significance was set at p = 0.05 a priori , and the statistical package for social sciences ( version 20 ) was used for all analysis ( spss inc . , il , usa ) . in respect to heart rate , results indicated no significant interactions or main effect due to the substances ingested . there was however a significant main effect for time ( f 3 , 27 = 266.7 , p = 0.0001 , p = 0.967 ) where heart rate was significantly increased throughout the exercise bout irrespective of substance ingested . mean se of heart rate ( bpm ) was 77 4 at rest compared to 151 4 , 158 4 , and 162 4 at 10 , 20 , and 30 minutes into the exercise bout , respectively . bonferroni comparisons indicated that heart rate was significantly different at all time points ( p = 0.001 in all cases ) . in the case of rpe , results indicated a significant substance time interaction ( f 3 , 27 = 6.12 , p = 0.003 , p = 0.405 , see figure 1 ) . rpe data were not significantly different at rest and at 10 and 20 minutes into the exercise bout between the two conditions . however , rpe in the placebo condition was significantly higher than that in the r. rosea condition 30 minutes into the exercise bout ( p = 0.003 ) . scores from the fatigue subscale of the brums indicated a significant main effect across time ( f 2 , 18 = 12.879 , p = 0.0001 , p = 0.589 ) where scores were significantly higher after exercise compared to before exercise ( p = 0.028 ) and compared to before exercise but after ingestion ( p = 0.008 ) . there were no other significant main effects of interactions for fatigue subscale data . in regard to the vigor subscale of the brums , there was no significant substance time interaction . there were however significant main effects for time ( f 2 , 18 = 4.435 , p = 0.027 , p = 0.350 ) and for the substance ingested ( f 2 , 9 = 11.692 , p = 0.008 , p = 0.565 ) . scores for vigor were significantly lower at after exercise compared to after ingestion but before exercise ( p = 0.04 ) . mean se for vigor scores was 6.9 0.56 before ingestion , 7.4 0.44 after ingestion but before exercise , and 5.6 0.74 after exercise . for the substance ingested , scores for vigor were significantly higher in the r. rosea condition ( p = 0.008 ) compared to the placebo . mean se for vigor was 7.6 0.52 in the r. rosea condition compared to 5.7 0.57 in the placebo condition . perceptions of arousal were also significantly different in the presence of r. rosea compared to placebo ( f 1 , 9 = 5.0 , p = 0.05 , p = 0.357 ) with scores for arousal being higher ( 3.1 0.19 ) in the presence of r. rosea compared to placebo ( 2.8 0.11 ) . when data for feeling of pleasure / displeasure were assessed , there was however a significant condition time effect ( f 2 , 18 = 12.795 , p = 0.0001 , p = 0.587 , see figure 2 ) . this followed a similar pattern to rpe with no significant difference evident before ingestion ( p = 0.591 ) or after ingestion but before exercise ( p = 0.08 ) . there was however significantly lower scores for pleasure after exercise in the placebo condition compared to the r. rosea condition ( p = 0.003 ) . energy expenditure was significantly higher during exercise ( f 2 , 18 = 130.921 , p < 0.001 , p = 0.936 ) ( see figure 3 ) , irrespective of the trial ( f 1 , 9 = 1.198 , p = 0.302 , p = 0.117 ) . the rate of total carbohydrate oxidation ( figure 4 ) was not significantly different between trials ( f 1 , 9 = 0.317 , p = 0.587 , p = 0.034 ) . carbohydrate oxidation remained relatively constant throughout exercise but was higher compared with the rest ( f 2 , 18 = 13.902 , p < 0.001 , p = 0.770 ) . the rate of fat oxidation ( figure 5 ) was higher than that at rest during exercise ( f 1 , 11 = 25.131 , p < 0.001 , p = 0.736 ) but there was no significant main effect due to the substances ingested ( f 1 , 9 = 0.950 , p = 0.355 , p = 0.095 ) . acute r. rosea ingestion has been reported to improve exercise performance [ 13 , 14 ] in performance based tasks . authors have suggested that this performance enhancement may result from r. rosea 's action as an opioid producer [ 2 , 13 ] . however , use of performance based exercise tasks ( e.g. , time trial ) in prior studies makes it difficult to assess the effect of the substance ingested apart from the exercise intensity in tasks where intensity will fluctuate . arguably , the sue of a constant load exercise protocol for 30 minutes is a more externally valid form of exercise for most individuals as it mirrors the minimum exercise recommendations for healthy adults . no study to date has assessed the effects of r. rosea ingestion on mood , perceptions of exertion , arousal , and feelings of pleasure / displeasure during steady state exercise . in the current study , rpe and affect were measured prior to , during , and after 30-minute submaximal cycling at a moderate exercise intensity after ingestion of 3 mgkg r. rosea of placebo . this study is novel in that results demonstrated dampened rpe , greater vigor , perception of arousal , and pleasure in the presence of r. rosea compared to placebo . in the case of rpe and feelings of pleasure , thus , this data indicate that acute r. rosea ingestion positively modifies affect during relatively brief , steady state exercise at moderate intensity . previous studies have speculated that the ingestion of r. rosea can improve exercise performance via altered energy metabolism , activated by the synthesis or resynthesis of atp in mitochondria and stimulated restorative energy processes after intense exercise . however , the results of the present study suggest that altered substrate utilization is not responsible for the improved performance . the discrepancy between the findings of the present study in this respect and those of prior work [ 15 , 16 ] may be due to varying preexercise fed state across studies . in the present study the frayn equation was employed to assess fat oxidation as this is the most widely applied equation for fat oxidation available . however , we acknowledge that other equations are also available and may be preferable in future work . it is also important to note that there is no gold standard to validate whole body substrate oxidation but that assessment of substrate oxidation using indirect calorimetry may be prone to large errors . therefore , the acute increase in endogenous opioid production may play a role in enhancing exercise performance in the presence of r. rosea and is evidenced through changes in perceived exertion and exercise affect rather than changes in physiological markers . prior research has tended to employ protocols involving time trial performance or time to exhaustion , neither of which is applicable to the typical exercising public . while the protocol employed in the present study may be valid when examining steady state exercise at a moderate intensity , it also might not fully address the typical training session undertaken by many recreational exercisers and athletes . however , given the lack of studies examining the effect of r. rosea on exercise performance such steady state protocols are useful in developing the extant literature in this area . we do acknowledge that by controlling exercise intensity based on a percentage of vo2max we are unable to determine if participants were exercising below , at , or above their lactate threshold . future research might subsequently benefit from using a standardised protocol to mimic the type of exercise session commonly undertaken in gyms ( e.g. , combined aerobic and resistance exercise ) in order to determine if r. rosea ingestion is practically useful in a more ecologically valid exercise task . the current study also examined participants who were recreationally active but were not specifically cycling trained . it has been suggested that less fit individuals may experience greater fatigue and discomfort during exercise which may reduce feelings of pleasure compared to more highly trained individuals . it may therefore be useful to compare the responses of participants of different training status in order to make more conclusive statements regarding the effect of r. rosea on variables such as perception of exertion , arousal , and pleasure . furthermore , a posteriori power calculations indicated a sample size of 28 would have been required to detect a difference of p = 0.05 at 95% power . however , due to the paucity of data on effects of r. rosea on exercise performance this study was exploratory in nature and subsequently used a relatively small number of participants . finally , the assessment of affect immediately on completion of the cycling bout might have resulted in elevated scores for feeling states due to the cessation of exercise as has been suggested previously . it may be that the trajectory of pleasure and displeasure during and after exercise exhibits two distinct phases . the first phase involves a decline or increase of affective responses during exercise , whereas the second phase involves an improvement or rebound of affective responses after exercise . as measures of affect were only taken on completion of the exercise bouts , the data presented here are only representative of the rebound phase of exercise in the presence of r. rosea and placebo . future research should therefore attempt to assess affect during exercise in addition to immediately on cessation in order to more effectively capture the time course of affective responses to exercise following ingestion of different substances . ingestion of r. rosea favourably influenced rpe and exercise affect without changes in energy expenditure or substrate utilization during 30 minutes of submaximal cycling performance at moderate intensity in regularly active adults . these changes support the efficacy of acute r. rosea ingestion in positively enhancing psychophysiological responses to submaximal exercise performance .

the aim of this study was to examine the effect of acute rhodiola rosea ( r. rosea ) ingestion on substrate utilisation , mood state , rpe , and exercise affect . ten males ( mean age s.d . = 26 6 years ) completed two 30-minute cycling trials at an intensity of 70% of vo2max following ingestion of either 3 mgkg1 body mass of r. rosea or placebo using a double - blind , crossover design . during exercise , heart rate and rpe were recorded . participants completed measures of mood state and exercise affect before and after exercise . expired air samples were taken during exercise to determine substrate utilisation . repeated measures analysis of variance indicated that rpe was significantly lower at 30 minutes into exercise versus placebo ( p = 0.003 ) . perceptions of arousal ( p = 0.05 ) and pleasure were significantly higher after exercise with r. rosea compared to placebo ( p = 0.003 ) . mood state scores for vigor were also higher in r. rosea condition compared to placebo ( p = 0.008 ) . there were no significant differences in energy expenditure , carbohydrate , or fat oxidation between conditions ( p > 0.05 ) . ingestion of r. rosea favourably influenced rpe and exercise affect without changes in energy expenditure or substrate utilization during 30-minute submaximal cycling performance .

1. Introduction 2. Method 3. Substrate Oxidation 4. Results 5. Discussion 6. Conclusions

rhodiola rosea ( r. rosea ) is an herb that grows in mountainous regions of north america , europe , and asia . some recent studies have supported the results of earlier work by identifying antioxidant and anti - inflammatory properties of r. rosea [ 3 , 4 ] . another work has suggested ingestion of r. rosea appears to be effective , either acutely or with daily supplementation , for reducing perceived fatigue and improving cognition [ 5 , 7 ] , as well as reducing markers of physiological and psychological stress [ 7 , 8 ] . although r. rosea ingestion has been identified as a means to reduce physical fatigue , its efficacy during exercise is unclear . animal based research has shown increased swim time to exhaustion in rats [ 3 , 9 ] but the impact of r. rosea ingestion on exercise performance in humans is equivocal . some studies have shown no effect of r. rosea ingestion on exercise performance [ 4 , 1012 ] whilst others have supported its use [ 13 , 14 ] . reported that an acute dose of r. rosea increased time to exhaustion during an incremental cycle ergometer protocol . conversely , in the same study , 4 weeks of r. rosea supplementation did not significantly improve cycling performance . this was the first study to show an acute impact of r. rosea ingestion on human exercise performance . examined the efficacy of a 3 mgkg body mass dose of r. rosea on 6-mile cycle time trial performance in 18 active women . they reported that r. rosea significantly decreased submaximal exercise heart rate , reduced rpe , and improved time trial performance time . subsequently suggested that acute r. rosea ingestion might be employed by active individuals while exercising to complete a set distance in the shortest time . such data are interesting ; as if r. rosea does positively impact on exercise , it may be safe and legal way to enhance exercise performance in athletes and the exercise experience in recreational gym users . demonstrated that an extract of r. rosea significantly prolonged the duration of exhaustive swimming in rats and stimulated atp synthesis in muscle during exercise . these findings led perfumi and mattioli to speculate that , in addition to enhancing the catecholaminergic system , the ability of r. rosea to increase performance involves an improvement in cellular energy metabolism . however , some studies have suggested that r. rosea acts to acutely increase endogenous opioid production or receptor sensitivity [ 2 , 13 ] which subsequently impacts on brain dopamine and serotonin and cardiac activity and attenuates the perception of effort at a given workload . however , as only two studies have examined acute r. rosea ingestion on exercise performance to date , further data is needed on this topic . employed performance based protocols to examine the effect of r. rosea ingestion . while these are valuable in understanding the effect of a nutritional supplement on performance responses , different protocols may be better placed to evidence any effect of r. rosea on exercise performance . for example , using steady state submaximal exercise in the presence of r. rosea and placebo would allow for power output to remain constant across a given period . moreover , when employing protocols where intensity is modified throughout ( as is the case with both time trials and time to exhaustion ) understanding any impact of the exercise protocol of substance ingested becomes more difficult because even small increases in exercise intensity have been shown to markedly change feelings of pleasure during exercise . furthermore , given the postulation that r. rosea acts through increased opioid production , it is perhaps surprising that few studies have examined its impact on affect . reported no effect of r. rosea ingestion on mood state , as assessed using the poms questionnaire . however , changes in mood state with exercise have been reported following ingestion of a range of substances including caffeine [ 18 , 19 ] and carbohydrate . as a consequence if nutritional manipulation enhances the pleasure response to exercise , according to hedonic theory , it may mean individuals are more likely to exercise . this is important as different exercise intensities and individual fitness will create variability in the affective response to exercise [ 2224 ] and the effect of nutritional manipulation on these responses is not well known . assuming that the affective response to exercise is the same in recreational exercises compared to high level performers may therefore lead to erroneous conclusions regarding the exercise - affect relationship . thus , while the extant literature examining the effect of r. rosea ingestion has focused on performance measures , the current study seeks to develop this work by employing a model that can be applied to recreational exercisers and is more applicable to the exercising public in general . the aim of this study was to examine the effect of acute r. rosea ingestion on substrate utilisation , mood state , rpe , perceptions of arousal , and pleasure / displeasure in a population of recreationally active men . it was hypothesized that ingestion of r. rosea would positively influence perceived exertion during exercise as well as enhancing mood and perceptions of arousal and pleasure / displeasure before and after cycling exercise . testing these hypotheses will address gaps in knowledge on the effect of r. rosea by examining their effects on an active but not performance oriented group , more typical of regularly active individuals in the general population . the study will also combine physiological and psychological measures to address gaps in the literature base by assessing substrate utilisation , heart rate , rpe , mood state , and perceptions of arousal and pleasure / displeasure to provide a multidisciplinary investigation of the effect of r. rosea during exercise . this study employed a within - participants double - blind cross - over design whereby participants visited the laboratory on 3 occasions at the same time of day in a well - rested and well - hydrated state . participants were excluded if they had a musculoskeletal or cardiovascular contraindication to exercise , were taking any medication that could impact mood / affect , engaged in less than 3 or more than 10 hours physical activity per week , or were engaged in competitive sports activity as part of their habitual physical activity . the incremental exercise test was performed on a mechanically braked cycle ergometer ( monark exercise ab , sweden ) to assess vo2max following previously published guidelines . this protocol , using 3-minute stages rather than a ramp protocol , was employed based on prior research suggesting that this form of protocol provides more reliable physiological measures , particular for the population characteristics examined in this study ( i.e. expired air was collected via the douglas bag technique during the final minute of each incremental exercise stage . samples were analyzed for oxygen and carbon dioxide content ( servomex , crowborough , england ) and expired air volume ( harvard dry gas meter , harvard apparatus , kent , england ) with values for oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) subsequently calculated . heart rate ( polar electro , kempele , finland ) and rating of perceived exertion ( rpe ) , using the borg 620 rpe scale , were recorded during the final 15 seconds of each workload . participants were judged to have reached vo2max if they presented at least 3 of the following : ( a ) a respiratory exchange ratio of greater than 1.1 , ( b ) a heart rate during the last stage of testing that was 10 beats of age predicted maximum heart rate , ( c ) an rpe of 18 or greater , ( d ) a plateau in vo2 with an increase in workload , and ( e ) volitional fatigue . on completion of the vo2max testing and following a period of at least 72 hours participants completed two 30-minute submaximal cycling trials at a workload of 70% vo2max in a fasted state . the duration and intensity of exercise were chosen as it sits within the american college of sports medicine guidelines for the prescription of exercise for health benefit . on a molecular basis , the main active ingredients of r. rosea appear to be tyrosol and its glucoside known as salidroside and following oral ingestion of 100 mgkg salidroside , the half - life of salidroside appears to be 1.32 0.22 h . therefore , conditions were randomised , separated by 4872 hours , and consisted of a r. rosea condition where 3 mgkg body mass of r. rosea ( indigo herbs , glastonbury , uk ) placed in a coloured , opaque gelatin capsule or a placebo ( 3 mgkg body mass of maltodextrin , ( myprotein , northwich , uk ) ) was ingested with 250 ml water . the dose of r. rosea used was based on the previous work of de bock et al . who observed improvements in endurance performance following acute ingestion of r. rosea . during each of the cycling trials , heart rate was monitored ( polar rs400 , polar electro oy , kempele , finland ) continuously and assessed every 10 minutes . the felt arousal scale ( fas ) was also used as a measure of arousal . mood state was also assessed using the fatigue and vigour subscales of the brunel mood state inventory ( brums ) . this is a well - established , reliable , and valid measure of mood state that has been previously employed to assess the mood state response to various exercise modes [ 18 , 35 , 36 ] . the fatigue and vigor subscales were chosen in particular as prior research [ 1 , 7 ] has suggested r. rosea may particularly influence feelings of fatigue and/or vigor . participants were introduced to these scales during their first visit to the laboratory ( prior to establishment of vo2max ) . prior to cycling ( but whilst seated on the exercise bike ) at 14 min and 29 min during each exercise bout expired air samples were collected using the douglas bag technique to estimate fat and carbohydrate ( cho ) oxidation rates . the collection time was 60 s and samples were analysed for oxygen and carbon dioxide content ( servomex , crowborough , england ) that was calibrated at two points before each assessment using gases of known concentrations ( 15.12% o2 , 5.1% co2 ) in accordance with manufacturers guidelines and expired air volume ( harvard dry gas meter , harvard apparatus , kent , england ) with values for oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) subsequently calculated . energy expenditure ( kcalmin ) and the oxidation rates for carbohydrate and fat ( gmin ) were then calculated according to the weir and frayn equations , respectively . a series of 2 ( substance ingested ) 4 ( time point ) ways repeated measures analysis of variance was used to examine any differences in heart rate and rpe . a series of 2 ( substance ingested ) x 3 ( time point , before ingestion , after ingestion but before exercise , and after exercise ) ways repeated measures analysis of variance was used to examine any differences in mood state and perceptions of arousal and pleasure / displeasure and substrate utilisation . where any significant differences were discovered , bonferroni pairwise multiple comparisons were used to determine where the differences lay . partial was used as a measure of effect size , statistical significance was set at p = 0.05 a priori , and the statistical package for social sciences ( version 20 ) was used for all analysis ( spss inc . in respect to heart rate , results indicated no significant interactions or main effect due to the substances ingested . there was however a significant main effect for time ( f 3 , 27 = 266.7 , p = 0.0001 , p = 0.967 ) where heart rate was significantly increased throughout the exercise bout irrespective of substance ingested . mean se of heart rate ( bpm ) was 77 4 at rest compared to 151 4 , 158 4 , and 162 4 at 10 , 20 , and 30 minutes into the exercise bout , respectively . bonferroni comparisons indicated that heart rate was significantly different at all time points ( p = 0.001 in all cases ) . in the case of rpe , results indicated a significant substance time interaction ( f 3 , 27 = 6.12 , p = 0.003 , p = 0.405 , see figure 1 ) . rpe data were not significantly different at rest and at 10 and 20 minutes into the exercise bout between the two conditions . however , rpe in the placebo condition was significantly higher than that in the r. rosea condition 30 minutes into the exercise bout ( p = 0.003 ) . scores from the fatigue subscale of the brums indicated a significant main effect across time ( f 2 , 18 = 12.879 , p = 0.0001 , p = 0.589 ) where scores were significantly higher after exercise compared to before exercise ( p = 0.028 ) and compared to before exercise but after ingestion ( p = 0.008 ) . there were no other significant main effects of interactions for fatigue subscale data . in regard to the vigor subscale of the brums , there was no significant substance time interaction . there were however significant main effects for time ( f 2 , 18 = 4.435 , p = 0.027 , p = 0.350 ) and for the substance ingested ( f 2 , 9 = 11.692 , p = 0.008 , p = 0.565 ) . scores for vigor were significantly lower at after exercise compared to after ingestion but before exercise ( p = 0.04 ) . mean se for vigor scores was 6.9 0.56 before ingestion , 7.4 0.44 after ingestion but before exercise , and 5.6 0.74 after exercise . for the substance ingested , scores for vigor were significantly higher in the r. rosea condition ( p = 0.008 ) compared to the placebo . mean se for vigor was 7.6 0.52 in the r. rosea condition compared to 5.7 0.57 in the placebo condition . perceptions of arousal were also significantly different in the presence of r. rosea compared to placebo ( f 1 , 9 = 5.0 , p = 0.05 , p = 0.357 ) with scores for arousal being higher ( 3.1 0.19 ) in the presence of r. rosea compared to placebo ( 2.8 0.11 ) . when data for feeling of pleasure / displeasure were assessed , there was however a significant condition time effect ( f 2 , 18 = 12.795 , p = 0.0001 , p = 0.587 , see figure 2 ) . this followed a similar pattern to rpe with no significant difference evident before ingestion ( p = 0.591 ) or after ingestion but before exercise ( p = 0.08 ) . there was however significantly lower scores for pleasure after exercise in the placebo condition compared to the r. rosea condition ( p = 0.003 ) . energy expenditure was significantly higher during exercise ( f 2 , 18 = 130.921 , p < 0.001 , p = 0.936 ) ( see figure 3 ) , irrespective of the trial ( f 1 , 9 = 1.198 , p = 0.302 , p = 0.117 ) . the rate of total carbohydrate oxidation ( figure 4 ) was not significantly different between trials ( f 1 , 9 = 0.317 , p = 0.587 , p = 0.034 ) . carbohydrate oxidation remained relatively constant throughout exercise but was higher compared with the rest ( f 2 , 18 = 13.902 , p < 0.001 , p = 0.770 ) . the rate of fat oxidation ( figure 5 ) was higher than that at rest during exercise ( f 1 , 11 = 25.131 , p < 0.001 , p = 0.736 ) but there was no significant main effect due to the substances ingested ( f 1 , 9 = 0.950 , p = 0.355 , p = 0.095 ) . acute r. rosea ingestion has been reported to improve exercise performance [ 13 , 14 ] in performance based tasks . , time trial ) in prior studies makes it difficult to assess the effect of the substance ingested apart from the exercise intensity in tasks where intensity will fluctuate . arguably , the sue of a constant load exercise protocol for 30 minutes is a more externally valid form of exercise for most individuals as it mirrors the minimum exercise recommendations for healthy adults . no study to date has assessed the effects of r. rosea ingestion on mood , perceptions of exertion , arousal , and feelings of pleasure / displeasure during steady state exercise . in the current study , rpe and affect were measured prior to , during , and after 30-minute submaximal cycling at a moderate exercise intensity after ingestion of 3 mgkg r. rosea of placebo . this study is novel in that results demonstrated dampened rpe , greater vigor , perception of arousal , and pleasure in the presence of r. rosea compared to placebo . in the case of rpe and feelings of pleasure , thus , this data indicate that acute r. rosea ingestion positively modifies affect during relatively brief , steady state exercise at moderate intensity . previous studies have speculated that the ingestion of r. rosea can improve exercise performance via altered energy metabolism , activated by the synthesis or resynthesis of atp in mitochondria and stimulated restorative energy processes after intense exercise . in the present study the frayn equation was employed to assess fat oxidation as this is the most widely applied equation for fat oxidation available . therefore , the acute increase in endogenous opioid production may play a role in enhancing exercise performance in the presence of r. rosea and is evidenced through changes in perceived exertion and exercise affect rather than changes in physiological markers . however , given the lack of studies examining the effect of r. rosea on exercise performance such steady state protocols are useful in developing the extant literature in this area . we do acknowledge that by controlling exercise intensity based on a percentage of vo2max we are unable to determine if participants were exercising below , at , or above their lactate threshold . future research might subsequently benefit from using a standardised protocol to mimic the type of exercise session commonly undertaken in gyms ( e.g. , combined aerobic and resistance exercise ) in order to determine if r. rosea ingestion is practically useful in a more ecologically valid exercise task . it has been suggested that less fit individuals may experience greater fatigue and discomfort during exercise which may reduce feelings of pleasure compared to more highly trained individuals . it may therefore be useful to compare the responses of participants of different training status in order to make more conclusive statements regarding the effect of r. rosea on variables such as perception of exertion , arousal , and pleasure . furthermore , a posteriori power calculations indicated a sample size of 28 would have been required to detect a difference of p = 0.05 at 95% power . however , due to the paucity of data on effects of r. rosea on exercise performance this study was exploratory in nature and subsequently used a relatively small number of participants . finally , the assessment of affect immediately on completion of the cycling bout might have resulted in elevated scores for feeling states due to the cessation of exercise as has been suggested previously . it may be that the trajectory of pleasure and displeasure during and after exercise exhibits two distinct phases . the first phase involves a decline or increase of affective responses during exercise , whereas the second phase involves an improvement or rebound of affective responses after exercise . as measures of affect were only taken on completion of the exercise bouts , the data presented here are only representative of the rebound phase of exercise in the presence of r. rosea and placebo . future research should therefore attempt to assess affect during exercise in addition to immediately on cessation in order to more effectively capture the time course of affective responses to exercise following ingestion of different substances . ingestion of r. rosea favourably influenced rpe and exercise affect without changes in energy expenditure or substrate utilization during 30 minutes of submaximal cycling performance at moderate intensity in regularly active adults . these changes support the efficacy of acute r. rosea ingestion in positively enhancing psychophysiological responses to submaximal exercise performance .

human african trypanosomiasis ( hat ) or african sleeping sickness is a serious life threatening disease . around 60 million people in 36 african countries are currently in constant threat of infection . although the reported number of cases has dropped over recent years , the actual number of unreported cases is estimated to be around 7000080000 . hat is caused by infection with trypanosoma brucei , a vector - borne parasite , which is transmitted by the bite of tsetse flies . the symptoms of the disease occur in two main stages . in the first stage , known as the hemolymphatic phase , the parasites multiply in blood , subcutaneous tissues , and lymph , causing headaches , fever , itching , joint pains , and swelling of lymph nodes . in the second stage , or neurological phase , this phase entails confusion , change of behavior , reduced coordination , sensory disturbances , disturbance of sleep cycle , and finally death . most available drugs for hat display severe toxic side effects , require long periods of administration , and/or are expensive due to the logistics to reach rural african areas . further , resistance to all in use drugs has been observed in the laboratory and/or in the field , resulting in an urgent requirement for better , safer , and inexpensive therapeutic alternatives to the current treatments . genetic knockdown studies have identified several proteins that are essential for the survival of the parasite , including members of the protein kinase ( pk ) family . in trypanosoma brucei pks are essential in many fundamental cellular processes , e.g. , proliferation , differentiation , and cell cycle control , and can therefore be considered as potential drug targets for the treatment of hat . in the t. brucei genome there are two kinases that are highly homologous to human glycogen synthase kinase 3 ( hsgsk3 ) : tbgsk3 short and tbgsk3 long . rna interference ( rnai ) knockdown of tbgsk3 has shown that tbgsk3 short is critical for cell growth , with a role in the control of mitosis and/or cytokinesis . the ability to selectively inhibit tbgsk3 over the off - target hsgsk3 is highly desirable because mouse knockout studies revealed that the disruption of the murine gsk3 gene causes embryonic lethality ; consequently , nonselective inhibitors are not applicable for use in infants and women of child bearing age . from a homology perspective , tbgsk3 is not only very closely related to hsgsk3 but also to other human pks such as cyclin dependent kinase 1 ( hscdk1 ) and cyclin dependent kinase 2 ( hscdk2).hscdk2 and hscdk1 are essential for g1/g2 progression and s / m - phase entry of the cell cycle . off - target inhibition of these human kinases will therefore result in cell cycle arrest and reduction of cellular proliferation and as such potentially lead to severe side effects . over the past decade , various groups and pharmaceutical companies have identified multiple series of hsgsk3 inhibitors . recently , astex therapeutics and researchers at the university of osaka have developed a series of aminopyrazoles that are potent inhibitors of hsgsk3. co - crystal structures of this series with hsgsk3 are not available to date ; however , complex structures with the closely related hscdk2 have been determined . in all structures , the pyrazole scaffold forms two hydrogen bonds to the hinge region of hscdk2 ( figure 1 ) . further , the nh group of the 3-position amide forms an additional hydrogen - bond interaction to the backbone of leu83 . a water - mediated hydrogen bond from the amide carbonyl oxygen atom to the backbone the r residues ( figure 2 ) access the gatekeeper region between the gatekeeper residue phe80 and the catalytic asp145 ( figure 1 ) . the r substituents occupy the hydrophobic pocket ii , formed by the backbone of the linker region , leu83 , phe82 , and side chains of ile10 , asp86 , and leu134 . finally , an intramolecular hydrogen bond between the r - nh and r - carbonyl group is present . the similarity of hscdk2 , hsgsk3 , and tbgsk3 indicates that aminopyrazoles will also bind into the atp - binding site of the latter enzyme . co - crystal structure ( 2vu3 ) of at7519 ( carbon atoms in gray ) bound to cdk2 . key : red sphere , water molecule ; black dashed lines , protein ligand and water ligand hydrogen bonds ; yellow stick , hydrogen atom .. generic binding mode of the r and r substituted aminopyrazole scaffold ( carbon atoms in gray ) . herein , we describe the design , synthesis , and biological evaluation of aminopyrazole inhibitors which bind to tbgsk3 short . the inhibitors were also tested against the closely related off - targets hsgsk3 and hscdk2 and evaluated against a panel of mammalian protein kinases . the most potent compound has nanomolar affinity for tbgsk3 short , is selective over hsgsk3 and hscdk2 , and clean in the kinase panel . by using computer - aided molecular modeling enzyme affinity correlated with inhibition of t. b. brucei proliferation , albeit a 100-fold offset in potency , was found . in light of these results , the aminopyrazole derivatives developed by astex therapeutics and yumiko uno et al . for inhibition of hscdk2 and hsgsk3 enzymes were chosen as a starting point for the investigation of tbgsk3 short inhibitors . aminopyrazoles analogues were generated by substituting at either r or r position ( figure 2 ) using two synthetic routes ( scheme 1a , b ) . reagents and conditions used in routes a and b : ( a ) trans-4-methoxycyclohexylamine , edc , hobt , dmf , rt ; ( b ) 10% pd / c , h2 , dmf , rt ; ( c ) rcooh , edc , hobt , dipea , rt ; ( d ) socl2 , meoh , 0c , rt ; ( e ) 10% pd / c , h2 , etoh , rt ; ( f ) 2,6-dimethoxybenzoylchloride , et3n , dioxane , rt ; ( g ) naoh , dioxane , h2o , rt ; ( h ) r2nh2 , polystyrene - bound carbodiimide , hobt , acetonitrile , mw , 100 c . a structural model of tbgsk3 was built to assess the differences in the binding sites of tbgsk3 short , hsgsk3 , and hscdk2 and to guide ligand design . an overlay of 42 hsgsk3 crystal structures showed that there is low flexibility in the atp binding site . only the regions including phe67 and arg141 showed some mobility . arg141 also spans a number of distinct conformations , including examples where it occupies space in the binding site ( 1j1b , 1j1c , 1o9u , 2o5k ) and therefore could influence docking results . however , to allow for ligands of a significant size , we have used examples with arg141 pointing out of the binding site . therefore , two homology models for tbgsk3 were generated representing both states of phe67 . as we were mainly interested in aminopyrazoles with less extended r groups , the crystal structure with phe67 pointing toward the hinge ( with structure 1r0e as a representative ) the selection of 1r0e instead of other members of this group ( with phe67 pointing toward the hinge ) was arbitrary . for this analysis , all residues that are located within 6 of the ligand bound to the template structure ( 1r0e ) were considered . the binding pockets of tbgsk3 and hsgsk3 differ by nine amino acid residues ( table 1 , figure 3a ) . of the amino acid side chains that point toward the ligand , the most significant differences are the replacement of tyr134 in hsgsk3 with phe103 , leu132 with met101 , gln72 with leu36 , and tyr140 with his109 . in total 16 out of 26 amino acids were found to be different ( table 1 , figure 3b ) , the most important of these differences being the replacement of lys20 in cdk2 with leu36 , phe80 with met101 , his84 with pro105 , lys89 with arg110 , and ala144 with cys170 . interestingly , most of the amino acid differences occurred in the hydrophobic pocket ii and the gatekeeper region . therefore , we decided to direct the optimization of the lead scaffold toward suitable interactions with amino acids which are located in these subpockets of the atp binding site . amino acids of hgsk3 or hscdk2 which differ in tbgsk3 short are shown in boldface . superposition of the binding sites of the homology model of tbgsk3 short ( blue carbon atoms ) with ( a ) the hsgsk3 crystal structure ( pdb code 1r0e ) and the solvent accessible surface of tbgsk3 short is shown in light blue . only residues that differ between the binding pockets are shown . the synthesis of r and r substituted aminopyrazole derivatives started from 4-nitro - pyrazole-3-carboxylic acid 1 and is described by two different routes ( scheme 1 ) based on previous work from wyatt et al . in route reduction of the subsequent intermediate 2 by hydrogenation in the presence of palladium on carbon generated amino pyrazole 3 . the conversion to compounds 4a z was accomplished by coupling of 3 with a suitable selection of carboxylic acids . in route b , after esterification of the carboxyl group of 1 , the nitro group of intermediate 5 was reduced to afford amine 6 . treatment of 6 with 2,6-dimethoxybenzoyl chloride under standard conditions , followed by base hydrolysis of the ester , provided acid 8 . in the final step , 8 was coupled with appropriate amines in a microwave reaction using polystyrene - bound carbodiimide to yield final compounds 9a x . twenty - six r substituted aminopyrazole analogues ( table 2 ) were made according to the synthetic route shown in scheme 1a . a range of r groups varying in size and polarity was chosen to probe whether the differences in the gate keeper region between tbgsk3 , hsgsk3 , and hscdk2 could be exploited to derive selective and potent tbgsk3 inhibitors ( figure 3 ) . concentration required to inhibit the growth of t. b. brucei in culture by 50% over 72 h. concentration required to inhibit the growth of mrc5 cells in culture by 50% over 72 h. all compounds showed good potency against tbgsk3 ( < 1 m ) . an unsubstituted phenyl ring ( 4f ) provided on average a 20-fold improvement of inhibition potency relative to saturated six - membered ring systems ( 4a and 4b ) and benzyl groups ( 4c , 4d , and 4e ) . in general , a variety of different aryl and heteroaryl rings ( 4g4y compared to 4a and 4b ) in the r position led to significantly improved potency against tbgsk3 . additionally , a wide variety of substituents were tolerated on the phenyl ring . in general , ortho - substituted phenyl rings gave the best improvement in activity compared to the unsubstituted phenyl group ( 4j , 4k , 4 m , and 4n ) . the methoxyphenyl moieties in 4j , 4 m , and 4n , which had tbgsk3 ic50 values of 4 , 2 , and 3 nm , respectively , were the most favorable substituents . interestingly , this was also the most selective compound for hscdk2 ( > 1000-fold ) . to rationalize the observed selectivity , all analogues were docked into the binding sites of tbgsk3 , hsgsk3 , and hscdk2 and their poses were visually analyzed . for most compounds , a binding mode similar to that observed for at7519 in hscdk2 ( figure 1 ) was predicted in tbgsk3 , hsgsk3 , and hscdk2 . one important difference between hscdk2 , tbgsk3 , and hsgsk3 is the gatekeeper residue ( table 1 , figure 3a , b ) . while hsgsk3 and tbgsk3 enzymes have leu or met , respectively , in this position , in hscdk2 phe is present . as a consequence , the gatekeeper region of hscdk2 ( located between phe80 and asp145 ) is more restricted compared to the other two enzymes . this resulted in a higher energy , out of plane conformation of the amide group of 4f when binding into this pocket ( figure 4 ) , while a low energy conformation was found when binding into t. brucei and human gsk3 ( not shown ) . further , without induced fit adaptations , the bulky r - substituents such as the 2,6-dimethoxybenzamide group of 4 m , the 2,4,6-trimethoxybenzamide groups 4n , and the phenylaminobenzamide groups of 4k and 4l can only be accommodated by the gatekeeper region of tbgsk3 and hsgsk3 but not the narrower hscdk2 gatekeeper region . these observations might explain the reduced binding affinity of 4f for hscdk2 compared to hsgsk3 . of note , this explanation is further supported by the report by wyatt et al . , which found that the aryl groups which are located in the same position need to twist in order to provide potent cdk2 activity docking results suggested that the phenyl ring of compound 4f needs to be significantly twisted out of plane by approximately 60 compared to the amide in order to fit into the gatekeeper region .. all r substituted compounds were tested for their ability to inhibit the proliferation of bloodstream form ( bsf ) t. b. brucei in culture . as an initial indication of potential toxicity , compounds 4a4z were additionally tested against proliferating human fetal lung fibroblast cells ( mrc5 cell line ) . four compounds ( 4 g , 4j , 4 m , and 4y ) had ec50 values < 1 m and a further 11 compounds had ec50 values <3 m against bsf t. b. brucei ( table 2 ) . the ec50 values correlated well with enzyme activity ( r = 0.73 , figure 5 ) . however , a 100-fold drop from enzyme to cellular activity was observed . selectivity over the mrc5 cells was achieved with compounds 4 m ( 60-fold ) , 4s ( > 19-fold ) , 4x ( 12-fold ) , and 4n ( > 9-fold ) , however , the majority of compounds showed a poor selectivity over mrc5 cells . correlation between the inhibition of recombinant tbgsk3 and bloodstream form t. b. brucei proliferation by r substituted aminopyrazole derivatives ( 4a z ) . as 4 m was the most potent inhibitor of tbgsk3 and proliferation of t. b. brucei cells , we retained the 2,6-dimethoxybenzamide group at position r for optimization of the r substituent . r substituted aminopyrazole analogues ( 9a9x ) were made according to the synthetic route shown in scheme 1b to explore the structural requirements for improvement of antiparasitic activity and selectivity over the closely related human kinases . the majority of variations led to potent tbgsk3 inhibitors , indicating that chemical diversity at this position was well tolerated ( table 3 ) . one of the sar trends observed was that six - membered saturated rings ( 9c ) and seven - membered saturated rings ( 9d ) were favored over their three- and four - membered equivalents ( 9a and 9b ) . further , it was noted that the replacement of the cyclohexane of 9c with a phenyl ring or 4-pyridine , to give 9k or 9l , gave a 6-fold decrease in potency against tbgsk3 . the 2-pyridine analogue ( 9 m ) , on the other hand , was much less active ( 50-fold ) against tbgsk3 . homologation of aromatic ( 9 g ) and saturated six - membered ( 9j ) rings by one carbon atom produced inhibitors with 1 nm activity for tbgsk3 . for aliphatic side chain derivatives 9r9w , the pentanyl and 1-isopropoxypropanyl analogues had ic50 values of 1 nm . the impact of replacing the amide group ( 3-position ) with carboxylic acid and ester groups was investigated with compounds 8 and 7 . compound 8 containing a carboxylate group in the 4-position showed a dramatic loss in activity ( ic50 > 50 m ) . the ester group of compound 7 on the other hand was better tolerated ( ic50 0.5 m ) . interestingly , compared with 4a4z , a majority of r substituted analogues ( 9a9x ) showed selectivity over hscdk2 and hsgsk3. these are the most selective tbgsk3 inhibitors described to date . concentration required to inhibit the growth of t. b. brucei in culture by 50% over 72 h. concentration required to inhibit the growth of mrc5 cells in culture by 50% over 72 h. the predicted binding mode of 9 g in tbgsk3 offers an explanation for the observed selectivity ( figure 6 ) . in the highest scoring docking pose , the core scaffold adopts a similar binding mode as observed for at7519 in hscdk2 ( figure 1 ) . in addition , the docking results suggested that the hydrophobic pocket ii of tbgsk3 was occupied by the n - benzylamide group of 9 g in such a way that its phenyl moiety formed t - shaped edge - to - face interactions with the side chain of phe103 and hydrophobic interactions with leu36 and ala26 . in hgsk3 , phe103 is replaced with a tyr ( table 1 , figure 3a ) , resulting in steric clash and electrostatic repulsion toward the benzyl moiety of 9 g . further , leu36 is substituted with gln72 in hgsk3 and lys20 in hscdk2 , diminishing hydrophobic interactions between the benzyl moiety of 9 g and these residues . overall , these changes together with differences in the gatekeeper region of hscdk2 ( see above ) are likely to be responsible for the high selectivity of 9 g for tbgsk3 over hgsk3 and hscdk2 . similar observations regarding the r group placement and selectivity were also made for compound 9h supporting this model . proposed binding mode of 9 g in the homology model of tbgsk3 ( blue carbon atoms ) overlaid on the hsgsk3 crystal structure ( pink carbon atoms ) . both ligand and protein are represented as sticks and color coded by atom types . ligand carbon atoms are shown in gray , protein carbon atoms of tbgsk3 are shown in blue , and hsgsk3 carbon atoms in salmon . hydrogen bonds and hydrophobic interactions are shown as black dotted lines , with interaction distances in angstroms . tbgsk3 amino acids which are involved in hydrophobic interactions with the benzyl group are marked in bold . the gold sphere represents the center of the phenyl ring .. the r substituted compounds were tested against bsf t. b. brucei and mrc5 cells . as for the r - substituted analogues , a good correlation between the ec50 and ic50 values and a 100-fold drop in activity between the biochemical and cell assay was observed ( figure 7 ) . compound 9c had an ec50 for t. b. brucei of 4 m ( table 3 ) . limited selectivity ( > 7-fold ) over mrc5 cells was achieved with compounds 9c , 9d , 9 g , 9s , and 9 t . it was found that the compounds showed selective inhibition of tbgsk3 over hsgsk3 ( > 20-fold ) and hscdk2 ( > 1200-fold ) . correlation between the inhibition of recombinant tbgsk3 and bloodstream from t. b. brucei proliferation using r substituted aminopyrazole derivatives ( 9a x ) . pk inhibitors frequently inhibit multiple kinases , often leading to off - target toxic effects . to assess the selectivity of the aminopyrazole inhibitors , remaining activity at 10 m concentration was measured for compounds 4f , 4 m , and 4y against a panel of 80 human pks and for compound 9 g against 124 human pks . compounds 4 m and 9 g were found to be highly specific ( table 4 ) . compound 4 m inhibited only two pks , namely gsk3 and cdk2 , at more than 80% . 9 g showed activity against three pks : gsk3 , mapkap - k2 , and mink1 at more than 80% . compound 4f was found to inhibit seven pks and compound 4y 15 pks by greater than 80% at 10 m . numbers represent average percentage of activity compared to the control at 10 m . in this table , only kinases with activity values < 20% are shown ( for full table see supporting information ) . in this work , we exploited the knowledge of the previously described aminopyrazoles inhibitors of hscdk2 and hsgsk3 to identify selective inhibitors of the tbgsk3 short isoform . this kinase has been shown using genetic manipulation studies to be essential for the survival of the t. b. brucei parasite . however , we wanted to confirm if antiparasitic activity could be gained using selective , small molecule inhibitors of tbgsk3 . the ability to selectively inhibit tbgsk3 over hsgsk3 and hscdk2 is essential to avoid potential side effects . therefore , more than 50 aminopyrazole derivatives were synthesized and screened against tbgsk3 , hsgsk3 , hscdk2 , and proliferating t. b. brucei and human cells in culture . the results ( table 2 and table 3 ) showed that almost all compounds were highly potent tbgsk3 inhibitors . the activity could be rationalized using the homology models and subsequent molecular docking studies ( figure 4 and figure 6 ) . the aminopyrazole derivatives make three h - bond interactions with the kinase hinge region , driving much of the potency of the compounds against the three kinases studied . selectivity could be derived from substitution at both r and r positions . from a homology perspective , hscdk2 is the most closely related kinase to hsgsk3. although , the enzymes only share approximately 33% amino acid identity , their atp binding pockets are highly conserved , resulting in the majority of known hscdk2 inhibitors also potently inhibiting hsgsk3. the results demonstrated that the required profile could be achieved , with several compounds with high affinity ( < 18 nm ) for tbgsk3 showing high selectivity ( > 500-fold ) over hscdk2 . docking studies provided a number of important insights into the binding modes and the selectivity profile of aminopyrazole derivatives . first , the docking results suggested that if the phenyl ring of compound 4f is planar with the amide group at r position it can not bind into the truncated gate keeper region of hscdk2 , defined by phe80 in hscdk2 , compared to met101 in tbgsk3 . to fit into this region of hscdk2 , the phenyl ring needs to significantly twist out of plane of the amide , with a torsion angle of approximately 60 , resulting in reduced binding affinity ( figure 4 ) . to stabilize this twist , di - ortho - substituents on the r phenyl group are required to cause a steric / electronic clash with the carbonyl of the amide bond . however , this region of the pocket in hscdk2 is narrow ( in a plane perpendicular to the hinge backbone and the pyrazole core ) , only allowing small ortho - substituents ( such as in compound 9 g ) on the phenyl group . in contrast , the wider gatekeeper regions of hgsk3 and tbgsk3 can tolerate large substituents such as the ortho - dimethoxy groups of compound 4 m or 4n and the ortho - phenylaminobenzamide groups of 4k or 4l . second , the highest increase in selectivity ( > 10000-fold ) over hscdk2 was achieved by accessing the hydrophobic pocket ii . exploitation of hydrophobic interactions in these two pockets not only reliably increased ligand - binding affinity but also impacted on the selectivity profile of these compounds . on the basis of the biological results of compound 9 g and structural modeling studies , we have shown that selectivity over hsgsk3 can be achieved by exploiting the phe103tyr , leu36gln , and ala26ile active site differences in the hydrophobic pocket ii of tbgsk3 enzyme ( figure 6 ) . taken together , the region between the gate keeper residue and the catalytic aspartate of the dfg loop , together with the hydrophobic pocket ii , are the key areas to exploit to achieve high selectivity over hscdk2 . the tbgsk3 short enzyme ic50 values correlated well with the t. b. brucei antiproliferative ec50 activities of the described substituted aminopyrazole inhibitors ( figures 5 and 7 ) , indicating that the compounds act on target . however , a 100-fold drop in cell activity was observed , compared to that in the tbgsk3 assay ( 1 m ) . the calculated physical properties ( mw < 473 ; log p 0.43.6 ; psa < 130 ) of the series of compounds suggest this loss of activity was not driven by lack of cellular penetration . in addition , the compound series was observed to be highly chemically stable under the range of synthetic conditions used during the chemistry campaign , suggesting that chemical degradation was not responsible for the loss of activity in the proliferation assay . although metabolism by the parasite can not be ruled out , the high degree of correlation between enzyme inhibition and antiparasitic activity suggests this is not the case , as it would be not expected that all compounds be metabolized to a constant extent . therefore , the drop of activity was probably due to the high atp concentration ( millimolar range ) in the cell compared to the kinase assay conditions . furthermore , chemical proteomic profiling conducted in parasite cell extracts confirmed that compound 4 m binds the endogenous tbgsk3 short with nanomolar affinity and very few other kinase targets with much lower affinity in the micromolar range . in this study , we have developed a series of substituted aminopyrazole amides as tbgsk3 short inhibitors starting from a compound series initially designed by astex therapeutics to inhibit hscdk2 and hsgsk3. sar investigation and optimization successfully provided 18 low nanomolar ( ic50 < 10 nm ) inhibitors of tbgsk3 with high selectivity ( > 10000-fold ) over hscdk2 . with compound 9 g , we have shown that good ( 330-fold ) selectivity over hsgsk3 can be achieved by targeting the hydrophobic pocket ii . in addition , 9 g proved to be highly selective against a panel of 124 human pks , showing > 90% inhibition at 10 m against only one pk , hsgsk3. molecular modeling has also shown that despite overall conservation in sequence and conformation between the three pks ( hsgsk3 , hscdk2 , and tbgsk3 ) , the binding pockets have distinct features that determine their specificity for particular compounds . further , we have shown that enzymatic inhibition correlates well with cell efficacy over a wide range of concentrations and a representative member of this series binds the endogenous tbgsk3 with nanomolar potency , indicating that compounds definitely act on target . however , a general 100-fold drop in activity between target and cellular activities resulted at best in compounds with low micromolar antiparasitic activity . taken together , this data suggests that specific atp competitive hinge binders of tbgsk3 short require low picomolar potency to obtain nanomolar antiproliferative activity against t. brucei . first , non - atp competitive approaches to inhibition of tbgsk3 , through irreversible hinge binders or allosteric inhibitors , could be pursued . however , these approaches have potential downsides , through the introduction of a reactive functionality or an increased chance of resistance causing mutations , respectively . second , a polypharmacology approach through the inhibition of a number of essential t. brucei kinases in addition to tbgsk3 could be investigated , although obtaining selectivity over human kinases would be more problematical . however , the aminopyrazole compounds ( 4a4z and 9a9x ) reported here represent an excellent start for chemistry optimization of selective tbgsk3 short inhibitors and an outstanding probe for studying the physiological functions of tbgsk3 short in t. brucei parasites . subsequently , modeler 9.2 was used to build homology models of tbgsk3 short , whereas the hsgsk3 crystal structure ( pdb code 1r0e ) served as template . modeler was run with default settings , and only the highest - scoring structure was used for further analysis and modeling . flexx 2.0.1 ( biosolveit gmbh ) was used to dock ligands flexible into protein binding sites . the active sites were defined as the areas within 7 of the co - crystallized ligands of hscdk2 ( pdb code 2vu3 ) and hsgsk3 ( pdb code 1r0e ) or the equivalent residues in the homology model of tbgsk3 . in all three structures , protonation states of amino acids and the orientations of the protons of hydroxyl and amine groups of active - site residues a highly conserved water molecule ( h2o 82 in 1r0e or h2o 2134 in 2vu3 ) was kept in all three protein structures used for docking . docking was carried out using default settings , and only the highest scoring binding modes were visually analyzed . all figures of protein binding sites were prepared using pymol . for compound potency determinations , compounds were solubilized in dmso at a top concentration of 3 mm and serially diluted to achieve 10-point titration of final assay concentrations from 30 m to 0.3 nm with a final dmso concentration of 1% ( v / v ) . the reaction mixtures contained 1 m biotinylated gsp2 substrate , 1 m atp , 3.7 kbq / well [ -33p]-atp and 2.5 nm tbgsk3 in the tbgsk3 kinase assay buffer . gsk3 inhibitors were screened for selectivity assessment also against hsgsk3. for hsgsk3 assay , the reaction mixes contained 1 m biotinylated gsp2 substrate , 2 m atp , 7.4 kbq / well [ -33p]-atp and 15 nm hsgsk3 in the tbgsk3 kinase assay buffer ( 25 mm tris - hcl , ph 7.5 , 10 mm mgcl2 , 5 mm dtt , 0.02% chaps , 2 u / ml heparin ) . for hscdk2/cyclin a assay , the reaction mixtures contained 1 mm cdk5 biotinylated peptide substrate ( biotin - c6-pktpkkakkl ) , 1 m atp , 7.4 kbq / well [ -p]-atp and 2 nm hscdk2/cyclin a in the kinase assay buffer ( 50 mm tris - hcl , ph 7.5 , 10 mm mgcl2 , 2 mm dtt , 100 mm nacl , 0.2 mm egta , 0.02% ( v / v ) brij35 ) . the statistical significance of the compound potency ( ic50 ) was based on the performance of standard molecules which have been tested to a high replication . in the case of tbgsk3 short assay the average pic50 value was 8.26 with a sd ( standard deviation ) of 0.23 . the minimum significant ratio ( msr ) of 0.4 was evaluated considering the following formula : where sd is the standard deviation and n the number of replicate values routinely used for the assay ( 2 in our case ) . this implies that a difference of > 0.4 in pic50 can be considered statistically significant for this assay . in the case of hsgsk3 assay , the standard compound gw8510 was tested 47 times across 5 independent runs , with an average pic50 value of 8.10 and a sd of 0.21 . this implies that a difference of > 0.3 in pic50 can be considered statistically significant for this assay . for the hscdk2 assay , the analysis was performed using two different standards ( gw8510 and staurosporine ) tested respectively five times in a single run and 19 times in 2 independent runs . this implies that a difference of > 0.3 in pic50 can be considered statistically significant for this assay . selected compounds were screened against a panel of mammalian kinases routinely run by the division of signal transduction therapy ( dstt ) at the university of dundee in duplicate at 10 m . enzymes included in the panel and assay conditions are reported in the literature . all biochemical assays are run below the km for the atp for each enzyme , allowing comparison of inhibition across the panel . measurement of inhibition of the proliferation of mrc5 ( human lung fibroblast ) cells and t. b. brucei bloodstream stage cells was performed using a modification of the cell viability assay previously described . compounds ( 50 m to 0.5 nm ) were incubated with 2 10 cells / well in 0.2 ml of the appropriate culture medium ( mem with 10% fetal bovine serum for mrc5 cells ) in clear 96-well plates . plates were incubated at 37 c in the presence of 5% co2 for 69 h. resazurin was then added to a final concentration of 50 m , and plates were incubated as above for a further 4 h before being read on a biotek flx800 fluorescent plate reader . h and c nmr spectra were recorded on either a bruker avance dpx 300 or 500 mhz spectrometer . chemical shifts ( ) are expressed in parts per million ( ppm ) and coupling constants ( j ) are in hertz ( hz ) . signal splitting patterns are described as singlet ( s ) , broad singlet ( br s ) , doublet ( d ) , triplet ( t ) , quartet ( q ) , quintuplet ( quin ) , sextuplet ( sex ) , septet ( sept ) , multiplet ( m ) , or combinations thereof . lcms ( liquid chromatography mass spectrometry ) analyses were performed with either an agilent hplc 1100 series connected to a bruker daltonics microtof or an agilent technologies 1200 series hplc connected to an agilent technologies 6130 quadrupole lcms , and both instruments were connected to an agilent diode array detector . lcms chromatographic separations were conducted with a phenomenex gemini c18 column , 50 mm 3.0 mm , 5 m particle size ; mobile phase / acetonitrile + 0.1% hcooh 80:20 to 5:95 over 3.5 min , and then held for 1.5 min ; flow rate 0.5 ml min . high resolution electrospray measurements ( hrms ) were performed with a bruker daltonics microtof mass spectrometer . thin layer chromatography ( tlc ) was carried out on merck silica gel 60 f254 plates using uv light and/or kmno4 for visualization . when applicable , all glassware was oven - dried overnight and all reactions were carried out under dry and inert conditions ( argon atmosphere ) . all in this work synthesized compounds had a measured purity of greater than 95% ( measured on analytical hplc - ms system ) . m data are given below to substantiate the purity and integrity of the compounds . h nmr , c nmr , and hrms experiments were also used to confirm compound identity and purity . a mixture of 4-nitro-3-pyrazolecarboxylic acid ( 1 ) ( 2.33 g , 14.8 mmol ) , trans-4-methoxy - cyclohexylamine ( 2.39 g , 18.5 mmol ) , edc ( 3.55 g , 18.5 mmol ) , and hobt ( 2.50 g , 18.5 mmol ) in dmf ( 75 ml ) was stirred at ambient temperature for 16 h. the mixture was reduced in vacuo and partitioned between saturated aqueous sodium bicarbonate and etoac . the organic layer was washed ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to give a yellow oil , which was purified by column chromatography , eluting 0100% etoac in petroleum ether to give 2 . h nmr ( dmso - d6 dmso - d6 ) ( ppm ) 14.02 ( s , 1h ) , 8.73 ( s , 1h ) , 8.57 ( d , j = 7.81 hz , 1h ) , 3.74 ( m , 1h ) , 3.24 ( s , 3h ) , 3.11 ( m , 1h ) , 1.95 ( dd , j = 55.8 , 10.9 hz , 4h ) , 1.27 ( m , 4h ) . c nmr ( dmso - d6 ) ( ppm ) 159.04 , 141.49 , 132.17 , 131.44 , 77.40 , 55.01 , 47.58 , 29.67 , 29.30 . lrms ( es ) : a solution of 2 ( 1.13 g , 4.2 mmol ) in dmf ( 100 ml ) was treated with 10% palladium on carbon then shaken under hydrogen at room temperature and atmospheric pressure for 5 h. the reaction mixture was diluted with etoac , filtered through celite , washing with further etoac , and the filtrate reduced in vacuo to give crude 3 as brown oil . yield : 982 mg , 98% . h nmr ( cd3od ) ( ppm ) 7.23 ( s , 1h ) , 3.84 ( m , 1h ) , 3.37 ( s , 3h ) , 3.23 ( m , 1h ) , 2.07 ( dd , j = 45.8 , 11.2 hz , 4h ) , 1.38 ( m , 4h ) . c nmr ( meod - d4 ) ( ppm ) 165.58 , 134.15 , 133.13 , 118.22 , 79.72 , 56.15 , 48.58 , 31.37 . lrms ( es ) : m / z 239 [ m + h ] . a mixture of benzoic acid ( 0.051 g , 0.42 mmol ) , 3 ( 0.1 g , 0.42 mmol ) , edc ( 0.096 g , 0.5 mmol ) , and hobt ( 0.068 g , 0.5 mmol ) in dmf ( 10 ml ) was stirred at ambient temperature for 16 h. the mixture was reduced in vacuo and partitioned between saturated aqueous sodium bicarbonate and etoac . the organic layer was washed ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to give a creamy solid 4f , which was purified by column chromatography . evaporation of the appropriate fraction yielded the desired compound as an amorphous solid . h nmr ( cdcl3 ) ( ppm ) 10.65 ( s , 1h ) , 8.52 ( s , 1h ) , 8.01 ( d , j = 7.2 hz , 2h ) , 7.57 ( t , j = 7.2 hz , 1h ) , 7.51 ( m , 2h ) , 6.86 ( d , j = 8.3 hz , 1h ) , 4.01 ( m , 1h ) , 3.39 ( s , 3h ) , 3.22 ( m , 1h ) , 2.16 ( m , 4h ) , 1.42 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 164.36 , 163.21 , 133.54 , 133.28 , 132.03 , 128.83 , 127.24 , 123.74 , 120.82 , 78.13 , 55.91 , 47.48 , 30.69 , 30.11 . hrms ( es ) : calcd for c18h23n4o3 [ m + h ] 343.1765 , found 343.1751 . h nmr ( cdcl3 ) ( ppm ) 9.86 ( s , 1h ) , 8.35 ( s , 1h ) , 6.83 ( d , j = 8.3 hz , 1h ) , 3.95 ( m , 1h ) , 3.39 ( s , 3h ) , 3.21 ( m , 1h ) , 2.44 ( m , 1h ) , 2.22 ( m , 2h ) , 2.11 ( m , 6h ) , 1.94 ( m , 2h ) , 1.81 ( m , 2h ) , 1.40 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 171.61 , 163.21 , 133.28 , 123.22 , 122.55 ( t , j = 239.4 hz ) , 120.71 , 78.07 , 55.91 , 47.50 , 42.74 , 32.85 ( t , j = 23.5 hz ) , 30.35 , 25.73 . hrms ( es ) : calcd for c18h27f2n4o3 [ m + h ] 385.2046 , found 385.2036 . h nmr ( cdcl3 ) ( ppm ) 9.86 ( s , 1h ) , 8.34 ( s , 1h ) , 6.81 ( d , j = 8.1 hz , 1h ) , 4.07 ( m , 2h ) , 3.95 ( m , 1h ) , 3.47 ( m , 2h ) , 3.39 ( s , 3h ) , 3.20 ( m , 1h ) , 2.59 ( m , 1h ) , 2.13 ( d , j = 11.0 hz , 4h ) , 1.91 ( m , 4h ) , 1.40 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 171.97 , 163.30 , 133.22 , 123.23 , 120.71 , 78.12 , 67.26 , 55.92 , 47.50 , 42.20 , 30.64 , 30.12 , 29.10 . lrms ( es ) : m / z 351 [ m + h ] . hrms ( es ) : calcd for c17h27n4o4 [ m + h ] 351.2027 , found 351.2011 . h nmr ( cd3od ) ( ppm ) 8.20 ( s , 1h ) , 7.38 ( t , 7.7 hz , 1h ) , 7.347.26 ( m , 1h ) , 7.187.05 ( m , 2h ) , 3.83 ( m , 1h ) , 3.80 ( s , 2h ) , 3.33 ( s , 3h ) , 3.16 ( m , 1h ) , 2.06 ( br d , j = 11.5 hz , 2h ) , 1.97 ( br d , j = 12.0 hz , 2h ) , 1.34 ( m , 4h ) . c nmr ( cd3od ) ( ppm ) 169.57 , 164.14 ( d , j = 159.5 hz ) , 134.23 , 132.98 , 130.64 ( d , j = 10.9 hz ) , 125.75 , 123.77 , 123.20 ( d , j = 18.2 hz ) , 121.96 , 116.48 ( d , j = 21.7 hz ) , 79.69 , 56.14 , 48.68 , 37.61 , 31.41 , 31.27 . hrms ( es ) : calcd for c19h24fn4o3 [ m + h ] 375.1827 , found 375.1817 . h nmr ( dmso - d6 ) ( ppm ) 13.13 ( s , 1h ) , 9.76 ( s , 1h ) , 8.15 ( s , 1h ) , 8.00 ( d , j = 8.3 hz , 1h ) , 7.317.23 ( m , 2h ) , 7.02 ( d , j = 8.0 hz , 1h ) , 6.93 ( t , j = 7.4 hz , 1h ) , 3.82 ( s , 3h ) , 3.793.70 ( m , 1h ) , 3.24 ( s , 3h ) , 3.07 ( m , 1h ) , 2.01 ( d , j = 11.4 hz , 2h ) , 1.79 ( d , j = 11.4 hz , 2h ) , 1.45 ( m , 2h ) , 1.21 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 167.30 , 162.43 , 157.02 , 132.38 , 130.90 , 128.59 , 123.21 , 122.30 , 120.44 , 119.70 , 110.88 , 77.70 , 55.41 , 55.04 , 46.70 , 38.17 , 30.26 , 29.73 . lrms(es ) : m / z 387 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2028 . h nmr ( dmso - d6 ) ( ppm ) 13.18 ( br s , 1h ) , 9.86 ( s , 1h ) , 8.14 ( s , 1h ) , 8.05 ( d , j = 7.9 hz , 1h ) , 7.51 ( d , j = 8.2 hz , 2h ) , 7.36 ( t , j = 8.2 hz , 1h ) , 4.10 ( s , 2h ) , 3.76 ( m , 1h ) , 3.24 ( s , 3h ) , 3.08 ( s , 1h ) , 2.01 ( m , 2h ) , 1.81 ( m , 2h ) , 1.45 ( m , 2h ) , 1.19 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 170.16 , 162.31 , 135.49 , 131.62 , 129.68 , 128.30 , 128.10 , 122.23 , 122.09 , 77.68 , 55.04 , 46.77 , 38.23 , 30.25 , 29.74 . hrms ( es ) : calcd for c19h23cl2n4o3 [ m + h ] 425.1142 , found 425.1147 . h nmr ( cdcl3 ) ( ppm ) 10.89 ( d , j = 12.2 hz , 1h ) , 8.45 ( s , 1h ) , 8.07 ( m , 1h ) , 7.44 ( m , 1h ) , 7.22 ( m , 1h ) , 7.13 ( m , 1h ) , 6.73 ( d , j = 8.3 hz , 1h ) , 3.96 ( m , 1h ) , 3.29 ( s , 3h ) , 3.11 ( m , 1h ) , 2.05 ( m , 4h ) , 1.31 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 162.80 , 160.84 ( d , j = 246.9 hz ) , 160.53 , 133.81 ( d , j = 10.9 hz ) , 131.77 , 124.79 , 123.19 , 121.42 , 120.65 ( d , j = 10.7 hz ) , 116.46 ( d , j = 21.8 hz ) , 78.19 , 55.88 , 47.27 , 30.75 , 30.14 . lrms ( es ) : m / z 361 [ m + h ] . hrms ( es ) : calcd for c18h22fn4o3 [ m + h ] 361.1670 , found 361.1645 . h nmr ( dmso - d6 ) ( ppm ) 13.40 ( s , 1h ) , 10.22 ( s , 1h ) , 8.32 ( s , 1h ) , 8.29 ( d , j = 8.6 hz , 1h ) , 7.907.88 ( m , 1h ) , 7.847.81 ( m , 1h ) , 7.787.75 ( m , 2h ) , 3.72 ( m , 1h ) , 3.23 ( s , 3h ) , 3.06 ( m , 1h ) , 2.00 ( br d , j = 12.5 hz , 2h ) , 1.76 ( br d , j = 12.5 hz , 2h ) , 1.45 ( m , 2h ) , 1.14 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 163.34 , 162.73 , 135.06 , 133.01 , 132.78 , 130.73 , 128.39 , 126.63 ( d , j = 5.2 hz ) 126.04 ( d , j = 29.1 hz ) , 123.57 ( d , j = 276.3 hz ) , 122.06 , 120.30 , 77.66 , 55.06 , 46.97 , 30.28 , 29.67 . lrms ( es ) : m / z 411 [ m + h ] . hrms ( es ) : calcd for c19h22f3n4o3 [ m + h ] 411.1639 , found 411.1621 . h nmr ( cdcl3 ) ( ppm ) 10.04 ( s , 1h ) , 8.39 ( s , 1h ) , 7.53 ( d , j = 7.6 hz , 1h ) , 7.39 ( t , j = 7.6 hz , 1h ) , 7.30 ( d , 7.7 hz , 1h ) , 7.25 ( t , j = 7.5 hz , 1h ) , 6.87 ( d , j = 8.3 hz , 1h ) , 3.92 ( m , 1h ) , 3.36 ( s , 3h ) , 3.16 ( m , 1h ) , 2.90 ( q , j = 7.6 hz , 2h ) , 2.08 ( br d , j = 10.0 hz , 4h ) , 1.35 ( m , 4h ) , 1.26 ( t , j = 7.6 hz , 3h ) . c nmr ( cdcl3 ) ( ppm ) 167.60 , 163.29 , 142.86 , 134.89 , 133.19 , 130.65 , 129.69 , 127.15 , 126.11 , 123.25 , 120.94 , 78.18 , 55.86 , 74.45 , 30.58 , 30.12 , 26.44 , 15.84 . hrms ( es ) : calcd for c20h27n4o3 [ m + h ] 371.2078 , found 371.2078 . h nmr : ( dmso - d6 ) ( ppm ) 13.25 ( s , 1h ) , 11.77 ( s , 1h ) , 8.44 ( s , 1h ) , 8.15 ( s , 1h ) , 8.04 ( s , 1h ) , 7.63 ( s , 1h ) , 7.29 ( s , 1h ) , 7.18 ( s , 1h ) , 4.15 ( s , 3h ) , 3.88 ( m , 1h ) , 3.30 ( s , 3h ) , 3.16 ( m , 1h ) , 2.08 ( m , 2h ) , 1.91 ( m , 2h ) , 1.55 ( m , 2h ) , 1.29 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.43 , 160.94 , 157.47 , 133.49 , 131.33 , 122.29 , 120.83 , 120.59 , 120.28 , 112.32 , 77.73 , 56.05 , 55.05 , 46.72 , 30.28 , 29.83 . lrms ( es ) : m / z 373 [ m + h ] . hrms ( es ) : calcd for c19h25n4o4 [ m + h ] 373.1870 , found 373.1873 . h nmr ( dmso - d6 ) ( ppm ) 13.32 ( br s , 1h ) , 10.93 ( s , 1h ) , 9.53 ( s , 1h ) , 8.31 ( s , 1h ) , 8.28 ( d , j = 8.5 hz , 1h ) , 7.68 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.43 ( m , 1h ) , 7.337.28 ( m , 3h ) , 7.167.14 ( m , 2h ) , 7.016.96 ( m , 2h ) , 3.82 ( m , 1h ) , 3.35 ( s , 1h ) , 3.25 ( s , 3h ) , 3.09 ( m , 1h ) , 2.03 ( br d , j = 12.0 hz , 2h ) , 1.81 ( br d , j = 12.0 hz , 2h ) , 1.48 ( m , 2h ) , 1.20 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 164.84 , 162.97 , 144.57 , 141.62 , 132.81 , 129.33 , 128.04 , 122.32 , 122.00 , 120.01 , 119.63 , 119.26 , 118.30 , 116.49 , 77.74 , 55.06 , 46.95 , 30.33 , 29.73 . lrms ( es ) : m / z 434 [ m + h ] . hrms ( es ) : calcd for c24h28n5o3 [ m + h ] 434.2187 , found 434.2165 . h nmr ( dmso - d6 ) ( ppm ) 13.35 ( br s , 1h ) , 10.89 ( s , 1h ) , 9.53 ( s , 1h ) , 8.33 ( s , 1h ) , 7.63 ( dd , j = 8.1 , 1.4 hz , 1h ) , 7.34 ( m , 1h ) , 7.117.09 ( m , 2h ) , 6.996.97 ( m , 1h ) , 6.87 ( m , 1h ) , 6.82 ( dd , j = 8.4 , 1.0 hz , 1h ) , 3.84 ( m , 1h ) , 3.35 ( s , 1h ) , 3.25 ( s , 3h ) , 3.09 ( m , 1h ) , 2.29 ( s , 3h ) , 2.14 ( s , 3h ) , 2.03 ( br d , j = 12.0 hz , 2h ) , 1.82 ( br d , j = 12.0 hz , 2h ) , 1.49 ( m , 2h ) , 1.21 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 165.36 , 163.08 , 147.05 , 138.87 , 137.74 , 132.95 , 132.72 , 130.25 , 127.52 , 125.96 , 125.74 , 122.41 , 120.79 , 119.98 , 117.44 , 115.36 , 114.63 , 77.74 , 55.06 , 46.95 , 30.33 , 29.75 , 20.25 , 13.64 . lrms ( es ) : m / z 462 [ m + h ] . hrms ( es ) : calcd for c26h32n5o3 [ m + h ] 462.2500 , found 462.2503 . h nmr ( dmso - d6 ) ( ppm ) 13.28 ( s , 1h ) , 9.75 ( s , 1h ) , 8.29 ( s , 1h ) , 8.20 ( d , j = 8.4 hz , 1h ) , 7.39 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.5 hz , 2h ) , 3.76 ( s , 6h ) , 3.70 ( m , 1h ) , 3.22 ( s , 3h ) , 3.06 ( m , 1h ) , 2.00 ( m , 2h ) , 1.76 ( m , 2h ) , 1.45 ( m , 2h ) , 1.13 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.79 , 161.21 , 156.88 , 132.21 , 131.08 , 122.52 , 119.81 , 115.03 , 104.28 , 77.66 , 55.81 , 55.08 , 46.93 , 30.29 , 29.66 . lrms ( es ) : . hrms ( es ) : calcd for c20h27n4o5 [ m + h ] 403.1976 , found 403.1960 . h nmr ( cd3od ) ( ppm ) 8.36 ( s , 1h ) , 6.17 ( s , 2h ) , 3.84 ( s , 3h ) , 3.82 ( s , 6h ) , 3.35 ( s , 3h ) , 3.22 ( m , 1h ) , 2.08 ( m , 4h ) , 1.36 ( m , 4h ) . c nmr ( cd3od ) ( ppm ) 164.90 , 164.72 , 164.35 , 160.74 , 134.07 , 124.36 , 122.58 , 108.39 , 92.09 , 79.74 , 57.05 , 56.89 , 56.62 , 48.71 , 31.61 , 31.40 . lrms ( es ) : m / z 433 [ m + h ] . hrms ( es ) : calcd for c21h29n4o6 [ m + h ] 433.2082 , found 433.2065 . h nmr ( dmso - d6 ) ( ppm ) 13.24 ( s , 1h ) , 10.89 ( d , j = 10.2 hz , 1h ) , 8.33 ( s , 1h ) , 8.09 ( q , j = 8.7 hz , 1h ) , 8.05 ( d , j = 8.4 hz , 1h ) , 7.39 ( m , 1h ) , 7.24 ( m , 1h ) , 3.82 ( m , 1h ) , 3.25 ( s , 3h ) , 3.10 ( m , 1h ) , 2.02 ( d , j = 10.3 hz , 2h ) , 1.84 ( d , j = 11.6 hz , 2h ) , 1.47 ( q , j = 12.0 hz , 2h ) , 1.22 ( q , j = 12.5 hz , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 164.3 ( q , j = 250.6 , 10.9 hz ) , 162.71 , 160.3 ( q , j = 250.8 , 14.5 hz ) , 158.03 , 133.26 , 133.18 , 132.82 , 122.12 , 120.50 , 117.39 , 117.31 , 112.59 , 112.42 , 104.96 , 104.75 , 104.52 , 79.12 , 78.84 , 78.58 , 77.71 , 55.07 , 46.89 , 30.09 , 29.76;. lrms ( es ) : m / z 379 [ m + h ] . hrms ( es ) : calcd for c18h21f2n4o3 [ m + h ] 379.1576 , found 379.1567 . h nmr ( dmso - d6 ) ( ppm ) 13.39 ( s , 1h ) , 10.77 ( s , 1h ) , 8.34 ( d , j = 8.5 hz , 1h ) , 8.30 ( s , 1h ) , 7.59 ( m , 1h ) , 7.52 ( m , 2h ) , 3.84 ( m , 1h ) , 3.24 ( s , 3h ) , 3.09 ( m , 1h ) , 2.02 ( d , j = 10.7 hz , 2h ) , 1.81 ( d , j = 10.7 hz , 2h ) , 1.49 ( q , j = 11.8 hz , 2h ) , 1.20 ( q , j = 11.8 hz , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.8 , 162.5(q , j = 250.7 , 14.4 hz ) , 160.3 , 137.16 127.75 , 126.59 , 124.12 , 122.13 , 118.94 , 110.2 ( q , j = 22.0 , 14.5 hz ) , 107.5 ( t , j = 27.0 hz ) , 77.71 , 55.06 , 46.99 , 30.29 , 29.76 . lrms ( es ) : m / z 379 [ m + h ] . hrms ( es ) : calcd for c18h21f2n4o3 [ m + h ] 379.1576 , found 379.1568 . h nmr ( cdcl3 ) ( ppm ) 10.34 ( s , 1h ) , 8.23 ( s , 1h ) , 7.60 ( d , j = 1.9 hz , 2h ) , 7.31 ( t , j = 1.8 hz , 1h ) , 7.05 ( s , 1h ) , 6.58 ( d , 1h ) , 3.77 ( m , 1h ) , 3.15 ( s , 3h ) , 2.98 ( m , 1h ) , 1.91 ( m , 4h ) , 1.18 ( m , 4h ) . c nmr ( dmso - d6 ) ( ppm ) 163.12 , 161.74 , 136.46 , 135.73 , 131.89 , 128.34 , 126.03 , 125.79 , 121.09 , 78.10 , 55.91 , 47.48 , 30.69 , 30.08 . lrms ( es ) : m / z 411 [ m + h ] . hrms ( es ) : calcd for c18h21cl2n4o3 [ m + h ] 411.0985 , found 411.0966 . h nmr ( cdcl3 ) ( ppm ) 10.65 ( s , 1h ) , 8.48 ( s , 1h ) , 8.02 ( d , j = 8.6 hz , 2h ) , 7.24 ( d , j = 8.6 hz , 2h ) , 6.84 ( d , j = 8.3 hz , 1h ) , 6.62 ( t , j = 73.2 hz , 1h ) , 4.00 ( m , 1h ) , 3.39 ( s , 3h ) , 3.22 ( m , 1h ) , 2.15 ( m , 4h ) , 1.42 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 163.28 , 163.20 , 154.0 ( t , j = 2.9 hz ) , 133.56 , 130.28 , 129.24 , 123.64 , 120.74 , 119.22 , 118.94 , 115.47 , 112.01 , 78.11 , 55.94 , 47.53 , 30.67 , 30.12 . hrms ( es ) : calcd for c19h23f2n4o4 [ m + h ] 409.1682 , found 409.1649 . h nmr ( cdcl3 ) ( ppm ) 10.44 ( s , 1h ) , 8.49 ( s , 1h ) , 7.89 ( d , j = 8.8 hz , 2h ) , 6.85 ( d , j = 8.3 hz , 1h ) , 6.62 ( d , j = 8.8 hz , 2h ) , 4.01 ( m , 1h ) , 3.39 ( m , 7h ) , 3.20 ( m , 1h ) , 2.15 ( m , 4h ) , 2.06 ( m , 4h ) , 1.41 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 164.74 , 163.48 , 150.26 , 133.26 , 128.99 , 124.26 , 120.52 , 119.42 , 111.20 , 78.20 , 55.92 , 47.66 , 47.39 , 30.73 , 30.18 , 25.46 . hrms ( es ) : calcd for c22h30n5o3 [ m + h ] 412.2343 , found 412.2338 . h nmr ( cdcl3 ) ( ppm ) 10.48 ( s , 1h ) , 8.47 ( s , 1h ) , 7.90 ( d , j = 8.8 hz , 2h ) , 6.94 ( d , j = 8.8 hz , 2h ) , 6.83 ( m , 1h ) , 4.00 ( m , 1h ) , 3.39 ( s , 3h ) , 3.37 ( t , j = 5 hz , 4h ) , 3.20 ( m , 1h ) , 2.60 ( t , j = 5 hz , 4h ) , 2.38 ( s , 3h ) , 2.14 ( m , 4h ) , 1.41 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 164.17 , 163.44 , 153.54 , 133.33 , 128.81 , 124.05 , 122.92 , 120.61 , 114.27 , 78.17 , 55.92 , 54.79 , 47.62 , 47.41 , 46.11 , 30.72 , 30.17 . lrms ( es ) : m / z 441 [ m + h ] . hrms ( es ) : calcd for c23h33n6o3 [ m + h ] 441.2609 , found 441.2597 . h nmr ( cdcl3 ) ( ppm ) 10.76 ( s , 1h ) , 8.53 ( s , 1h ) , 7.70 ( d , j = 7.5 hz , 1h ) , 7.66 ( d , j = 8.4 hz , 1h ) , 7.59 ( d , j = 0.9 hz , 1h ) , 7.46 ( t , j = 7.8 hz , 1h ) , 7.33 ( t , j = 7.5 hz , 1h ) , 6.85 ( d , j = 8.4 hz , 1h ) , 4.07 ( m , 1h ) , 3.40 ( s , 3h ) , 3.22 ( m , 1h ) , 2.16 ( m , 4h ) , 1.43 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 163.03 , 156.21 , 155.22 , 148.18 , 133.76 , 129.23 , 127.56 , 127.24 , 123.77 , 122.59 , 121.20 , 112.41 , 111.23 , 78.15 , 55.91 , 47.41 , 30.74 , 30.14 . hrms ( es ) : calcd for c20h23n4o4 [ m + h ] 383.1714 , found 383.1703 . h nmr ( dmso - d6 ) ( ppm ) 13.30 ( s , 1h ) , 11.01 ( s , 1h ) , 8.83 ( d , j = 7.0 hz , 1h ) , 8.37 ( s , 1h ) , 8.15 ( d , j = 8.4 hz , 1h ) , 7.82 ( d , j = 8.9 hz , 1h ) , 7.34 ( t , j = 7.7 hz , 1h ) , 7.09 ( t , j = 6.9 hz , 1h ) , 3.85 ( m , 1h ) , 3.26 ( s , 3h ) , 3.11 ( m , 1h ) , 2.04 ( d , j = 10.3 hz , 2h ) , 1.84 ( d , j = 10.7 hz , 2h ) , 1.49 ( m , 2h ) , 1.23 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.70 , 158.15 , 146.85 , 141.15 , 132.80 , 128.99 , 124.60 , 122.00 , 120.08 , 119.14 , 114.53 , 97.79 , 77.74 , 55.06 , 46.95 , 30.33 , 29.79 . lrms ( es ) : m / z 383 [ m + h ] . hrms ( es ) : calcd for c19h23n6o3 [ m + h ] 383.1826 , found 383.1812 . h nmr ( meod - d4 ) ( ppm ) 9.57 ( d , j = 6.9 hz , 1h ) , 8.31 ( d , j = 8.3 hz , 2h ) , 7.74 ( d , j = 9.0 hz , 1h ) , 7.57 ( td , j = 6.9 , 1.2 hz , 1h ) , 7.19 ( t , j = 6.9 , 1.2 hz , 1h ) , 3.94 ( m , 1h ) , 3.39 ( s , 3h ) , 3.27 ( m , 1h ) , 2.12 ( m , 4h ) , 1.50 ( m , 2h ) , 1.39 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.89 , 156.52 , 147.37 , 136.35 , 127.61 , 127.41 , 122.12 , 119.89 , 117.57 , 117.46 , 114.43 , 77.71 , 55.07 , 46.90 , 30.30 , 29.76 . lrms ( es ) : m / z 383 [ m + h ] . hrms ( es ) : calcd for c19h23n6o3 [ m + h ] 383.1826 , found 383.1811 . h nmr ( cdcl3 ) ( ppm ) 10.88 ( s , 1h ) , 10.24 ( s , 1h ) , 9.58 ( d , j = 6.9 hz , 1h ) , 8.51 ( s , 1h ) , 7.67 ( d , j = 9.0 hz , 1h ) , 7.41 ( t , j = 7.9 hz , 1h ) , 6.99 ( t , j = 6.9 hz , 1h ) , 6.80 ( d , j = 7.9 hz , 1h ) , 4.01 ( m , 1h ) , 3.39 ( s , 3h ) , 3.21 ( m , 1h ) , 3.02 ( s , 3h ) , 2.14 ( m , 4h ) , 1.41 ( m , 4h ) . c nmr ( dmso - d6 ) ( ppm ) 162.73 , 157.27 , 146.18 , 145.49 , 127.61 , 127.42 , 122.35 , 120.04 , 116.31 , 114.46 , 113.62 , 77.74 , 55.06 , 46.77 , 30.24 , 29.81 , 16.52 . lrms ( es ) : m / z 397 [ m + h ] . hrms ( es ) : calcd for c20h25n6o3 [ m + h ] 397.1983 , found 397.1972 . h nmr ( cdcl3 ) : ( ppm ) 10.61 ( s , 1h ) , 8.49 ( s , 1h ) , 7.84 ( m , 2h ) , 7.48 ( t , j = 7.5 hz , 2h ) , 7.38 ( t , j = 7.4 hz , 1h ) , 7.31 ( t , j = 3.6 hz , 1h ) , 6.85 ( d , j = 8.5 hz , 1h ) , 6.80 ( d , j = 3.6 hz , 1h ) , 4.07 ( m , 1h ) , 3.40 ( s , 3h ) , 3.22 ( m , 1h ) , 2.16 ( m , 4h ) , 1.42 ( m , 4h ) . c nmr ( cdcl3 ) ( ppm ) 163.03 , 156.39 , 155.72 , 146.33 , 133.74 , 129.49 , 128.94 , 128.80 , 124.72 , 122.85 , 120.91 , 117.13 , 107.30 , 78.18 , 55.88 , 47.25 , 30.78 , 30.14 . lrms ( es ) : m / z 409 [ m + h ] . hrms ( es ) : calcd for c22h25n4o4 [ m + h ] 409.1870 , found 409.1832 . h nmr ( dmso - d6 ) ( ppm ) 13.30 ( s , 1h ) , 11.20 ( s , 1h ) , 8.68 ( s , 1h ) , 8.38 ( s , 1h ) , 8.30 ( m , 2h ) , 8.14 ( d , j = 8.4 hz , 1h ) , 7.587.51 ( m , 3h ) , 3.83 ( m , 1h ) , 3.26 ( s , 3h ) , 3.11 ( m , 1h ) , 2.04 ( d , j = 10.5 hz , 2h ) , 1.84 ( d , j = 11.0 hz , 2h ) , 1.49 ( q , j = 12.5 hz , 2h ) , 1.23 ( q , j = 11.5 hz , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.64 , 157.34 , 151.99 , 150.43 , 132.90 , 130.23 , 128.50 , 128.07 , 127.92 , 126.62 , 121.89 , 120.02 , 77.74 , 55.06 , 47.00 , 30.33 , 29.76 . lrms ( es ) : m / z 410 [ m + h ] . hrms ( es ) : calcd for c21h24n5o4 [ m + h ] 410.1823 , found 410.1807 . a 100 ml three - necked round - bottomed flask equipped with a magnetic stirring bar and fitted with a dropping funnel was charged with 4-nitro-1h - pyrazole-3-carboxylic acid ( 4.0 g , 25.5 mmol ) and methanol ( 40 ml ) . the flask was cooled to 0 c , and thionyl chloride ( 2.1 ml , 28.9 mmol ) was added to the vigorously stirred solution over a period of 10 min . the mixture was stirred for an additional 12 h at room temperature , after which time tlc indicated complete consumption of the starting acid . the reaction mixture was concentrated under reduced pressure at 40 c and the residue treated with toluene and reconcentrated ( 3 20 ml ) under reduced pressure at 40 c to give methyl ester 5 as an off - white solid . h nmr ( dmso - d6 ) ( ppm ) 14.39 ( br s , 1h ) , 9.98 ( s , 1h ) , 3.90 ( s , 3h ) . c nmr ( dmso - d6 ) ( ppm ) 161.15 , 138.13 , 133.20 , 130.90 , 52.84 . lrms ( es ) : m / z 172 [ m + h ] . a 100 ml round - bottomed flask equipped with digital thermometer and stirrer was charged with 10% palladium on carbon ( 0.621 g ) under argon . in a separate vessel , a slurry of methyl ester 5 ( 4.42 g , 25.8 mmol ) in ethanol ( 45 ml ) was warmed to 35 c to effect dissolution and the solution added to the catalyst under argon . following a nitrogen hydrogen purge sequence , an atmosphere of hydrogen was introduced and the reaction mixture maintained at 30 c until the reaction completion ( 6 h ) was noted by h nmr analysis . following a purge cycle , the reaction mixture under argon was filtered and the liquors concentrated under reduced pressure to give amine 6 as a solid . h nmr ( dmso - d6 ) ( ppm ) 12.83 ( br s , 1h ) , 7.10 ( s , 1h ) , 4.83 ( br s , 2h ) , 3.78 ( s , 3h ) . c nmr ( dmso - d6 ) ( ppm ) 160.39 , 136.94 , 128.43 , 115.59 , 50.88 . lrms ( es ) : a solution of amine 6 ( 3.57 g , 25.3 mmol ) in 1,4-dioxane ( 50 ml ) under argon was treated with triethylamine ( 4.3 ml , 31 mmol ) followed by 2,6-dimethoxybenzoyl chloride ( 6.13 g , 30.6 mmol ) such that the internal temperature was maintained in the range 2025 c . the reaction mixture was stirred at 25 c until the reaction was complete ( 12 h ) by tlc analysis . the reaction mixture was filtered , the filter - cake washed with 1,4-dioxane , and the combined filtrates progressed to next stage without further isolation . to obtain analytical data for compound 7 and also to determine the yield of this reaction , a 2 g sample was taken out of the homogeneous filtrate solution ( total weight of this solution is 91 g ) . the crude product ( 192 mg ) was purified by column chromatography ( dcm / meoh ) . evaporation of the appropriate fractions yielded finally the desired compound 7 as an amorphous solid ( 161 mg ) . a 5 mg sample was used for to obtain analytical data ; the rest was redissolved for use in the next reaction . h nmr ( dmso - d6 ) 13.68 ( br s , 1h ) , 9.16 ( s , 1h ) , 8.31 ( s , 1h ) , 7.41 ( t , j = 8.4 hz , 1h ) , 6.76 ( d , j = 8.4 hz , 2h ) , 3.83 ( s , 3h ) , 3.77 ( s , 6h ) . c nmr ( dmso - d6 ) ( ppm ) 163.86 , 161.55 , 157.07 , 131.27 , 129.97 , 123.61 , 120.41 , 114.66 , 104.35 , 55.84 , 51.63 . hrms ( es ) : calcd for c14h16n3o5 [ m + h ] 306.1084 , found 306.1081 . to a solution of sodium hydroxide ( 3.32 g , 83 mmol ) in water ( 20 ml ) was charged a solution of ester 7 in one portion ( 7.33 g , 24.0 mmol ; the solution of crude 7 from the previous reaction , plus 156 mg of redissolved pure 7 ) . the reaction mixture was stirred at 25 c until completion as determined by tlc analysis . the reaction mixture was concentrated under reduced pressure at 45 c , the oily residue diluted with water and acidified to ph 1 with concentrated hydrochloric acid , such that the temperature was maintained below 30 c . the resulting precipitate was collected by filtration , washed with water , pulled dry on the filter , and subsequently washed with heptanes . the filter cake was charged to a 200 ml rotary evaporator flask and drying completed azeotropically with toluene . h nmr ( dmso - d6 ) 13.44 ( br s , 2h ) , 9.17 ( br s , 1h ) , 8.29 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.76 ( d , j = 8.4 hz , 2h ) , 3.77 ( s , 6h ) . hrms ( es ) : calcd for c13h14n3o5 [ m + h ] 292.0928 , found 292.0920 . a mixture of carboxylic acid ( 50 mg , 0.17 mmol , 1.2 equiv ) , amine ( 14 mg , 0.14 mmol , 1.0 equiv ) , hydroxybenzotriazole ( 19 mg , 0.14 mmol , 1.0 equiv ) , polymer supported - carbodiimide ( 105 mg , 0.14 mmol , 1.0 equiv ) , and acetonitrile was heated by microwave irradiation for 10 min at 100 c . the final product ( 9c ) was isolated from the reaction mixture by filtering through a short column of si - carbonate under gravity , which scavenged the excess carboxylic acid and hydroxybenzotriazole . removal of the solvent under reduced pressure yielded the required compounds as amorphous solids . h nmr ( cd3od ) ( ppm ) 8.33 ( s , 1h ) , 7.42 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 3.86 ( s , 6h ) , 3.82 ( m , 1h ) , 1.88 ( m , 4h ) , 1.68 ( d , j = 12.8 hz , 1h ) , 1.40 ( m , 4h ) , 1.27 ( m , 1h ) . c nmr ( cd3od ) ( ppm ) 165.02 , 164.71 , 159.24 , 134.17 , 132.97 , 123.89 , 121.82 , 115.64 , 105.24 , 56.50 , 49.36 , 33.77 , 26.58 , 26.19 . lrms ( es ) : m / z 373 [ m + h ] . hrms ( es ) : calcd for c19h25n4o4 [ m + h ] 373.1870 , found 373.1850 . h nmr ( cd3od ) ( ppm ) 8.32 ( s , 1h ) , 7.42 ( t , j = 8.4 hz , 1h ) , 6.76 ( d , j = 8.4 hz , 2h ) , 3.86 ( s , 6h ) , 2.79 ( m , 1h ) , 0.80 ( m , 2h ) , 0.65 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 161.32 , 161.20 , 131.10 , 122.44 , 122.30 , 115.01 , 104.28 , 55.82 , 22.13 , and 22.02 ( d , rotamers ) , 5.55 . hrms ( es ) : calcd for c16h19n4o4 [ m + h ] 331.1401 , found 331.1385 . h nmr ( dmso - d6 ) ( ppm ) 13.28 ( s , 1h ) , 9.71 ( s , 1h ) , 8.60 ( d , j = 8.1 hz , 1h ) , 8.30 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.74 ( d , j = 8.4 hz , 2h ) , 4.37 ( sex , j = 8.3 , 1h ) , 3.76 ( s , 6h ) , 2.13 ( m , 4h ) , 1.63 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 162.53 , 161.23 , 156.93 , 132.14 , 131.07 , 122.61 , 119.84 , 115.07 , 104.32 , 55.82 , 54.86 , 43.39 , 29.81 , 14.62 . lrms ( es ) : m / z 345 [ m + h ] . hrms ( es ) : calcd for c17h21n4o4 [ m + h ] 345.1557 , found 345.1548 . h nmr ( cdcl3 ) ( ppm ) 12.19 ( br s , 1h ) , 9.94 ( s , 1h ) , 8.41 ( s , 1h ) , 7.29 ( t , j = 8.5 hz , 1h ) , 6.98 ( d , j = 8.1 hz , 1h ) , 6.57 ( d , j = 8.5 hz , 2h ) , 4.05 ( m , 1h ) , 3.80 ( s , 6h ) , 2.001.94 ( m , 2h ) , 1.661.45 ( m , 10h ) . c nmr ( cdcl3 ) ( ppm ) 161.75 , 160.98 , 155.95 , 131.18 , 129.57 , 121.16 , 119.75 , 112.84 , 102.24 , 54.19 , 48.30 , 33.28 , 26.12 , 22.39 . lrms ( es ) : m / z 387 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2043 . h nmr ( dmso - d6 ) ( ppm ) 11.80 ( br s , 1h ) , 9.72 ( s , 1h ) , 8.28 ( s , 1h ) , 8.00 ( br d , j = 6.9 hz , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 3.76 ( s , 6h ) , 3.66 ( m , 1h ) , 2.21 ( br s , 1h ) , 2.15 ( br s , 1h ) , 1.631.37 ( m , 5h ) , 1.160.99 ( m , 3h ) . c nmr ( cd3od ) ( ppm ) 165.11 , 164.82 , 159.28 , 133.97 , 133.00 , 124.01 , 122.33 , 115.79 , 105.34 , 56.59 , 53.91 , 43.80 , 40.38 , 37.06 , 36.28 , 29.28 , 27.54 . lrms ( es ) : m / z 385 [ m + h ] . hrms ( es ) : calcd for c20h25n4o4 [ m + h ] 385.1870 , found 385.1857 . h nmr ( dmso - d6 ) ( ppm ) 13.30 ( s , 1h ) , 9.66 ( s , 1h ) , 9.55 ( s , 1h ) , 8.31 ( s , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.6 hz , 2h ) , 3.76 ( s , 6h ) , 3.62 ( m , 4h ) , 2.84 ( m , 4h ) . c nmr ( dmso - d6 ) ( ppm ) 161.26 , 161.18 , 156.93 , 131.41 , 131.10 , 122.90 , 119.80 , 115.01 , 104.32 , 65.98 , 55.82 , 54.31 . lrms ( es ) : m / z 376 [ m + h ] . hrms ( es ) : calcd for c17h22n5o5 [ m + h ] 376.1615 , found 376.1620 . h nmr ( dmso - d6 ) ( ppm ) 13.15 ( brs , 1h ) , 9.70 ( s , 1h ) , 8.99 ( t , j = 6.3 hz , 1h ) , 8.32 ( s , 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 7.317.29 ( m , 4h ) , 7.22 ( m , 1h ) , 6.74 ( d , j = 8.4 hz , 2h ) , 4.41 ( d , j = 6.4 hz , 2h ) , 3.75 ( s , 6h ) . c nmr ( dmso - d6 ) ( ppm ) 163.46 , 161.29 , 156.93 , 139.45 , 131.95 , 131.10 , 128.21 , 127.26 , 126.71 , 122.58 , 120.18 , 115.01 , 104.32 , 55.82 , 41.66 . lrms ( es ) : m / z 381 [ m + h ] . hrms ( es ) : calcd for c20h21n4o4 [ m + h ] 381.1557 , found 381.1543 . h nmr ( dmso - d6 ) ( ppm ) 13.33 ( br s , 1h ) , 9.66 ( s , 1h ) , 8.96 ( t , j = 5.9 , 1h ) , 8.50 ( m , 1h ) , 8.34 ( s , 1h ) , 7.74 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 7.30 ( d , j = 7.8 hz , 1h ) , 7.25 ( m , 1h ) , 6.73 ( d , j = 8.4 hz , 2h ) , 4.53 ( d , j = 5.6 hz , 2h ) , 3.75 ( s , 6h ) . c nmr ( dmso - d6 ) ( ppm ) 163.65 , 161.32 , 158.14 , 156.93 , 148.75 , 136.68 , 131.89 , 131.10 , 122.61 , 122.06 , 120.84 , 120.18 , 114.98 , 104.32 , 55.82 , 43.54 . lrms ( es ) : h nmr ( dmso - d6 ) ( ppm ) 13.30 ( br s , 1h ) , 9.68 ( s , 1h ) , 8.72 ( t , j = 5.0 hz , 1h ) , 8.39 ( d , j = 5.0 hz , 1h ) , 8.34 ( s , 1h ) , 7.60 ( d , j = 7.6 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 7.23 ( dd , j = 7.6 , 5.0 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 4.54 ( d , j = 5.0 hz , 2h ) , 3.76 ( s , 6h ) , 2.29 ( s , 3h ) . c nmr ( dmso - d6 ) ( ppm ) 163.28 , 161.29 , 156.96 , 153.98 , 145.81 , 137.64 , 132.10 , 131.13 , 130.64 , 122.47 , 122.24 , 120.04 , 114.98 , 104.32 , 55.84 , 40.96 , 17 , 13 . lrms ( es ) : m / z 396 [ m + h ] . hrms ( es ) : calcd for c20h22n5o4 [ m + h ] 396.1666 , found 396.1656 . h nmr ( cd3od ) ( ppm ) 8.35 ( s. 1h ) , 7.37 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 3.82 ( s , 6h ) , 3.17 ( d , j = 7.0 hz , 2h ) , 1.771.64 ( m , 5h ) , 1.55 ( m , 1h ) , 1.281.13 ( m , 3h ) , 1.000.89 ( m , 2h ) . c nmr ( cd3od ) ( ppm ) 165.64 , 165.00 , 159.22 , 134.15 , 132.99 , 123.89 , 122.10 , 115.69 , 105.31 , 56.58 , 46.09 , 39.42 , 32.00 , 27.57 , 27.03 . lrms ( es ) : m / z 386 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2008 . yield : 48 mg ( solid ) , 76% . h nmr ( dmso - d6 ) ( ppm ) 13.51 ( s , 1h ) , 10.31 ( s , 1h ) , 9.65 ( s , 1h ) , 8.40 ( s , 1h ) , 7.78 ( d , j = 7.7 hz , 2h ) , 7.41 ( t , j = 8.5 hz , 1h ) , 7.31 ( t , j = 7.8 hz , 2h ) , 7.09 ( t , j = 7.3 hz , 1h ) , 6.76 ( d , j = 8.5 hz , 2h ) , 3.77 ( s , 6h ) . c nmr ( dmso - d6 ) ( ppm ) 162.29 , 161.40 , 157.02 , 138.10 , 132.32 , 131.19 , 128.49 , 123.81 , 123.02 , 120.69 , 120.37 , 114.98 , 104.40 , 55.88 . lrms ( es ) : m / z 367 [ m + h ] . hrms ( es ) : calcd for c19h19n4o4 [ m + h ] 367.1401 , found 367.1402 . h nmr ( dmso - d6 ) ( ppm ) 13.63 ( s , 1h ) , 10.73 ( s , 1h ) , 9.55 ( s , 1h ) , 8.43 ( d , j = 6.4 hz , 3h ) , 7.83 ( d , j = 5.5 hz , 2h ) , 7.42 ( t , j = 8.3 hz , 1h ) , 6.77 ( d , j = 8.4 hz , 2h ) , 3.78 ( s , 6h ) . hrms ( es ) : calcd for c18h18n5o4 [ m + h ] 368.1353 , found 368.1347 . h nmr ( dmso - d6 ) ( ppm ) 13.61 ( s , 1h ) , 9.63 ( s , 1h ) , 9.49 ( s , 1h ) , 8.42 ( s , 1h ) , 8.38 ( br d , j = 4.6 hz , 1h ) , 8.09 ( d , j = 8.4 hz , 1h ) , 7.83 ( m , 1h ) , 7.42 ( t , j = 8.4 hz , 1h ) , 7.18 ( m , 1h ) , 6.77 ( d , j = 8.5 hz , 2h ) , 3.78 ( s , 6h ) . c nmr ( dmso - d6 ) ( ppm ) 161.87 , 161.55 , 157.01 , 150.45 , 148.29 , 138.41 , 131.62 , 131.30 , 123.04 , 120.90 , 120.15 , 114.66 , 113.86 , 104.35 , 55.84 . lrms ( es ) : m / z 368 [ m + h ] . hrms ( es ) : calcd for c18h18n5o4 [ m + h ] 368.1353 , found 368.1335 . h nmr ( dmso - d6 ) ( ppm ) 13.21 ( br s , 1h ) , 9.75 ( s , 1h ) , 8.28 ( s , 1h ) , 8.09 ( d , j = 8.4 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 4.53 ( br d , j = 3.8 hz , 1h ) , 3.76 ( s , 6h ) , 3.66 ( m , 1h ) , 3.403.33 ( m , 1h ) , 1.82 ( m , 2h ) , 1.73 ( m , 2h ) , 1.44 ( m , 2h ) , 1.19 ( m , 2h ) . c nmr ( dmso - d6 ) 162.67 , 161.25 , 156.91 , 131.07 , 122.56 , 115.10 , 104.32 , 68.11 , 55.82 , 47.00 , 34.19 , 30.00 . lrms ( es ) : m / z 389 [ m + h ] . hrms ( es ) : calcd for c19h25n4o5 [ m + h ] 389.1819 , found 389.1800 . h nmr ( cd3od ) ( ppm ) 8.36 ( s , 0.5h ) , 8.33 ( s , 0.5h ) , 7.39 ( t , j = 8.5 hz , 1h ) , 6.73 ( d , j = 8.5 hz , 2h ) , 4.05 ( m , 0.5h ) , 3.84 ( s , 6h ) , 3.76 ( m , 0.5h ) , 1.961.32 ( m , 8h ) , 1.06 ( m , 1h ) , 0.98 ( d , j = 6.5 hz , 1.5 h ) , 0.92 ( d , j = 6.6 hz , 1.5h ) . c nmr ( cd3od ) ( ppm ) 165.06 , 165.02 , 164.76 , 164.64 , 159.25 , 134.07 , 134.01 , 132.97 , 123.99 , 123.91 , 122.30 , 122.12 , 115.70 , 115.66 , 105.39 , 105.29 , 56.54 , 49.52 , 46.86 , 35.15 , 33.67 , 33.12 , 31.53 , 31.01 , 30.09 , 22.63 , 21.25 . lrms ( es ) : m / z 387 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2007 . h nmr ( cd3od ) ( ppm ) 8.35 ( s , 1h ) , 7.37 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 4.083.99 ( m , 3h ) , 3.82 ( s , 6h ) , 2.90 ( m , 2h ) , 1.89 ( m , 2h ) , 1.51 ( m , 2h ) , 1.46 ( s , 9h ) . c nmr ( cd3od ) ( ppm ) 165.00 , 164.89 , 159.22 , 156.39 , 134.00 , 132.99 , 124.03 , 122.16 , 115.72 , 105.35 , 81.17 , 56.61 , 47.67 , 44.39 , and 43.61 ( br d , rotamers ) , 32.69 , 28.79 . lrms ( es ) : m / z 474 [ m + h ] . hrms ( es ) : calcd for c23h32n5o6 [ m + h ] 474.2347 , found 474.2324 . h nmr ( cd3od ) ( ppm ) 8.32 ( s , 1h ) , 7.36 ( t , j = 8.4 hz , 1h ) , 7.327.22 ( m , 5h ) , 6.68 ( d , j = 8.4 hz , 2h ) , 4.52 ( m , 1h ) , 3.78 ( s , 6h ) , 3.58 ( d , j = 2.5 hz , 2h ) , 2.76 ( m , 2h ) , 2.58 ( dd , j = 10.0 , 4.3 , 1h ) , 2.43 ( q , j = 8.1 hz , 1h ) , 2.27 ( m , 1h ) , 1.75 ( m , 1h ) . c nmr ( cd3od ) ( ppm ) 165.06 , 165.00 , 159.22 , 139.26 , 133.92 , 132.99 , 130.33 , 129.50 , 128.49 , 124.01 , 122.16 , 115.67 , 105.32 , 61.26 , 61.17 , 56.58 , 53.83 , 49.19 , 32.54 . lrms ( es ) : m / z 450 [ m + h ] . hrms ( es ) : calcd for c24h28n5o4 [ m + h ] 450.2136 , found 450.2113 . h nmr ( cd3od ) ( ppm ) 8.35 ( s. 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 6.72 ( d , 8.4 j = 8.4 hz , 2h ) , 3.83 ( s , 6h ) , 3.53 ( s , 4h ) , 3.37 ( s , 3h ) . c nmr ( cd3od ) ( ppm ) 165.76 , 165.05 , 159.26 , 134.10 , 132.99 , 115.71 , 105.32 , 72.07 , 59.03 , 56.56 , 39.52 . lrms ( es ) : m / z 349 [ m + h ] . hrms ( es ) : calcd for c16h21n4o5 [ m + h ] 349.1506 , found 349.1504 . h nmr ( dmso - d6 ) ( ppm ) 13.23 ( br s , 1h ) , 9.74 ( s , 1h ) , 8.38 ( br s , 1h ) , 8.29 ( s , 1h ) , 7.39 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.5 hz , 2h ) , 3.76 ( s , 6h ) , 3.19 ( m , 2h ) , 1.49 ( quint , j = 7.2 hz , 2h ) , 1.311.22 ( m , 4h ) , 0.85 ( t , j = 7.0 hz , 3h ) . c nmr ( dmso - d6 ) ( ppm ) 163.39 , 161.20 , 156.93 , 132.25 , 131.07 , 122.35 , 119.79 , 115.05 , 104.32 , 55.82 , 37.95 , 28.77 , 28.57 , 21.78 , 13.87 . lrms ( es ) : m / z 361 [ m + h ] . hrms ( es ) : calcd for c18h25n4o4 [ m + h ] 361.1870 , found 361.1868 . h nmr ( cd3od ) ( ppm ) 8.35 ( s , 1h ) , 7.38 ( t , j = 8.5 hz , 1h ) , 6.71 ( d , j = 8.5 hz , 2h ) , 3.83 ( s , 6h ) , 3.58 ( sept , j = 6.1 hz , 1h ) , 3.53 ( t , j = 6.1 hz , 2h ) , 3.45 ( t , j = 6.6 hz , 2h ) , 1.83 ( quin , j = 6.4 hz , 2h ) , 1.15 ( d , j = 6.1 hz , 6h ) . c nmr ( cd3od ) ( ppm ) 165.58 , 165.00 , 159.22 , 134.17 , 132.99 , 123.82 , 121.99 , 115.69 , 105.32 , 73.08 , 67.50 , 56.61 , 38.06 , 30.72 , 22.49 . lrms ( es ) : m / z 391 [ m + h ] . hrms ( es ) : calcd for c19h27n4o5 [ m + h ] 391.1976 , found 391.1961 . h nmr ( cd3od ) ( ppm ) 8.23 ( s , 1h ) , 7.26 ( t , j = 8.4 hz , 1h ) , 6.59 ( d , j = 8.4 hz , 2h ) , 3.71 ( s , 6h ) , 3.26 ( t , j = 7.5 hz , 2h ) , 2.26 ( t , j = 7.5 hz , 2h ) , 2.11 ( s , 6h ) , 1.65 ( q , j = 7.5 hz , 2h ) . c nmr ( cd3od ) ( ppm ) 165.70 , 164.94 , 159.22 , 134.15 , 132.99 , 123.92 , 122.05 , 115.75 , 105.35 , 58.25 , 56.61 , 45.52 , 38.23 , 28.32 . lrms ( es ) : m / z 376 [ m + h ] . hrms ( es ) : calcd for c18h26n5o4 [ m + h ] 376.1979 , found 376.1965 . h nmr ( cd3od ) ( ppm ) 8.34 ( s , 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 3.83 ( s , 6h ) , 3.51 ( t , j = 6.9 hz , 2h ) , 2.69 ( t , j = 6.9 hz , 2h ) , 2.59 ( m , 4h ) , 1.80 ( m , 4h ) . c nmr ( cd3od ) ( ppm ) 165.69 , 164.97 , 159.22 , 134.09 , 132.99 , 123.89 , 122.01 , 115.72 , 105.32 , 56.58 , 56.32 , 55.08 , 38.64 , 24.31 . hrms ( es ) : calcd for c19h26n5o4 [ m + h ] 388.1979 , found 388.1965 . h nmr ( cd3od ) ( ppm ) 8.34 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.73 ( d , j = 8.4 hz , 2h ) , 3.84 ( s , 6h ) , 3.70 ( t , j = 4.5 hz , 4h ) , 3.5 ( t , j = 6.6 , 2h ) , 2.57 ( t , j = 6.6 hz , 2h ) , 2.52 ( br t , j = 4.5 hz , 4 hz ) . c nmr ( cd3od ) ( ppm ) 165.67 , 165.00 , 159.23 , 134.06 , 132.96 , 123.86 , 122.02 , 115.72 , 105.32 , 67.80 , 58.60 , 56.55 , 54.67 , 36.50 . lrms ( es ) : m / z 404 [ m + h ] . hrms ( es ) : calcd for c19h26n5o5 [ m + h ] 404.1928 , found 404.1929 .

glycogen synthase kinase 3 ( gsk3 ) is a genetically validated drug target for human african trypanosomiasis ( hat ) , also called african sleeping sickness . we report the synthesis and biological evaluation of aminopyrazole derivatives as trypanosoma brucei gsk3 short inhibitors . low nanomolar inhibitors , which had high selectivity over the off - target human cdk2 and good selectivity over human gsk3 enzyme , have been prepared . these potent kinase inhibitors demonstrated low micromolar levels of inhibition of the trypanosoma brucei brucei parasite grown in culture .

Introduction Results Discussion Conclusion Experimental Section

human african trypanosomiasis ( hat ) or african sleeping sickness is a serious life threatening disease . hat is caused by infection with trypanosoma brucei , a vector - borne parasite , which is transmitted by the bite of tsetse flies . in the t. brucei genome there are two kinases that are highly homologous to human glycogen synthase kinase 3 ( hsgsk3 ) : tbgsk3 short and tbgsk3 long . the ability to selectively inhibit tbgsk3 over the off - target hsgsk3 is highly desirable because mouse knockout studies revealed that the disruption of the murine gsk3 gene causes embryonic lethality ; consequently , nonselective inhibitors are not applicable for use in infants and women of child bearing age . off - target inhibition of these human kinases will therefore result in cell cycle arrest and reduction of cellular proliferation and as such potentially lead to severe side effects . co - crystal structures of this series with hsgsk3 are not available to date ; however , complex structures with the closely related hscdk2 have been determined . the r substituents occupy the hydrophobic pocket ii , formed by the backbone of the linker region , leu83 , phe82 , and side chains of ile10 , asp86 , and leu134 . herein , we describe the design , synthesis , and biological evaluation of aminopyrazole inhibitors which bind to tbgsk3 short . for inhibition of hscdk2 and hsgsk3 enzymes were chosen as a starting point for the investigation of tbgsk3 short inhibitors . the synthesis of r and r substituted aminopyrazole derivatives started from 4-nitro - pyrazole-3-carboxylic acid 1 and is described by two different routes ( scheme 1 ) based on previous work from wyatt et al . in route b , after esterification of the carboxyl group of 1 , the nitro group of intermediate 5 was reduced to afford amine 6 . the methoxyphenyl moieties in 4j , 4 m , and 4n , which had tbgsk3 ic50 values of 4 , 2 , and 3 nm , respectively , were the most favorable substituents . this resulted in a higher energy , out of plane conformation of the amide group of 4f when binding into this pocket ( figure 4 ) , while a low energy conformation was found when binding into t. brucei and human gsk3 ( not shown ) . , which found that the aryl groups which are located in the same position need to twist in order to provide potent cdk2 activity docking results suggested that the phenyl ring of compound 4f needs to be significantly twisted out of plane by approximately 60 compared to the amide in order to fit into the gatekeeper region .. all r substituted compounds were tested for their ability to inhibit the proliferation of bloodstream form ( bsf ) t. b. brucei in culture . selectivity over the mrc5 cells was achieved with compounds 4 m ( 60-fold ) , 4s ( > 19-fold ) , 4x ( 12-fold ) , and 4n ( > 9-fold ) , however , the majority of compounds showed a poor selectivity over mrc5 cells . correlation between the inhibition of recombinant tbgsk3 and bloodstream form t. b. brucei proliferation by r substituted aminopyrazole derivatives ( 4a z ) . r substituted aminopyrazole analogues ( 9a9x ) were made according to the synthetic route shown in scheme 1b to explore the structural requirements for improvement of antiparasitic activity and selectivity over the closely related human kinases . further , it was noted that the replacement of the cyclohexane of 9c with a phenyl ring or 4-pyridine , to give 9k or 9l , gave a 6-fold decrease in potency against tbgsk3 . concentration required to inhibit the growth of t. b. brucei in culture by 50% over 72 h. concentration required to inhibit the growth of mrc5 cells in culture by 50% over 72 h. the predicted binding mode of 9 g in tbgsk3 offers an explanation for the observed selectivity ( figure 6 ) . the gold sphere represents the center of the phenyl ring .. the r substituted compounds were tested against bsf t. b. brucei and mrc5 cells . correlation between the inhibition of recombinant tbgsk3 and bloodstream from t. b. brucei proliferation using r substituted aminopyrazole derivatives ( 9a x ) . pk inhibitors frequently inhibit multiple kinases , often leading to off - target toxic effects . to assess the selectivity of the aminopyrazole inhibitors , remaining activity at 10 m concentration was measured for compounds 4f , 4 m , and 4y against a panel of 80 human pks and for compound 9 g against 124 human pks . this kinase has been shown using genetic manipulation studies to be essential for the survival of the t. b. brucei parasite . therefore , more than 50 aminopyrazole derivatives were synthesized and screened against tbgsk3 , hsgsk3 , hscdk2 , and proliferating t. b. brucei and human cells in culture . the aminopyrazole derivatives make three h - bond interactions with the kinase hinge region , driving much of the potency of the compounds against the three kinases studied . docking studies provided a number of important insights into the binding modes and the selectivity profile of aminopyrazole derivatives . however , this region of the pocket in hscdk2 is narrow ( in a plane perpendicular to the hinge backbone and the pyrazole core ) , only allowing small ortho - substituents ( such as in compound 9 g ) on the phenyl group . on the basis of the biological results of compound 9 g and structural modeling studies , we have shown that selectivity over hsgsk3 can be achieved by exploiting the phe103tyr , leu36gln , and ala26ile active site differences in the hydrophobic pocket ii of tbgsk3 enzyme ( figure 6 ) . taken together , the region between the gate keeper residue and the catalytic aspartate of the dfg loop , together with the hydrophobic pocket ii , are the key areas to exploit to achieve high selectivity over hscdk2 . the tbgsk3 short enzyme ic50 values correlated well with the t. b. brucei antiproliferative ec50 activities of the described substituted aminopyrazole inhibitors ( figures 5 and 7 ) , indicating that the compounds act on target . sar investigation and optimization successfully provided 18 low nanomolar ( ic50 < 10 nm ) inhibitors of tbgsk3 with high selectivity ( > 10000-fold ) over hscdk2 . with compound 9 g , we have shown that good ( 330-fold ) selectivity over hsgsk3 can be achieved by targeting the hydrophobic pocket ii . first , non - atp competitive approaches to inhibition of tbgsk3 , through irreversible hinge binders or allosteric inhibitors , could be pursued . second , a polypharmacology approach through the inhibition of a number of essential t. brucei kinases in addition to tbgsk3 could be investigated , although obtaining selectivity over human kinases would be more problematical . the statistical significance of the compound potency ( ic50 ) was based on the performance of standard molecules which have been tested to a high replication . all biochemical assays are run below the km for the atp for each enzyme , allowing comparison of inhibition across the panel . measurement of inhibition of the proliferation of mrc5 ( human lung fibroblast ) cells and t. b. brucei bloodstream stage cells was performed using a modification of the cell viability assay previously described . the organic layer was washed ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to give a yellow oil , which was purified by column chromatography , eluting 0100% etoac in petroleum ether to give 2 . h nmr ( cd3od ) ( ppm ) 7.23 ( s , 1h ) , 3.84 ( m , 1h ) , 3.37 ( s , 3h ) , 3.23 ( m , 1h ) , 2.07 ( dd , j = 45.8 , 11.2 hz , 4h ) , 1.38 ( m , 4h ) . a mixture of benzoic acid ( 0.051 g , 0.42 mmol ) , 3 ( 0.1 g , 0.42 mmol ) , edc ( 0.096 g , 0.5 mmol ) , and hobt ( 0.068 g , 0.5 mmol ) in dmf ( 10 ml ) was stirred at ambient temperature for 16 h. the mixture was reduced in vacuo and partitioned between saturated aqueous sodium bicarbonate and etoac . the organic layer was washed ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to give a creamy solid 4f , which was purified by column chromatography . h nmr ( dmso - d6 ) ( ppm ) 13.13 ( s , 1h ) , 9.76 ( s , 1h ) , 8.15 ( s , 1h ) , 8.00 ( d , j = 8.3 hz , 1h ) , 7.317.23 ( m , 2h ) , 7.02 ( d , j = 8.0 hz , 1h ) , 6.93 ( t , j = 7.4 hz , 1h ) , 3.82 ( s , 3h ) , 3.793.70 ( m , 1h ) , 3.24 ( s , 3h ) , 3.07 ( m , 1h ) , 2.01 ( d , j = 11.4 hz , 2h ) , 1.79 ( d , j = 11.4 hz , 2h ) , 1.45 ( m , 2h ) , 1.21 ( m , 2h ) . h nmr ( dmso - d6 ) ( ppm ) 13.40 ( s , 1h ) , 10.22 ( s , 1h ) , 8.32 ( s , 1h ) , 8.29 ( d , j = 8.6 hz , 1h ) , 7.907.88 ( m , 1h ) , 7.847.81 ( m , 1h ) , 7.787.75 ( m , 2h ) , 3.72 ( m , 1h ) , 3.23 ( s , 3h ) , 3.06 ( m , 1h ) , 2.00 ( br d , j = 12.5 hz , 2h ) , 1.76 ( br d , j = 12.5 hz , 2h ) , 1.45 ( m , 2h ) , 1.14 ( m , 2h ) . c nmr ( dmso - d6 ) ( ppm ) 164.3 ( q , j = 250.6 , 10.9 hz ) , 162.71 , 160.3 ( q , j = 250.8 , 14.5 hz ) , 158.03 , 133.26 , 133.18 , 132.82 , 122.12 , 120.50 , 117.39 , 117.31 , 112.59 , 112.42 , 104.96 , 104.75 , 104.52 , 79.12 , 78.84 , 78.58 , 77.71 , 55.07 , 46.89 , 30.09 , 29.76;. h nmr ( cdcl3 ) ( ppm ) 10.34 ( s , 1h ) , 8.23 ( s , 1h ) , 7.60 ( d , j = 1.9 hz , 2h ) , 7.31 ( t , j = 1.8 hz , 1h ) , 7.05 ( s , 1h ) , 6.58 ( d , 1h ) , 3.77 ( m , 1h ) , 3.15 ( s , 3h ) , 2.98 ( m , 1h ) , 1.91 ( m , 4h ) , 1.18 ( m , 4h ) . h nmr ( meod - d4 ) ( ppm ) 9.57 ( d , j = 6.9 hz , 1h ) , 8.31 ( d , j = 8.3 hz , 2h ) , 7.74 ( d , j = 9.0 hz , 1h ) , 7.57 ( td , j = 6.9 , 1.2 hz , 1h ) , 7.19 ( t , j = 6.9 , 1.2 hz , 1h ) , 3.94 ( m , 1h ) , 3.39 ( s , 3h ) , 3.27 ( m , 1h ) , 2.12 ( m , 4h ) , 1.50 ( m , 2h ) , 1.39 ( m , 2h ) . h nmr ( cd3od ) ( ppm ) 8.33 ( s , 1h ) , 7.42 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 3.86 ( s , 6h ) , 3.82 ( m , 1h ) , 1.88 ( m , 4h ) , 1.68 ( d , j = 12.8 hz , 1h ) , 1.40 ( m , 4h ) , 1.27 ( m , 1h ) . h nmr ( dmso - d6 ) ( ppm ) 13.28 ( s , 1h ) , 9.71 ( s , 1h ) , 8.60 ( d , j = 8.1 hz , 1h ) , 8.30 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.74 ( d , j = 8.4 hz , 2h ) , 4.37 ( sex , j = 8.3 , 1h ) , 3.76 ( s , 6h ) , 2.13 ( m , 4h ) , 1.63 ( m , 2h ) . h nmr ( dmso - d6 ) ( ppm ) 13.33 ( br s , 1h ) , 9.66 ( s , 1h ) , 8.96 ( t , j = 5.9 , 1h ) , 8.50 ( m , 1h ) , 8.34 ( s , 1h ) , 7.74 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 7.30 ( d , j = 7.8 hz , 1h ) , 7.25 ( m , 1h ) , 6.73 ( d , j = 8.4 hz , 2h ) , 4.53 ( d , j = 5.6 hz , 2h ) , 3.75 ( s , 6h ) . lrms ( es ) : h nmr ( dmso - d6 ) ( ppm ) 13.30 ( br s , 1h ) , 9.68 ( s , 1h ) , 8.72 ( t , j = 5.0 hz , 1h ) , 8.39 ( d , j = 5.0 hz , 1h ) , 8.34 ( s , 1h ) , 7.60 ( d , j = 7.6 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 7.23 ( dd , j = 7.6 , 5.0 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 4.54 ( d , j = 5.0 hz , 2h ) , 3.76 ( s , 6h ) , 2.29 ( s , 3h ) . h nmr ( dmso - d6 ) ( ppm ) 13.63 ( s , 1h ) , 10.73 ( s , 1h ) , 9.55 ( s , 1h ) , 8.43 ( d , j = 6.4 hz , 3h ) , 7.83 ( d , j = 5.5 hz , 2h ) , 7.42 ( t , j = 8.3 hz , 1h ) , 6.77 ( d , j = 8.4 hz , 2h ) , 3.78 ( s , 6h ) . h nmr ( cd3od ) ( ppm ) 8.35 ( s , 1h ) , 7.38 ( t , j = 8.5 hz , 1h ) , 6.71 ( d , j = 8.5 hz , 2h ) , 3.83 ( s , 6h ) , 3.58 ( sept , j = 6.1 hz , 1h ) , 3.53 ( t , j = 6.1 hz , 2h ) , 3.45 ( t , j = 6.6 hz , 2h ) , 1.83 ( quin , j = 6.4 hz , 2h ) , 1.15 ( d , j = 6.1 hz , 6h ) . h nmr ( cd3od ) ( ppm ) 8.23 ( s , 1h ) , 7.26 ( t , j = 8.4 hz , 1h ) , 6.59 ( d , j = 8.4 hz , 2h ) , 3.71 ( s , 6h ) , 3.26 ( t , j = 7.5 hz , 2h ) , 2.26 ( t , j = 7.5 hz , 2h ) , 2.11 ( s , 6h ) , 1.65 ( q , j = 7.5 hz , 2h ) .

colorectal cancer ( crc ) is the third most frequent cancer worldwide and the fourth most common cause of cancer deaths . growing evidence supports that 15%20% of crc arise through the serrated pathway , which is characterized by widespread gene inactivation via hypermethylation of promoter regions ( the cpg island methylator phenotype ) , braf mutations , and frequent microsatellite instability . colorectal serrated polyps are histologically typical for a serrated or sawtooth - like appearance of the crypt epithelium and have been recognized as precursor lesions for crc in western countries over the last decade , especially for interval or missed crc . currently , serrated polyps are classified by the world health organization ( who ) into three distinct subtypes : hyperplastic polyp ( hp ) , sessile serrated adenoma / polyp ( ssa / p ) with or without cytological dysplasia , and traditional serrated adenoma ( tsa ) , and each has specific features . autopsy studies demonstrated the variable prevalence of serrated lesions but collectively indicate that 25%50% of caucasians have one or more serrated lesions . recent studies in western countries reporting on the prevalence of colorectal serrated polyps in patients with average risk of crc showed that hp might account for 70%95% of all serrated polyps , ssa / p for 5%25% , and tsa for < 2% . the overall prevalence of serrated polyps increases only slightly with age during adulthood , and these lesions are most common in the sigmoid colon and rectum , but the distribution varies by histological subtype . hp , typically 5 mm in size , appears more in the distal colon and rectum , as well as tsa . as the second frequent subset in serrated polyps , ssa / p occurs predominantly in the proximal colon and in older women . as hp and ssa / p have the similarity in color and vessel distribution , some important features can help identify ssa / p including flat morphology , red - colored surface , mucus cap , lateral growth of crypts at the base , dilation in the lower third of crypts , and hyperserration of the crypt bases , sometimes with branching . tsa is a relatively rare lesion and the only member of the serrated class that is uniformly dysplastic . in addition , studies also suggested that large serrated lesions , proximal serrated polyps , or proximal hyperplasic polyps were associated with synchronous advanced neoplasia ( an ) . however , few asian studies have investigated the comprehensive clinical features of colorectal serrated polyps . to address this issue and also provide more evidence for the management of colorectal serrated polyps in clinical practice , we evaluated the detection rate and the features of serrated polyps in a chinese symptomatic patient population . all consecutive symptomatic patients who underwent routine colonoscopy at the digestive endoscopy center of tianjin medical university general hospital , between january 2010 and december 2014 , were investigated . the indications for colonoscopy were due to various symptoms , such as abdominal pain , diarrhea , and change of bowel habits . the pathological sections of suspected colorectal serrated polyps were reevaluated and reclassified by one experienced pathologist ( wen - jing song ) using the who criteria . the demographic and clinicopathological data of all colorectal serrated polyps with a final diagnosis and classification were further collected , including patient age and gender , lesion size , polyp number , and location . the size of colorectal serrated polyps was determined on the basis of endoscopic descriptions , and large serrated polyps ( lsps ) were defined as a diameter 10 mm . the proximal polyps refer to the polyps which locate in the cecum , ascending colon , and transverse colon including the splenic flexure , and distal polyps define as polyps in the descending colon , sigmoid colon , and rectum . the an included adenocarcinomas , adenomas with high - grade dysplasia ( hgd ) , adenomas with any villous histology , or adenomas 10 mm . exclusion criteria were as follows : ( 1 ) < 20 years old , ( 2 ) patients with any kinds of polyposis syndromes , ( 3 ) patients with a history of crc or inflammatory bowel disease , ( 4 ) patients with a history of colonic resection or polypectomy , ( 5 ) patients with any emergent and therapeutic colonoscopy , and ( 6 ) patients with inadequate bowel preparation and had incomplete colonoscopy . informed consents for colonoscopy were granted from all the patients before the procedure , and ethical approval was obtained from the ethics committee of tianjin medical university general hospital . colonoscopy ( olympus cf - q260 , olympus optical co. , tokyo , japan ) was used for all procedures by the experienced endoscopists . patients were orally lavaged , and watery diarrhea excretion before the procedure indicated adequate intestinal preparation . all collected specimens were fixed in 10% formalin and then fixed for a minimum of 4 h. hematoxylin and eosin staining was used for histopathological evaluation and classification . all the serrated lesions were divided into three subtypes : hp , ssa / p with or without dysplasia , and tsa . the histopathological criteria used for diagnosis were those described by rex et al . and east et al . the defining histologic feature of hp is a sawtooth pattern of epithelial infolding in the upper half of the crypt with a lack of cytologic dysplasia . anchor-shaped crypts above the muscularis mucosae , serration in the lower third of the crypts with and without branching of the crypts , inverted crypts below the muscularis mucosae , and columnar dilation in the lower third . distinguishing the tsa from the other polyps is the unique characteristics described as a serrated architecture and at least a focal area with tall columnar cells having elongated nuclei and an abundant eosinophilic cytoplasm . the budding of proliferative crypts situated perpendicular to the long axis of filiform or villous structures ( ectopic crypt formation ) also helps identify tsa . all statistical analyses were performed using spss 19.0 ( spss inc , chicago , il , usa ) for windows . risks of colorectal serrated polyps were compared by chi - square test or fisher 's exact test . means and standard deviation were calculated for continuous variables . logistic regression analysis was used to evaluate the odds ratio ( or ) and 95% confidence interval ( ci ) for colorectal synchronous an ( san ) . age , gender , size , location , and dysplasia were selected as possible confounding factors . all consecutive symptomatic patients who underwent routine colonoscopy at the digestive endoscopy center of tianjin medical university general hospital , between january 2010 and december 2014 , were investigated . the indications for colonoscopy were due to various symptoms , such as abdominal pain , diarrhea , and change of bowel habits . the pathological sections of suspected colorectal serrated polyps were reevaluated and reclassified by one experienced pathologist ( wen - jing song ) using the who criteria . the demographic and clinicopathological data of all colorectal serrated polyps with a final diagnosis and classification were further collected , including patient age and gender , lesion size , polyp number , and location . the size of colorectal serrated polyps was determined on the basis of endoscopic descriptions , and large serrated polyps ( lsps ) were defined as a diameter 10 mm . the proximal polyps refer to the polyps which locate in the cecum , ascending colon , and transverse colon including the splenic flexure , and distal polyps define as polyps in the descending colon , sigmoid colon , and rectum . the an included adenocarcinomas , adenomas with high - grade dysplasia ( hgd ) , adenomas with any villous histology , or adenomas 10 mm . exclusion criteria were as follows : ( 1 ) < 20 years old , ( 2 ) patients with any kinds of polyposis syndromes , ( 3 ) patients with a history of crc or inflammatory bowel disease , ( 4 ) patients with a history of colonic resection or polypectomy , ( 5 ) patients with any emergent and therapeutic colonoscopy , and ( 6 ) patients with inadequate bowel preparation and had incomplete colonoscopy . informed consents for colonoscopy were granted from all the patients before the procedure , and ethical approval was obtained from the ethics committee of tianjin medical university general hospital . colonoscopy ( olympus cf - q260 , olympus optical co. , tokyo , japan ) was used for all procedures by the experienced endoscopists . patients were orally lavaged , and watery diarrhea excretion before the procedure indicated adequate intestinal preparation . all collected specimens were fixed in 10% formalin and then fixed for a minimum of 4 h. hematoxylin and eosin staining was used for histopathological evaluation and classification . all the serrated lesions were divided into three subtypes : hp , ssa / p with or without dysplasia , and tsa . the histopathological criteria used for diagnosis were those described by rex et al . and east et al . the defining histologic feature of hp is a sawtooth pattern of epithelial infolding in the upper half of the crypt with a lack of cytologic dysplasia . anchor-shaped crypts above the muscularis mucosae , serration in the lower third of the crypts with and without branching of the crypts , inverted crypts below the muscularis mucosae , and columnar dilation in the lower third . distinguishing the tsa from the other polyps is the unique characteristics described as a serrated architecture and at least a focal area with tall columnar cells having elongated nuclei and an abundant eosinophilic cytoplasm . the budding of proliferative crypts situated perpendicular to the long axis of filiform or villous structures ( ectopic crypt formation ) also helps identify tsa . all statistical analyses were performed using spss 19.0 ( spss inc , chicago , il , usa ) for windows . risks of colorectal serrated polyps were compared by chi - square test or fisher 's exact test . means and standard deviation were calculated for continuous variables . logistic regression analysis was used to evaluate the odds ratio ( or ) and 95% confidence interval ( ci ) for colorectal synchronous an ( san ) . age , gender , size , location , and dysplasia were selected as possible confounding factors . a total of 28,981 symptomatic patients undergoing colonoscopy from 2010 to 2014 were collected in this study . the mean age was 53.2 15.0 years , and 14,332 of the patients ( 49.5% ) were males . the main indications for colonoscopy were abdominal discomfort in 47.2% patients , abdominal pain in 15.9% patients , the change of bowel habits in 10.3% patients , and diarrhea in 9.3% patients . a total of 9191 individuals ( 31.7% ) were found with at least one colorectal polyp and 149 individuals had at least one serrated polyp . among the 149 patients , the overall prevalence of colorectal serrated polyps in the current study was about 0.53% ( 153/28,981 ) . among the 153 serrated polyps , hp , ssa / p , and tsa accounted for 41.2% ( 63/153 ) , 7.2% ( 11/153 ) , and 51.6% ( 79/153 ) , respectively . the demographic characteristics of the patients with colorectal serrated polyps are listed in table 1 . colorectal serrated polyps appeared more in males ( = 4.785 , p < 0.05 ) and in patients 50 years old ( = 5.593 , p < 0.05 ) . the mean age of the patients with serrated polyps was 57.4 13.6 years , and hp , ssa / p , and tsa were 56.2 13.0 years , 60.3 9.4 years , and 58.0 14.4 years , respectively . however , there was no significant difference in the subtype 's distribution in gender and age [ table 2 ] . as shown in figure 1 , the detection rates of serrated adenomas ( including ssa / p and tsa ) in different age decades showed a steadily increased trend with age while hps showed a light peak in 6069 years old . the characteristics of three subtypes of colorectal serrated polyps in a symptomatic patient population csp : colorectal serrated polyps ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; san : synchronous advanced neoplasia ; sd : standard deviation . distribution of colorectal serrated polyps according to gender and age csp : colorectal serrated polyp ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the serrated polyps in the present study tended to have a small diameter ( < 10 mm ) . furthermore , lsps ( diameter 10 mm ) have a pooled prevalence of 0.2% ( 21/9191 ) of all polyps , and the proportion of lsps in all serrated polyps was 13.7% ( 21/153 ) . serrated polyps ( 53.6% in the distal colon ) , hps ( 60.3% in distal colon ) , ssa / ps ( 54.5% in proximal colon ) , and tsas ( 50.6% in proximal colon ) showed no significant difference in anatomic location which was different from previous studies . in total , 98.9% ( 89/90 ) serrated adenomas were found with dysplasia , and the low - grade dysplasia was more commonly found . a total of 14 colorectal serrated polyps were found associated with san which included adenomas with a size larger than 10 mm , villous component , and hgd and crc [ table 1 ] . these included 5 serrated polyps coexisted with invasive adenocarcinoma , 1 with hgd , 1 with villous or tubulovillous adenoma , and 7 with adenoma 10 mm in size . logistic regression analysis indicated that lsps ( or : 3.446 , 95% ci : 1.01011.750 , p < 0.05 ) had a significant association with san [ table 3 ] . remarkably , large hps ( or : 11.417 , 95% ci : 1.47488.405 , characteristics of colorectal serrated polyps associated with synchronous advanced neoplasia csp : colorectal serrated polyps ; san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; sd : standard deviation . association of colorectal serrated subtypes and synchronous advanced neoplasia san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . a total of 28,981 symptomatic patients undergoing colonoscopy from 2010 to 2014 were collected in this study . the mean age was 53.2 15.0 years , and 14,332 of the patients ( 49.5% ) were males . the main indications for colonoscopy were abdominal discomfort in 47.2% patients , abdominal pain in 15.9% patients , the change of bowel habits in 10.3% patients , and diarrhea in 9.3% patients . a total of 9191 individuals ( 31.7% ) were found with at least one colorectal polyp and 149 individuals had at least one serrated polyp . among the 149 patients , the overall prevalence of colorectal serrated polyps in the current study was about 0.53% ( 153/28,981 ) . among the 153 serrated polyps , hp , ssa / p , and tsa accounted for 41.2% ( 63/153 ) , 7.2% ( 11/153 ) , and 51.6% ( 79/153 ) , respectively . the demographic characteristics of the patients with colorectal serrated polyps are listed in table 1 . colorectal serrated polyps appeared more in males ( = 4.785 , p < 0.05 ) and in patients 50 years old ( = 5.593 , p < 0.05 ) . the mean age of the patients with serrated polyps was 57.4 13.6 years , and hp , ssa / p , and tsa were 56.2 13.0 years , 60.3 9.4 years , and 58.0 14.4 years , respectively . however , there was no significant difference in the subtype 's distribution in gender and age [ table 2 ] . as shown in figure 1 , the detection rates of serrated adenomas ( including ssa / p and tsa ) in different age decades showed a steadily increased trend with age while hps showed a light peak in 6069 years old . the characteristics of three subtypes of colorectal serrated polyps in a symptomatic patient population csp : colorectal serrated polyps ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; san : synchronous advanced neoplasia ; sd : standard deviation . distribution of colorectal serrated polyps according to gender and age csp : colorectal serrated polyp ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the serrated polyps in the present study tended to have a small diameter ( < 10 mm ) . furthermore , lsps ( diameter 10 mm ) have a pooled prevalence of 0.2% ( 21/9191 ) of all polyps , and the proportion of lsps in all serrated polyps was 13.7% ( 21/153 ) . serrated polyps ( 53.6% in the distal colon ) , hps ( 60.3% in distal colon ) , ssa / ps ( 54.5% in proximal colon ) , and tsas ( 50.6% in proximal colon ) showed no significant difference in anatomic location which was different from previous studies . in total , 98.9% ( 89/90 ) serrated adenomas were found with dysplasia , and the low - grade dysplasia was more commonly found . a total of 14 colorectal serrated polyps were found associated with san which included adenomas with a size larger than 10 mm , villous component , and hgd and crc [ table 1 ] . these included 5 serrated polyps coexisted with invasive adenocarcinoma , 1 with hgd , 1 with villous or tubulovillous adenoma , and 7 with adenoma 10 mm in size . logistic regression analysis indicated that lsps ( or : 3.446 , 95% ci : 1.01011.750 , p < 0.05 ) had a significant association with san [ table 3 ] . remarkably , large hps ( or : 11.417 , 95% ci : 1.47488.405 , characteristics of colorectal serrated polyps associated with synchronous advanced neoplasia csp : colorectal serrated polyps ; san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; sd : standard deviation . association of colorectal serrated subtypes and synchronous advanced neoplasia san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the current study suggested a lower detection rate of colorectal serrated polyps of 0.53% ( 153/28,981 ) and a different distribution of subtypes compared with available previous reports . these also led to a lower overall detection rate of lsps ; however , the proportion of lsps among colorectal serrated polyps corresponded with lately reports . after being fully recognized and classified in 2005 , colorectal serrated polyps were always reported to have a flat or sessile appearance and paler than surrounding mucosa under white light colonoscopy . this may contribute to a high missing rate even when the polyp detection rate and withdraw time correspond with standards recommended worldwide . it has been reported that the prevalence of proximal colorectal serrated polyps in western countries ranged from 1% to 18% ( average 13% ) . the endoscopy detection rates of hp ( 5.7%44% ) and ssa / p ( 1%14% ) also varied significantly among different studies . to the best of our knowledge , some studies in chinese population also suggested the similar low prevalence of ssa / ps . various factors including the patients population , colonoscopy quality , pathologic validation , variation between centers , and ethnicity might cause these differences . in addition , inadequate communication between pathologists and endoscopists , ignorance of inconspicuous rectal polyps , and lack of validated classification criterion may also contribute to the missing rate in chinese patients . lately , a body of evidence indicated that the western diet habit ( with higher total energy intake and red - meat intake ) was a high risk of development of colorectal polyps in both number and size , as well as the left - sided advanced serrated lesions . further investigation is needed to find the exact reason for the lower prevalence of colorectal serrated polyps and different distribution of subtypes . compared with the prevalence of conventional adenomas which sharply increased with age , the age - specific prevalence of serrated polyps was found to increase steadily with age in the current study which was similar with some recent findings . we also found the prevalence of hps in different age decades reached a light peak at 6069 years old and serrated adenomas steadily increased with age . however , it is still controversial that some studies indicated that increasing age was not materially associated with risk of serrated polyps . therefore , further study is needed to determine whether aging is significantly associated with the development of colorectal serrated polyps . to investigate the predictors of san in colorectal serrated polyps in chinese symptomatic patient population , the current study analyzed multiple factors including age , gender , size , location , and dysplasia . by logistic regression analysis previous studies in asymptomatic screening population reported that large and proximal serrated polyps were the independent predictors of san . multiple ssas , hyperplastic polyposis , proximal and large hps , as well as proximal and large ssa / ps , were further distinguished to be associated with san . however , a large , nationwide , population - based , multicenter , randomized study in average - risk individuals showed that lsps , but not proximal serrated polyps , were the independent risk factor of san . the investigation of genetic or environmental risk factors may be needed to confirm the exact predictors . first , the study chose a simply and widely used and accepted method ( white light colonoscopy ) , so the results could coincide with the common prevalence standards . second , we chose a recent period to reduce the influences of inadequate knowledge and less attention of serrated polyps . third , the relatively large sample size was based on the results of 28,981 colonoscopy of consecutive patients referred to clinic in a comprehensive clinical center . first , the present study was a retrospective study , so the observational design was an important limitation . second , the current study was conducted in a tertiary endoscopic center , so selection bias might not be ignored . third , our population was from symptomatic patients undergoing colonoscopy , while previous reports might include average - risk , asymptomatic patients aged 50 years who underwent screening colonoscopy , and thus these comparisons among different studies should be made with caution . in conclusion , our data showed that the detection rate of colorectal serrated polyps in a chinese symptomatic patient population was low , and distribution pattern of the three subtypes is different from previous reports . the detection rates with age increasing of serrated adenomas showed a steadily increased trend while hps showed a light peak in 6069 years old . moreover , lsps , especially large hps , might be associated with an increased risk of san . prospective studies may be necessary to evaluate the accurate prevalence and subtypes distribution of colorectal serrated polyps in chinese population . this article may provide a reference for the future studies about chinese colorectal serrated polyps . this study was supported by the grants from the national natural science foundation of china ( no . this study was supported by the grants from the national natural science foundation of china ( no .

background : colorectal serrated polyp is considered as histologically heterogeneous lesions with malignant potential in western countries . however , few asian studies have investigated the comprehensive clinical features of serrated polyps in symptomatic populations . the aim of the study was to evaluate the features of colorectal serrated polyps in a chinese symptomatic population.methods:data from all consecutive symptomatic patients were documented from a large colonoscopy database and were analyzed . chi - square test or fisher 's exact test and logistic regression analysis were used for the data processing.results:a total of 9191 ( 31.7% ) patients were detected with at least one colorectal polyp . the prevalence of serrated polyps was 0.53% ( 153/28,981 ) . the proportions of hyperplastic polyp ( hp ) , sessile serrated adenoma / polyp ( ssa / p ) , and traditional serrated adenoma ( tsa ) of all serrated polyps were 41.2% , 7.2% , and 51.6% , respectively , which showed a lower proportion of hp and ssa / p and a higher proportion of tsa . serrated polyps appeared more in males and elder patients while there was no significant difference in the subtype distribution in gender and age . the proportions of large and proximal serrated polyps were 13.7% ( 21/153 ) and 46.4% ( 71/153 ) , respectively . in total , 98.9% ( 89/90 ) serrated adenomas were found with dysplasia . moreover , 14 patients with serrated polyps were found with synchronous advanced colorectal neoplasia , and large serrated polyps ( lsps ) ( odds ratio : 3.446 , 95% confidence interval : 1.01011.750 , p < 0.05 ) , especially large hps , might have an association with synchronous advanced neoplasia ( an).conclusions : the overall detection rate of colorectal serrated polyps in chinese symptomatic patient population was low , and distribution pattern of three subtypes is different from previous reports . moreover , lsps , especially large hps , might be associated with an increased risk of synchronous an .

I M Patients Endoscopic procedure and pathological evaluation Statistical analysis R Detected rate of colorectal serrated polyps Clinical features of colorectal serrated polyps in symptomatic patients Association of colorectal serrated polyps and synchronous advanced neoplasia D Financial support and sponsorship Conflicts of interest

growing evidence supports that 15%20% of crc arise through the serrated pathway , which is characterized by widespread gene inactivation via hypermethylation of promoter regions ( the cpg island methylator phenotype ) , braf mutations , and frequent microsatellite instability . colorectal serrated polyps are histologically typical for a serrated or sawtooth - like appearance of the crypt epithelium and have been recognized as precursor lesions for crc in western countries over the last decade , especially for interval or missed crc . currently , serrated polyps are classified by the world health organization ( who ) into three distinct subtypes : hyperplastic polyp ( hp ) , sessile serrated adenoma / polyp ( ssa / p ) with or without cytological dysplasia , and traditional serrated adenoma ( tsa ) , and each has specific features . recent studies in western countries reporting on the prevalence of colorectal serrated polyps in patients with average risk of crc showed that hp might account for 70%95% of all serrated polyps , ssa / p for 5%25% , and tsa for < 2% . the overall prevalence of serrated polyps increases only slightly with age during adulthood , and these lesions are most common in the sigmoid colon and rectum , but the distribution varies by histological subtype . as the second frequent subset in serrated polyps , ssa / p occurs predominantly in the proximal colon and in older women . as hp and ssa / p have the similarity in color and vessel distribution , some important features can help identify ssa / p including flat morphology , red - colored surface , mucus cap , lateral growth of crypts at the base , dilation in the lower third of crypts , and hyperserration of the crypt bases , sometimes with branching . in addition , studies also suggested that large serrated lesions , proximal serrated polyps , or proximal hyperplasic polyps were associated with synchronous advanced neoplasia ( an ) . however , few asian studies have investigated the comprehensive clinical features of colorectal serrated polyps . to address this issue and also provide more evidence for the management of colorectal serrated polyps in clinical practice , we evaluated the detection rate and the features of serrated polyps in a chinese symptomatic patient population . all consecutive symptomatic patients who underwent routine colonoscopy at the digestive endoscopy center of tianjin medical university general hospital , between january 2010 and december 2014 , were investigated . the pathological sections of suspected colorectal serrated polyps were reevaluated and reclassified by one experienced pathologist ( wen - jing song ) using the who criteria . the demographic and clinicopathological data of all colorectal serrated polyps with a final diagnosis and classification were further collected , including patient age and gender , lesion size , polyp number , and location . the size of colorectal serrated polyps was determined on the basis of endoscopic descriptions , and large serrated polyps ( lsps ) were defined as a diameter 10 mm . the proximal polyps refer to the polyps which locate in the cecum , ascending colon , and transverse colon including the splenic flexure , and distal polyps define as polyps in the descending colon , sigmoid colon , and rectum . exclusion criteria were as follows : ( 1 ) < 20 years old , ( 2 ) patients with any kinds of polyposis syndromes , ( 3 ) patients with a history of crc or inflammatory bowel disease , ( 4 ) patients with a history of colonic resection or polypectomy , ( 5 ) patients with any emergent and therapeutic colonoscopy , and ( 6 ) patients with inadequate bowel preparation and had incomplete colonoscopy . all the serrated lesions were divided into three subtypes : hp , ssa / p with or without dysplasia , and tsa . the defining histologic feature of hp is a sawtooth pattern of epithelial infolding in the upper half of the crypt with a lack of cytologic dysplasia . anchor-shaped crypts above the muscularis mucosae , serration in the lower third of the crypts with and without branching of the crypts , inverted crypts below the muscularis mucosae , and columnar dilation in the lower third . risks of colorectal serrated polyps were compared by chi - square test or fisher 's exact test . logistic regression analysis was used to evaluate the odds ratio ( or ) and 95% confidence interval ( ci ) for colorectal synchronous an ( san ) . all consecutive symptomatic patients who underwent routine colonoscopy at the digestive endoscopy center of tianjin medical university general hospital , between january 2010 and december 2014 , were investigated . the pathological sections of suspected colorectal serrated polyps were reevaluated and reclassified by one experienced pathologist ( wen - jing song ) using the who criteria . the demographic and clinicopathological data of all colorectal serrated polyps with a final diagnosis and classification were further collected , including patient age and gender , lesion size , polyp number , and location . the size of colorectal serrated polyps was determined on the basis of endoscopic descriptions , and large serrated polyps ( lsps ) were defined as a diameter 10 mm . the proximal polyps refer to the polyps which locate in the cecum , ascending colon , and transverse colon including the splenic flexure , and distal polyps define as polyps in the descending colon , sigmoid colon , and rectum . exclusion criteria were as follows : ( 1 ) < 20 years old , ( 2 ) patients with any kinds of polyposis syndromes , ( 3 ) patients with a history of crc or inflammatory bowel disease , ( 4 ) patients with a history of colonic resection or polypectomy , ( 5 ) patients with any emergent and therapeutic colonoscopy , and ( 6 ) patients with inadequate bowel preparation and had incomplete colonoscopy . all the serrated lesions were divided into three subtypes : hp , ssa / p with or without dysplasia , and tsa . the defining histologic feature of hp is a sawtooth pattern of epithelial infolding in the upper half of the crypt with a lack of cytologic dysplasia . anchor-shaped crypts above the muscularis mucosae , serration in the lower third of the crypts with and without branching of the crypts , inverted crypts below the muscularis mucosae , and columnar dilation in the lower third . risks of colorectal serrated polyps were compared by chi - square test or fisher 's exact test . logistic regression analysis was used to evaluate the odds ratio ( or ) and 95% confidence interval ( ci ) for colorectal synchronous an ( san ) . a total of 9191 individuals ( 31.7% ) were found with at least one colorectal polyp and 149 individuals had at least one serrated polyp . among the 149 patients , the overall prevalence of colorectal serrated polyps in the current study was about 0.53% ( 153/28,981 ) . among the 153 serrated polyps , hp , ssa / p , and tsa accounted for 41.2% ( 63/153 ) , 7.2% ( 11/153 ) , and 51.6% ( 79/153 ) , respectively . the demographic characteristics of the patients with colorectal serrated polyps are listed in table 1 . colorectal serrated polyps appeared more in males ( = 4.785 , p < 0.05 ) and in patients 50 years old ( = 5.593 , p < 0.05 ) . the mean age of the patients with serrated polyps was 57.4 13.6 years , and hp , ssa / p , and tsa were 56.2 13.0 years , 60.3 9.4 years , and 58.0 14.4 years , respectively . however , there was no significant difference in the subtype 's distribution in gender and age [ table 2 ] . as shown in figure 1 , the detection rates of serrated adenomas ( including ssa / p and tsa ) in different age decades showed a steadily increased trend with age while hps showed a light peak in 6069 years old . the characteristics of three subtypes of colorectal serrated polyps in a symptomatic patient population csp : colorectal serrated polyps ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; san : synchronous advanced neoplasia ; sd : standard deviation . distribution of colorectal serrated polyps according to gender and age csp : colorectal serrated polyp ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the serrated polyps in the present study tended to have a small diameter ( < 10 mm ) . furthermore , lsps ( diameter 10 mm ) have a pooled prevalence of 0.2% ( 21/9191 ) of all polyps , and the proportion of lsps in all serrated polyps was 13.7% ( 21/153 ) . serrated polyps ( 53.6% in the distal colon ) , hps ( 60.3% in distal colon ) , ssa / ps ( 54.5% in proximal colon ) , and tsas ( 50.6% in proximal colon ) showed no significant difference in anatomic location which was different from previous studies . in total , 98.9% ( 89/90 ) serrated adenomas were found with dysplasia , and the low - grade dysplasia was more commonly found . a total of 14 colorectal serrated polyps were found associated with san which included adenomas with a size larger than 10 mm , villous component , and hgd and crc [ table 1 ] . logistic regression analysis indicated that lsps ( or : 3.446 , 95% ci : 1.01011.750 , p < 0.05 ) had a significant association with san [ table 3 ] . remarkably , large hps ( or : 11.417 , 95% ci : 1.47488.405 , characteristics of colorectal serrated polyps associated with synchronous advanced neoplasia csp : colorectal serrated polyps ; san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; sd : standard deviation . association of colorectal serrated subtypes and synchronous advanced neoplasia san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . a total of 9191 individuals ( 31.7% ) were found with at least one colorectal polyp and 149 individuals had at least one serrated polyp . among the 149 patients , the overall prevalence of colorectal serrated polyps in the current study was about 0.53% ( 153/28,981 ) . among the 153 serrated polyps , hp , ssa / p , and tsa accounted for 41.2% ( 63/153 ) , 7.2% ( 11/153 ) , and 51.6% ( 79/153 ) , respectively . the demographic characteristics of the patients with colorectal serrated polyps are listed in table 1 . colorectal serrated polyps appeared more in males ( = 4.785 , p < 0.05 ) and in patients 50 years old ( = 5.593 , p < 0.05 ) . the mean age of the patients with serrated polyps was 57.4 13.6 years , and hp , ssa / p , and tsa were 56.2 13.0 years , 60.3 9.4 years , and 58.0 14.4 years , respectively . however , there was no significant difference in the subtype 's distribution in gender and age [ table 2 ] . as shown in figure 1 , the detection rates of serrated adenomas ( including ssa / p and tsa ) in different age decades showed a steadily increased trend with age while hps showed a light peak in 6069 years old . the characteristics of three subtypes of colorectal serrated polyps in a symptomatic patient population csp : colorectal serrated polyps ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; san : synchronous advanced neoplasia ; sd : standard deviation . distribution of colorectal serrated polyps according to gender and age csp : colorectal serrated polyp ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the serrated polyps in the present study tended to have a small diameter ( < 10 mm ) . furthermore , lsps ( diameter 10 mm ) have a pooled prevalence of 0.2% ( 21/9191 ) of all polyps , and the proportion of lsps in all serrated polyps was 13.7% ( 21/153 ) . serrated polyps ( 53.6% in the distal colon ) , hps ( 60.3% in distal colon ) , ssa / ps ( 54.5% in proximal colon ) , and tsas ( 50.6% in proximal colon ) showed no significant difference in anatomic location which was different from previous studies . in total , 98.9% ( 89/90 ) serrated adenomas were found with dysplasia , and the low - grade dysplasia was more commonly found . a total of 14 colorectal serrated polyps were found associated with san which included adenomas with a size larger than 10 mm , villous component , and hgd and crc [ table 1 ] . logistic regression analysis indicated that lsps ( or : 3.446 , 95% ci : 1.01011.750 , p < 0.05 ) had a significant association with san [ table 3 ] . remarkably , large hps ( or : 11.417 , 95% ci : 1.47488.405 , characteristics of colorectal serrated polyps associated with synchronous advanced neoplasia csp : colorectal serrated polyps ; san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma ; sd : standard deviation . association of colorectal serrated subtypes and synchronous advanced neoplasia san : synchronous advanced neoplasia ; or : odd ratio ; ci : confidence interval ; hp : hyperplastic polyp ; ssa / p : sessile serrated adenoma / polyp ; tsa : traditional serrated adenoma . the current study suggested a lower detection rate of colorectal serrated polyps of 0.53% ( 153/28,981 ) and a different distribution of subtypes compared with available previous reports . these also led to a lower overall detection rate of lsps ; however , the proportion of lsps among colorectal serrated polyps corresponded with lately reports . after being fully recognized and classified in 2005 , colorectal serrated polyps were always reported to have a flat or sessile appearance and paler than surrounding mucosa under white light colonoscopy . it has been reported that the prevalence of proximal colorectal serrated polyps in western countries ranged from 1% to 18% ( average 13% ) . the endoscopy detection rates of hp ( 5.7%44% ) and ssa / p ( 1%14% ) also varied significantly among different studies . lately , a body of evidence indicated that the western diet habit ( with higher total energy intake and red - meat intake ) was a high risk of development of colorectal polyps in both number and size , as well as the left - sided advanced serrated lesions . further investigation is needed to find the exact reason for the lower prevalence of colorectal serrated polyps and different distribution of subtypes . compared with the prevalence of conventional adenomas which sharply increased with age , the age - specific prevalence of serrated polyps was found to increase steadily with age in the current study which was similar with some recent findings . we also found the prevalence of hps in different age decades reached a light peak at 6069 years old and serrated adenomas steadily increased with age . however , it is still controversial that some studies indicated that increasing age was not materially associated with risk of serrated polyps . therefore , further study is needed to determine whether aging is significantly associated with the development of colorectal serrated polyps . to investigate the predictors of san in colorectal serrated polyps in chinese symptomatic patient population , the current study analyzed multiple factors including age , gender , size , location , and dysplasia . by logistic regression analysis previous studies in asymptomatic screening population reported that large and proximal serrated polyps were the independent predictors of san . multiple ssas , hyperplastic polyposis , proximal and large hps , as well as proximal and large ssa / ps , were further distinguished to be associated with san . however , a large , nationwide , population - based , multicenter , randomized study in average - risk individuals showed that lsps , but not proximal serrated polyps , were the independent risk factor of san . third , our population was from symptomatic patients undergoing colonoscopy , while previous reports might include average - risk , asymptomatic patients aged 50 years who underwent screening colonoscopy , and thus these comparisons among different studies should be made with caution . in conclusion , our data showed that the detection rate of colorectal serrated polyps in a chinese symptomatic patient population was low , and distribution pattern of the three subtypes is different from previous reports . the detection rates with age increasing of serrated adenomas showed a steadily increased trend while hps showed a light peak in 6069 years old . moreover , lsps , especially large hps , might be associated with an increased risk of san . prospective studies may be necessary to evaluate the accurate prevalence and subtypes distribution of colorectal serrated polyps in chinese population . this article may provide a reference for the future studies about chinese colorectal serrated polyps .

fascial or myofascial unwinding is a process in which a client undergoes a spontaneous reaction in response to the therapist s touch . fascial unwinding can be used to release fascial restriction by encouraging the body or parts of the body to move to areas of ease . mosby s dictionary of complementary and alternative medicine defines fascial unwinding as a manual technique in which the therapist passively moves some part of the client s body , with constant feedback as to the sensations of motion being given by the client . furthermore , the glossary of osteopathic terminology states that fascial unwinding is a manual technique involving constant feedback to the osteopathic practitioner who is passively moving a portion of the patient s body in response to the sensation of movement . its forces are localized using the sensations of ease and bind over wider regions . from these two definitions , we can therefore infer that fascial unwinding is a type of indirect myofascial release ( mfr ) technique . the client responds to the induction with spontaneous bending , rotating , and twisting of the upper or lower limbs or the whole body in either a rhythmic or a chaotic pattern . the phenomenon of unwinding , in which parts of the body move spontaneously and involuntarily , can appear mystical , and yet its therapeutic effects are known both anecdotally and clinically . the therapist acts as a catalyst or facilitator by placing the client s body in certain configurations that allow it to unwind and release . the release is set in motion with the therapist s touch , but the client soon takes control . motion is usually induced in the body by lifting and holding certain body parts to remove the influence of gravity and to overcome the reactive proprioceptive postural tone a technique often used when working with the limbs . according to kern , when the effect of gravity is removed , any strain patterns held in the tissues become more easily clarified . in an alternative method , the therapist adds compression to the joints in the area or holds a part of the body in a relaxed position . the unwinding process can be carried out either on the body as a whole or on specific body parts such as arms , legs , the neck , and even the jaw . for example , in arm and shoulder unwinding , the client lies supine while the therapist lifts an arm . the arm is then supported under the therapist s elbow and wrist , and a light compression toward the shoulder joint can be added . after a while although many research papers relating to fascia currently hypothesize about how mfr works , unwinding is still a grey area : the mechanism is not well known . the most accepted explanation for unwinding is that tissues can hold memories that are independent of the nervous system , but there are no scientific research studies to back that claim . although scientific evidence shows that fascia can contract and relax , an ability to unwind has not been shown . ward suggested that the seemingly random movements that characterize unwinding reflect a variety of interacting electromechanical events affecting central , peripheral , autonomic , and even physiologic functions . he added that amid much speculation , satisfactory scientific descriptions for the events are lacking . in an endeavor to understand the process of unwinding , a literature review was conducted . a hypothesis that aims to explain the mechanism of the process was subsequently developed . searches using the terms fascial unwinding or myofascial unwinding were carried out in mainstream research databases : pubmed and isi web of knowledge ( table 1 ) . google scholar , a search engine that looks for scholarly literature across many disciplines and sources , including theses , books , abstracts , and magazine articles , identified only 28 articles . articles for various manual therapy techniques in pubmed , isi web of knowledge , and google scholar as at february 2009 articles , manual therapy books , and scientific papers were reviewed and synthesized to find a possible explanation for the unwinding process . based on that synthesis , i propose a hypothetical model to describe how and why unwinding occurs . ward stated that the osteopathic origins of unwinding are unclear ; however , the procedures have been described for decades by many osteopathic practitioners . most indirect manipulative techniques such as unwinding are based on a common concept that the role of the practitioner is to encourage the inherent corrective activity or homeostatic physiologic mechanisms . in some references , viola m. frymann , an osteopathic physician , is credited with developing the technique and coining the term unwinding . sills preferred the term macro - motions to describe the process in which a client s body expresses large motions . he avoided the term unwinding because of its connotations with release . although unwinding originated in osteopathic schools , it is now also widely offered in classes that teach cranial or craniosacral therapy and mfr . by attending these classes , massage therapists obtain training in fascial unwinding techniques that they can apply in the context of their massage treatments . the discussion herein is limited to the phenomenon of physical unwinding , which aims to release musculoskeletal pain and to allow greater range of motion . in some cases , emotional release can also occur or be induced during the unwinding process , but this secondary effect is not the subject of this paper . although specific techniques can be used to initiate the unwinding process , unwinding itself can be viewed as a process that may occur when those techniques , or others , are applied to the body . additionally , the unwinding process can occur around any articulation or group of articulations : for example , between the head and the neck , between regions or segments of the vertebral column , or between a limb and the torso . generally , unwinding is explained in bodywork literature as being based on the simple principle of the body s ability for self - correction from mechanical disturbances . that is , unwinding occurs because tissues hold memories of trauma and the unwinding process allows the body to adjust to a new position of ease . sills stated that the motions often signal the letting go of frozen stress responses and unresolved trauma . in contrast to the tissue memory theory , ideomotor movement was suggested by dorko as a possible explanation of fascial unwinding . ideomotor actions are unconscious , involuntary movements that are performed by a person and that may be caused by prior expectations , suggestions , or preconceptions . ideomotor action has two important characteristics : first , the person is not aware of causing the movements , and therefore the movements are ascribed to an external force or power ; and second , the movement feels unnatural , and thus the external forces perceived are usually regarded as being mystical or paranormal in nature . automatic writing , dowsing , and using ouija boards to make contact with those in the spirit world have been attributed to the effects of this phenomenon . according to dorko , activities in which movement is thought to be caused by forces that transcend our senses or that are described as metaphysical in nature can be suspected to begin with movement that is not consciously planned . in this paper , i propose a model based on scientific literature to explain the process and mechanism of fascial unwinding ( fig . the model is based on the theories of ideomotor action by carpenter and dorko , fascia neurobiologic theory by schleip , and the psychology of consciousness by halligan and oakley . a hypothetical model for fascial unwinding ( based on schleip ) . the therapist s sensitivity and fine palpation skills form the most important part of these conditions , but it is also imperative that the client be able to relax and let go of his or her body . in the first stage the initiation or induction phase the therapist working on a client will introduce touch or stretching onto the tissue . touch stimulates the fascia s mechanoreceptors and , in turn , arouses a parasympathetic nervous system response . as a result of this latter response , the client is in a state of deep relaxation and calm , sometimes followed with rapid eye movement , twitching , or deep breathing a state that can be observed clinically . in this state , the central nervous system responds to this proprioceptive input by allowing the muscles to perform actions that decrease tone or that create movement in a joint or limb , making it move into an area of ease . at this point , ideomotor action pertains to involuntary muscle movement , which can manifest in various ways and is directed at the central nervous system . these reflexes occur unconsciously , indicating dissociation between voluntary action and conscious experience . in clinical situations , the client is unaware of the unconscious movement and thinks that it is generated by the therapist . this unconscious movement or stretching sensation stimulates a response in the tissue , providing a feedback to the central nervous system as outlined in the theory of ideomotor action . the process is repeated until the client is relaxed or has reached a still point or state of ease . this general hypothesis is based on clinical observations and is backed up by scientific literature . however , it should be noted that not everyone will respond to the unwinding process and not everyone will go through the same process in the same way . schleip presents a comprehensive review of the neurobiology of fascia and provides a theory on how mfr works . pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the autonomic nervous system response includes an altered global muscle tonus , a change in local vasodilatation and tissue viscosity , and a lowered tonus of intrafascial smooth muscle cells . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . gentler types of myofascial stretching and cranial techniques have also long been acknowledged to affect the parasympathetic nervous system . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . recent studies have used heart rate variability , respiratory rate , skin conductance , and skin temperature as measures of physiologic change . zullow and reisman indicated an increase in parasympathetic activity resulting from the compression of the fourth intracranial ventricle ( cv4 ) maneuver and sacral holds , as measured by heart rate variability . using heart rate variability measurement , henley et al . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system fernandez - perez et al . examined the effect on physiologic changes of introducing three myofascial induction or craniosacral techniques . heart rate and systolic blood pressure were altered during the course of the techniques , and the effects were observed for up to 20 minutes after the intervention . boyd showed that a combination of touch and cv4 technique is responsible for a decrease in brain wave activity primarily in the frequency ranges associated with active nervous system function . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . nevertheless , on examination of heart rate variability , those authors found that 3 of 10 subjects may have responded in a manner consistent with an increase in parasympathetic activity during the treatment phase , leading them to hypothesize that there may be both dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . were the first to document unwinding as an ideomotor - based manual therapy in the treatment of a patient with chronic neck pain . their research showed that a reduction in pain intensity and perceived disability can be achieved with the introduction of ideomotor treatment . ideomotor action or ideomotion is the influence of suggestion in modifying and directing muscular movement independently of volitiona definition given by psychologist and physiologist william carpenter in 1852 . carpenter used ideomotor action as an explanation for various phenomena that were being credited to new physical forces , spiritual intervention , or other supernatural causes . later , in 1890 , william james proposed a broader meaning : that ideomotor activity is the basic process underlying all volitional behavior . ideomotor action also pertains to body movements that can arise in observers watching other people perform certain actions . in other words , for example , asking a subject to think of an activity can be enough to set the muscles required for that activity into action . in the mid-1800s , carpenter characterized ideomotor action as the third law of reflex . at that time , reflex movements were partitioned into two types : excitomotor ( such as breathing and swallowing ) and sensory - motor ( such as surprise reactions to a loud noise ) . carpenter imagined the processing of stimuli and the generation of action as a bottom - up path of information processing ( fig . 2 ) . during the normal course of processing , external impressions evoke sensations , ideas , emotions , and intellectual processes that determine the will . carpenter suggested that , should the processing path be interrupted at any point , a reflex action occurs . his path of information processing comprises three levels . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . on the next processing level , sensory - motor reflexes set in , controlled by the sensory ganglia . and finally , on the highest level , the ideomotor reflexes occur when the will , as the highest controller , is turned off in some way . when the ideomotor reflexes occur , ideas and emotions gain direct access to action execution through these reflexes ( fig . a more contemporary study used functional magnetic resonance imaging of the brain to investigate the neural basis of ideomotor action . according to contemporary ideomotor theory , the typical reason for performing an action is to produce an effect in the environment . to perform an intentional action the ideomotor principle is based on two conditions : first , movements and their ensuing effects are required to become associated , so that prediction of an effect for any given movement is possible . second , this association is bidirectional , so that anticipation of the required movement directly triggers the actions that have been learned to produce those effects . , both types of learning may be present in playing the piano , but sensorimotor mapping would associate the finger movement to the sight of the musical note , whereas ideomotor learning would associate the finger movement to the hearing of the tone . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . mccarthy et al . suggested that the resolution of muscular pain depends on an appropriate motor response and that this response should proceed as the result of instinctive , in - built mechanisms . mccarthy et al . therefore hypothesized that the corrective ideomotor movement expression may lead to improvements in pain intensity and disability in symptomatic clients whose instinctive responses have been suppressed . in the field of psychology , halligan and oakley suggested that all the thoughts , activities , ideas , feelings , attitudes , and beliefs traditionally considered to be the contents of consciousness are produced by unconscious processes just like actions and perceptions . only later , when they enter consciousness , does a person become aware of them as outputs . in the model by halligan and oakley ( fig . 3 ) , all the information - processing activities of the brain referred to as unconscious , there must be some kind of decision - making device a central executive structure ( ces ) . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . only the selected information would be allowed to enter level 1 , to produce a conscious experience . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . unwinding or ideomotor process thus represents dissociation between voluntary action and conscious experience . the trial also found that the participant typically estimates that the movement occurs earlier in time when it is a voluntary movement than when the same movement is made involuntarily . this finding suggests that the processes that precede voluntary movement include an unconscious planning stage that leads to an anticipatory awareness of the impending movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . they are not accompanied by anticipatory awareness and are therefore experienced as occurring much closer to the time of the actual physical movement . ward stated that the osteopathic origins of unwinding are unclear ; however , the procedures have been described for decades by many osteopathic practitioners . most indirect manipulative techniques such as unwinding are based on a common concept that the role of the practitioner is to encourage the inherent corrective activity or homeostatic physiologic mechanisms . in some references , viola m. frymann , an osteopathic physician , is credited with developing the technique and coining the term unwinding . sills preferred the term macro - motions to describe the process in which a client s body expresses large motions . he avoided the term unwinding because of its connotations with release . although unwinding originated in osteopathic schools , it is now also widely offered in classes that teach cranial or craniosacral therapy and mfr . by attending these classes , massage therapists obtain training in fascial unwinding techniques that they can apply in the context of their massage treatments . the discussion herein is limited to the phenomenon of physical unwinding , which aims to release musculoskeletal pain and to allow greater range of motion . in some cases , emotional release can also occur or be induced during the unwinding process , but this secondary effect is not the subject of this paper . although specific techniques can be used to initiate the unwinding process , unwinding itself can be viewed as a process that may occur when those techniques , or others , are applied to the body . additionally , the unwinding process can occur around any articulation or group of articulations : for example , between the head and the neck , between regions or segments of the vertebral column , or between a limb and the torso . generally , unwinding is explained in bodywork literature as being based on the simple principle of the body s ability for self - correction from mechanical disturbances . that is , unwinding occurs because tissues hold memories of trauma and the unwinding process allows the body to adjust to a new position of ease . sills stated that the motions often signal the letting go of frozen stress responses and unresolved trauma . in contrast to the tissue memory theory , ideomotor movement was suggested by dorko as a possible explanation of fascial unwinding . ideomotor actions are unconscious , involuntary movements that are performed by a person and that may be caused by prior expectations , suggestions , or preconceptions . ideomotor action has two important characteristics : first , the person is not aware of causing the movements , and therefore the movements are ascribed to an external force or power ; and second , the movement feels unnatural , and thus the external forces perceived are usually regarded as being mystical or paranormal in nature . automatic writing , dowsing , and using ouija boards to make contact with those in the spirit world have been attributed to the effects of this phenomenon . according to dorko , activities in which movement is thought to be caused by forces that transcend our senses or that are described as metaphysical in nature can be suspected to begin with movement that is not consciously planned . in this paper , i propose a model based on scientific literature to explain the process and mechanism of fascial unwinding ( fig . the model is based on the theories of ideomotor action by carpenter and dorko , fascia neurobiologic theory by schleip , and the psychology of consciousness by halligan and oakley . a hypothetical model for fascial unwinding ( based on schleip ) . the therapist s sensitivity and fine palpation skills form the most important part of these conditions , but it is also imperative that the client be able to relax and let go of his or her body . in the first stage the initiation or induction phase the therapist working on a client will introduce touch or stretching onto the tissue . touch stimulates the fascia s mechanoreceptors and , in turn , arouses a parasympathetic nervous system response . as a result of this latter response , the client is in a state of deep relaxation and calm , sometimes followed with rapid eye movement , twitching , or deep breathing a state that can be observed clinically . in this state , the central nervous system responds to this proprioceptive input by allowing the muscles to perform actions that decrease tone or that create movement in a joint or limb , making it move into an area of ease . at this point , ideomotor action pertains to involuntary muscle movement , which can manifest in various ways and is directed at the central nervous system . these reflexes occur unconsciously , indicating dissociation between voluntary action and conscious experience . in clinical situations , the client is unaware of the unconscious movement and thinks that it is generated by the therapist . this unconscious movement or stretching sensation stimulates a response in the tissue , providing a feedback to the central nervous system as outlined in the theory of ideomotor action . the process is repeated until the client is relaxed or has reached a still point or state of ease . this general hypothesis is based on clinical observations and is backed up by scientific literature . however , it should be noted that not everyone will respond to the unwinding process and not everyone will go through the same process in the same way . schleip presents a comprehensive review of the neurobiology of fascia and provides a theory on how mfr works . pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the autonomic nervous system response includes an altered global muscle tonus , a change in local vasodilatation and tissue viscosity , and a lowered tonus of intrafascial smooth muscle cells . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . gentler types of myofascial stretching and cranial techniques have also long been acknowledged to affect the parasympathetic nervous system . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . recent studies have used heart rate variability , respiratory rate , skin conductance , and skin temperature as measures of physiologic change . zullow and reisman indicated an increase in parasympathetic activity resulting from the compression of the fourth intracranial ventricle ( cv4 ) maneuver and sacral holds , as measured by heart rate variability . using heart rate variability measurement , henley et al . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system . fernandez - perez et al . examined the effect on physiologic changes of introducing three myofascial induction or craniosacral techniques . heart rate and systolic blood pressure were altered during the course of the techniques , and the effects were observed for up to 20 minutes after the intervention . boyd showed that a combination of touch and cv4 technique is responsible for a decrease in brain wave activity primarily in the frequency ranges associated with active nervous system function . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . nevertheless , on examination of heart rate variability , those authors found that 3 of 10 subjects may have responded in a manner consistent with an increase in parasympathetic activity during the treatment phase , leading them to hypothesize that there may be both responders and non - responders to treatment . dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . were the first to document unwinding as an ideomotor - based manual therapy in the treatment of a patient with chronic neck pain . their research showed that a reduction in pain intensity and perceived disability can be achieved with the introduction of ideomotor treatment . ideomotor action or ideomotion is the influence of suggestion in modifying and directing muscular movement independently of volitiona definition given by psychologist and physiologist william carpenter in 1852 . carpenter used ideomotor action as an explanation for various phenomena that were being credited to new physical forces , spiritual intervention , or other supernatural causes . later , in 1890 , william james proposed a broader meaning : that ideomotor activity is the basic process underlying all volitional behavior . ideomotor action also pertains to body movements that can arise in observers watching other people perform certain actions . in other words , for example , asking a subject to think of an activity can be enough to set the muscles required for that activity into action . in the mid-1800s , carpenter characterized ideomotor action as the third law of reflex . at that time , reflex movements were partitioned into two types : excitomotor ( such as breathing and swallowing ) and sensory - motor ( such as surprise reactions to a loud noise ) . carpenter imagined the processing of stimuli and the generation of action as a bottom - up path of information processing ( fig . 2 ) . during the normal course of processing , external impressions evoke sensations , ideas , emotions , and intellectual processes that determine the will . carpenter suggested that , should the processing path be interrupted at any point , a reflex action occurs . his path of information processing comprises three levels . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . on the next processing level , sensory - motor reflexes set in , controlled by the sensory ganglia . and reflexes occur when the will , as the highest controller , is turned off in some way . when the ideomotor reflexes occur , ideas and emotions gain direct access to action execution through these reflexes ( fig . a more contemporary study used functional magnetic resonance imaging of the brain to investigate the neural basis of ideomotor action . according to contemporary ideomotor theory , the typical reason for performing an action is to produce an effect in the environment . to perform an intentional action , the consequences or effects of a particular movement the ideomotor principle is based on two conditions : first , movements and their ensuing effects are required to become associated , so that prediction of an effect for any given movement is possible . second , this association is bidirectional , so that anticipation of the required movement directly triggers the actions that have been learned to produce those effects . , both types of learning may be present in playing the piano , but sensorimotor mapping would associate the finger movement to the sight of the musical note , whereas ideomotor learning would associate the finger movement to the hearing of the tone . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . mccarthy et al . suggested that the resolution of muscular pain depends on an appropriate motor response and that this response should proceed as the result of instinctive , in - built mechanisms . mccarthy et al . therefore hypothesized that the corrective ideomotor movement expression may lead to improvements in pain intensity and disability in symptomatic clients whose instinctive responses have been suppressed . in the field of psychology , halligan and oakley suggested that all the thoughts , activities , ideas , feelings , attitudes , and beliefs traditionally considered to be the contents of consciousness are produced by unconscious processes just like actions and perceptions . only later , when they enter consciousness , does a person become aware of them as outputs . in the model by halligan and oakley ( fig . 3 ) , all the information - processing activities of the brain referred to as unconscious parts are designated level 2 . within this level , there must be some kind of decision - making device a central executive structure ( ces ) . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . only the selected information would be allowed to enter level 1 , to produce a conscious experience . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . mental process from thoughts to action ( halligan and oakley ) . haggard et al . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . unwinding or ideomotor process thus represents dissociation between voluntary action and conscious experience . the trial also found that the participant typically estimates that the movement occurs earlier in time when it is a voluntary movement than when the same movement is made involuntarily . this finding suggests that the processes that precede voluntary movement include an unconscious planning stage that leads to an anticipatory awareness of the impending movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . they are not accompanied by anticipatory awareness and are therefore experienced as occurring much closer to the time of the actual physical movement . schleip presents a comprehensive review of the neurobiology of fascia and provides a theory on how mfr works . in this theory , fascia and the autonomic nervous system pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the autonomic nervous system response includes an altered global muscle tonus , a change in local vasodilatation and tissue viscosity , and a lowered tonus of intrafascial smooth muscle cells . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . gentler types of myofascial stretching and cranial techniques have also long been acknowledged to affect the parasympathetic nervous system . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . recent studies have used heart rate variability , respiratory rate , skin conductance , and skin temperature as measures of physiologic change . zullow and reisman indicated an increase in parasympathetic activity resulting from the compression of the fourth intracranial ventricle ( cv4 ) maneuver and sacral holds , as measured by heart rate variability . using heart rate variability measurement , henley et al . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system . fernandez - perez et al . examined the effect on physiologic changes of introducing three myofascial induction or craniosacral techniques . heart rate and systolic blood pressure were altered during the course of the techniques , and the effects were observed for up to 20 minutes after the intervention . boyd showed that a combination of touch and cv4 technique is responsible for a decrease in brain wave activity primarily in the frequency ranges associated with active nervous system function . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . nevertheless , on examination of heart rate variability , those authors found that 3 of 10 subjects may have responded in a manner consistent with an increase in parasympathetic activity during the treatment phase , leading them to hypothesize that there may be both dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . were the first to document unwinding as an ideomotor - based manual therapy in the treatment of a patient with chronic neck pain . their research showed that a reduction in pain intensity and perceived disability can be achieved with the introduction of ideomotor treatment . ideomotor action or ideomotion is the influence of suggestion in modifying and directing muscular movement independently of volitiona definition given by psychologist and physiologist william carpenter in 1852 . carpenter used ideomotor action as an explanation for various phenomena that were being credited to new physical forces , spiritual intervention , or other supernatural causes . later , in 1890 , william james proposed a broader meaning : that ideomotor activity is the basic process underlying all volitional behavior . ideomotor action also pertains to body movements that can arise in observers watching other people perform certain actions . in other words , for example , asking a subject to think of an activity can be enough to set the muscles required for that activity into action . in the mid-1800s , carpenter characterized ideomotor action as the third law of reflex . at that time , reflex movements were partitioned into two types : excitomotor ( such as breathing and swallowing ) and sensory - motor ( such as surprise reactions to a loud noise ) . carpenter imagined the processing of stimuli and the generation of action as a bottom - up path of information processing ( fig . 2 ) . during the normal course of processing , external impressions evoke sensations , ideas , emotions , and intellectual processes that determine the will . carpenter suggested that , should the processing path be interrupted at any point , a reflex action occurs . his path of information processing comprises three levels . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . on the next processing level , sensory - motor reflexes set in , controlled by the sensory ganglia . and finally , on the highest level , the ideomotor reflexes occur when the will , as the highest controller , is turned off in some way . when the ideomotor reflexes occur , ideas and emotions gain direct access to action execution through these reflexes ( fig . a more contemporary study used functional magnetic resonance imaging of the brain to investigate the neural basis of ideomotor action . according to contemporary ideomotor theory , the typical reason for performing an action is to produce an effect in the environment . to perform an intentional action the ideomotor principle is based on two conditions : first , movements and their ensuing effects are required to become associated , so that prediction of an effect for any given movement is possible . second , this association is bidirectional , so that anticipation of the required movement directly triggers the actions that have been learned to produce those effects . , both types of learning may be present in playing the piano , but sensorimotor mapping would associate the finger movement to the sight of the musical note , whereas ideomotor learning would associate the finger movement to the hearing of the tone . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . mccarthy et al . suggested that the resolution of muscular pain depends on an appropriate motor response and that this response should proceed as the result of instinctive , in - built mechanisms . mccarthy et al . therefore hypothesized that the corrective ideomotor movement expression may lead to improvements in pain intensity and disability in symptomatic clients whose instinctive responses have been suppressed . in the field of psychology , the consciousness model is used to explain ideomotor action . halligan and oakley suggested that all the thoughts , activities , ideas , feelings , attitudes , and beliefs traditionally considered to be the contents of consciousness are produced by unconscious processes just like actions and perceptions . only later , when they enter consciousness , does a person become aware of them as outputs . in the model by halligan and oakley ( fig . 3 ) , all the information - processing activities of the brain referred to as unconscious , there must be some kind of decision - making device a central executive structure ( ces ) . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . only the selected information would be allowed to enter level 1 , to produce a conscious experience . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . used with permission . in a hypnosis trial , haggard et al . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . unwinding or ideomotor process thus represents dissociation between voluntary action and conscious experience . the trial also found that the participant typically estimates that the movement occurs earlier in time when it is a voluntary movement than when the same movement is made involuntarily . this finding suggests that the processes that precede voluntary movement include an unconscious planning stage that leads to an anticipatory awareness of the impending movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . they are not accompanied by anticipatory awareness and are therefore experienced as occurring much closer to the time of the actual physical movement . a model built upon the neurobiologic , ideomotor action , and consciousness theories is proposed to explain the mechanism of unwinding . touch , stretching , and manual therapy can induce relaxation in the parasympathetic nervous system . they also activate the central nervous system , which is involved in the modulation of muscle tone as well as movement . this activation stimulates the response to stretching : muscles find areas and positions of ease , the client experiences less pain or is more relaxed , thereby introducing the ideomotor action . the ideomotor action is generated through normal voluntary motor control systems , but is altered and experienced as an involuntary reaction . the stretching sensation provides a feedback to the nervous system , which in turn will generate the movements again . in bodywork literature , it is generally accepted that fascia or connective tissues can hold onto memories and trauma . but the fact that a specific touch or body positioning can trigger a specific memory associated with the touch or positioning does nt mean that the particular memory is stored in those tissues or cells . scientific evidence supports the effects of touch and positioning as potential triggers for a memory , but the memory is still stored within the central nervous system . this occurrence is called state - dependent memory ( coming from the observation that memory in one state of consciousness can not be recalled until the person returns to the same state ) . this paper demonstrates that it is possible to develop a useful working model for the unwinding process based on the existing scientific literature . however , additional evidence and testable hypotheses are required to move this concept beyond the realm of what is often considered to be pseudo - science . to test the proposed hypotheses , investigations involving the two main processes stimulation of the sympathetic nervous system and analysis of ideomotor action are required . ideomotor action provides a sound explanation , but it also has another implication that the perceived movement may be generated by the therapist while palpating . a therapist s non - conscious perceptions such as expectations , degree of empathy , and so on can influence decision - making and leave open the possibility of the therapist falling into a self - fulfilling prophecy or expectancy confirmation effects.as noted by upledger , i know that the intention of the therapist has a lot to do with it . the effect of myofascial manipulation has been studied from cellular to tissue to whole - body levels . although fascia can contract and relax , studies have demonstrated that it is impossible to generate immediate and permanent lengthening or unwinding of fascia by mechanical means . creating such changes requires a large amount of force , with longer durations of stretching . stimulation of mechanoreceptors is the most likely trigger of a release that instigates the unwinding process . furthermore , application of force by manual therapists at the surface of the body over the tensor fascia lata and plantar fascia is not within the range required to mechanically stretch the fascia , although over softer tissues such as nasal fascia , the applied forces are sufficient . finally , to relate unwinding to schleip s bodywork model , the process can be viewed as an application of the neurobiologic concept using the self - regulation dynamic system theory . the therapist works as a facilitator inducing the parasympathetic system ; paying attention to the state of the autonomic nervous system ; creating unusual sensations with subtle stimulation , including immediate feedback ; and involving active macro - movement participation .

background : fascial or myofascial unwinding is a process in which a client undergoes a spontaneous reaction in response to the therapist s touch . it can be induced by using specific techniques that encourage a client s body to move into areas of ease . unwinding is a popular technique in massage therapy , but its mechanism is not well understood . in the absence of a scientific explanation or hypothesis of the mechanism of action , it can be interpreted as mystical.purpose:this paper proposes a model that builds on the neurobiologic , ideomotor action , and consciousness theories to explain the process and mechanism of fascial unwinding.hypothetical model : during fascial unwinding , the therapist stimulates mechanoreceptors in the fascia by applying gentle touch and stretching . touch and stretching induce relaxation and activate the parasympathetic nervous system . they also activate the central nervous system , which is involved in the modulation of muscle tone as well as movement . as a result , the central nervous system is aroused and thereby responds by encouraging muscles to find an easier , or more relaxed , position and by introducing the ideomotor action . although the ideomotor action is generated via normal voluntary motor control systems , it is altered and experienced as an involuntary response.conclusions:fascial unwinding occurs when a physically induced suggestion by a therapist prompts ideomotor action that the client experiences as involuntary . this action is guided by the central nervous system , which produces continuous action until a state of ease is reached . consequently , fascial unwinding can be thought of as a neurobiologic process employing the self - regulation dynamic system theory .

INTRODUCTION METHODS RESULTS Origins and Concepts A Hypothetical Model Neurobiologic Fascia Theory Unwinding as an Ideomotor Action Consciousness Model DISCUSSION AND CONCLUSIONS

fascial or myofascial unwinding is a process in which a client undergoes a spontaneous reaction in response to the therapist s touch . fascial unwinding can be used to release fascial restriction by encouraging the body or parts of the body to move to areas of ease . mosby s dictionary of complementary and alternative medicine defines fascial unwinding as a manual technique in which the therapist passively moves some part of the client s body , with constant feedback as to the sensations of motion being given by the client . furthermore , the glossary of osteopathic terminology states that fascial unwinding is a manual technique involving constant feedback to the osteopathic practitioner who is passively moving a portion of the patient s body in response to the sensation of movement . from these two definitions , we can therefore infer that fascial unwinding is a type of indirect myofascial release ( mfr ) technique . the client responds to the induction with spontaneous bending , rotating , and twisting of the upper or lower limbs or the whole body in either a rhythmic or a chaotic pattern . the phenomenon of unwinding , in which parts of the body move spontaneously and involuntarily , can appear mystical , and yet its therapeutic effects are known both anecdotally and clinically . the therapist acts as a catalyst or facilitator by placing the client s body in certain configurations that allow it to unwind and release . the release is set in motion with the therapist s touch , but the client soon takes control . in an alternative method , the therapist adds compression to the joints in the area or holds a part of the body in a relaxed position . the unwinding process can be carried out either on the body as a whole or on specific body parts such as arms , legs , the neck , and even the jaw . for example , in arm and shoulder unwinding , the client lies supine while the therapist lifts an arm . the arm is then supported under the therapist s elbow and wrist , and a light compression toward the shoulder joint can be added . after a while although many research papers relating to fascia currently hypothesize about how mfr works , unwinding is still a grey area : the mechanism is not well known . the most accepted explanation for unwinding is that tissues can hold memories that are independent of the nervous system , but there are no scientific research studies to back that claim . a hypothesis that aims to explain the mechanism of the process was subsequently developed . searches using the terms fascial unwinding or myofascial unwinding were carried out in mainstream research databases : pubmed and isi web of knowledge ( table 1 ) . sills preferred the term macro - motions to describe the process in which a client s body expresses large motions . by attending these classes , massage therapists obtain training in fascial unwinding techniques that they can apply in the context of their massage treatments . although specific techniques can be used to initiate the unwinding process , unwinding itself can be viewed as a process that may occur when those techniques , or others , are applied to the body . additionally , the unwinding process can occur around any articulation or group of articulations : for example , between the head and the neck , between regions or segments of the vertebral column , or between a limb and the torso . generally , unwinding is explained in bodywork literature as being based on the simple principle of the body s ability for self - correction from mechanical disturbances . in contrast to the tissue memory theory , ideomotor movement was suggested by dorko as a possible explanation of fascial unwinding . ideomotor action has two important characteristics : first , the person is not aware of causing the movements , and therefore the movements are ascribed to an external force or power ; and second , the movement feels unnatural , and thus the external forces perceived are usually regarded as being mystical or paranormal in nature . according to dorko , activities in which movement is thought to be caused by forces that transcend our senses or that are described as metaphysical in nature can be suspected to begin with movement that is not consciously planned . in this paper , i propose a model based on scientific literature to explain the process and mechanism of fascial unwinding ( fig . the model is based on the theories of ideomotor action by carpenter and dorko , fascia neurobiologic theory by schleip , and the psychology of consciousness by halligan and oakley . the therapist s sensitivity and fine palpation skills form the most important part of these conditions , but it is also imperative that the client be able to relax and let go of his or her body . in the first stage the initiation or induction phase the therapist working on a client will introduce touch or stretching onto the tissue . touch stimulates the fascia s mechanoreceptors and , in turn , arouses a parasympathetic nervous system response . as a result of this latter response , the client is in a state of deep relaxation and calm , sometimes followed with rapid eye movement , twitching , or deep breathing a state that can be observed clinically . in this state , the central nervous system responds to this proprioceptive input by allowing the muscles to perform actions that decrease tone or that create movement in a joint or limb , making it move into an area of ease . at this point , ideomotor action pertains to involuntary muscle movement , which can manifest in various ways and is directed at the central nervous system . in clinical situations , the client is unaware of the unconscious movement and thinks that it is generated by the therapist . this unconscious movement or stretching sensation stimulates a response in the tissue , providing a feedback to the central nervous system as outlined in the theory of ideomotor action . the process is repeated until the client is relaxed or has reached a still point or state of ease . however , it should be noted that not everyone will respond to the unwinding process and not everyone will go through the same process in the same way . pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system fernandez - perez et al . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . nevertheless , on examination of heart rate variability , those authors found that 3 of 10 subjects may have responded in a manner consistent with an increase in parasympathetic activity during the treatment phase , leading them to hypothesize that there may be both dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . , both types of learning may be present in playing the piano , but sensorimotor mapping would associate the finger movement to the sight of the musical note , whereas ideomotor learning would associate the finger movement to the hearing of the tone . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . sills preferred the term macro - motions to describe the process in which a client s body expresses large motions . by attending these classes , massage therapists obtain training in fascial unwinding techniques that they can apply in the context of their massage treatments . although specific techniques can be used to initiate the unwinding process , unwinding itself can be viewed as a process that may occur when those techniques , or others , are applied to the body . generally , unwinding is explained in bodywork literature as being based on the simple principle of the body s ability for self - correction from mechanical disturbances . in contrast to the tissue memory theory , ideomotor movement was suggested by dorko as a possible explanation of fascial unwinding . ideomotor action has two important characteristics : first , the person is not aware of causing the movements , and therefore the movements are ascribed to an external force or power ; and second , the movement feels unnatural , and thus the external forces perceived are usually regarded as being mystical or paranormal in nature . in this paper , i propose a model based on scientific literature to explain the process and mechanism of fascial unwinding ( fig . the therapist s sensitivity and fine palpation skills form the most important part of these conditions , but it is also imperative that the client be able to relax and let go of his or her body . as a result of this latter response , the client is in a state of deep relaxation and calm , sometimes followed with rapid eye movement , twitching , or deep breathing a state that can be observed clinically . in this state , the central nervous system responds to this proprioceptive input by allowing the muscles to perform actions that decrease tone or that create movement in a joint or limb , making it move into an area of ease . at this point , ideomotor action pertains to involuntary muscle movement , which can manifest in various ways and is directed at the central nervous system . in clinical situations , the client is unaware of the unconscious movement and thinks that it is generated by the therapist . this unconscious movement or stretching sensation stimulates a response in the tissue , providing a feedback to the central nervous system as outlined in the theory of ideomotor action . the process is repeated until the client is relaxed or has reached a still point or state of ease . however , it should be noted that not everyone will respond to the unwinding process and not everyone will go through the same process in the same way . pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . were the first to document unwinding as an ideomotor - based manual therapy in the treatment of a patient with chronic neck pain . carpenter used ideomotor action as an explanation for various phenomena that were being credited to new physical forces , spiritual intervention , or other supernatural causes . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . to perform an intentional action , the consequences or effects of a particular movement the ideomotor principle is based on two conditions : first , movements and their ensuing effects are required to become associated , so that prediction of an effect for any given movement is possible . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . in this theory , fascia and the autonomic nervous system pressure from myofascial manipulation involves the stimulation of intrafascial mechanoreceptors whose signals are then processed by the central nervous system and autonomic nervous system . the response of the central nervous system changes the tonus of some related striated muscle fibers . the indirect stimulation of the autonomic nervous system ( that is , the parasympathetic nervous system ) , which results in global muscle relaxation and a more peaceful state of mind , represents the heart of the changes that are so vital to many manual therapies . gentler types of myofascial stretching and cranial techniques have also long been acknowledged to affect the parasympathetic nervous system . bertolucci observed that , when a client is being treated with a muscle repositioning technique , the client begins to show involuntary motor reactions several studies have evaluated the physiologic changes in the autonomic nervous system that occur as a result of craniosacral and mfr interventions , clinically - known techniques that can trigger the unwinding process . demonstrated that cervical mfr shifts sympathovagal balance from the sympathetic to the parasympathetic nervous system . boyd showed that a combination of touch and cv4 technique is responsible for a decrease in brain wave activity primarily in the frequency ranges associated with active nervous system function . however , milnes and moran demonstrated that the application of the cv4 technique in asymptomatic individuals had minimal physiologic effect in a range of autonomic response variables ( heart rate variability , respiration rate , galvanic skin resistance , and skin temperature ) . nevertheless , on examination of heart rate variability , those authors found that 3 of 10 subjects may have responded in a manner consistent with an increase in parasympathetic activity during the treatment phase , leading them to hypothesize that there may be both dorko was the first to suggest that fascial unwinding can be simply explained as an ideomotor movement . on the lower levels of the path are the excitomotor reflexes , which are controlled by the spinal cord . and finally , on the highest level , the ideomotor reflexes occur when the will , as the highest controller , is turned off in some way . according to contemporary ideomotor theory , the typical reason for performing an action is to produce an effect in the environment . in unwinding , when it is initiated , the movement is guided by and associated with the stretching sensation , to find areas of ease of movement and freedom from pain . this result is contrary to that in stretching , in which the movement is guided by the therapist s hand or instruction . in the field of psychology , the consciousness model is used to explain ideomotor action . the ces identifies the most important task the brain is carrying out at any moment and selects the information that best describes the current state of the brain in relation to the chosen task . during unwinding , because the autonomic nervous system is in a relaxed state , the ces does not select information for entry to level 1 , and the client remains unaware of it . showed that an ideomotor response produced by suggestion is generated by normal voluntary motor control systems , but is experienced as involuntary , resulting in a conscious experience close to that of a passive movement . movements that are produced reflexively or by unwinding , on the other hand , lack the central planning stage , because the client is in a relaxed state . a model built upon the neurobiologic , ideomotor action , and consciousness theories is proposed to explain the mechanism of unwinding . touch , stretching , and manual therapy can induce relaxation in the parasympathetic nervous system . they also activate the central nervous system , which is involved in the modulation of muscle tone as well as movement . this activation stimulates the response to stretching : muscles find areas and positions of ease , the client experiences less pain or is more relaxed , thereby introducing the ideomotor action . the ideomotor action is generated through normal voluntary motor control systems , but is altered and experienced as an involuntary reaction . the stretching sensation provides a feedback to the nervous system , which in turn will generate the movements again . scientific evidence supports the effects of touch and positioning as potential triggers for a memory , but the memory is still stored within the central nervous system . ideomotor action provides a sound explanation , but it also has another implication that the perceived movement may be generated by the therapist while palpating . a therapist s non - conscious perceptions such as expectations , degree of empathy , and so on can influence decision - making and leave open the possibility of the therapist falling into a self - fulfilling prophecy or expectancy confirmation effects.as noted by upledger , i know that the intention of the therapist has a lot to do with it . furthermore , application of force by manual therapists at the surface of the body over the tensor fascia lata and plantar fascia is not within the range required to mechanically stretch the fascia , although over softer tissues such as nasal fascia , the applied forces are sufficient . finally , to relate unwinding to schleip s bodywork model , the process can be viewed as an application of the neurobiologic concept using the self - regulation dynamic system theory . the therapist works as a facilitator inducing the parasympathetic system ; paying attention to the state of the autonomic nervous system ; creating unusual sensations with subtle stimulation , including immediate feedback ; and involving active macro - movement participation .

hamster anti murine fas ( jo2 ) was purchased from pharmingen ( san diego , ca ) . indirect staining was carried out as previously described ( 21 ) using 10 cells and 0.5 g anti - fas or isotype - matched control and a 1:50 dilution secondary antibody . fas staining was determined on cells that had been fixed overnight in 1% paraformaldehyde in pbs using a facscan ( becton dickinson , mountain view , ca ) with the windows set to exclude dead cells and debris . fas sensitivity was determined using the cd4 t hybridoma line , 3a9 ( 22 ) . 3a9 cells were activated by exposure to 10 ng / ml pma ( sigma chemical co. , st . louis , mo ) and 3 g / ml ionomycin ( sigma chemical co. ) for 4 h to upregulate fasl . after activation , 3a9 cells were washed three times and plated in quadruplicate in 96-well microtiter plates with cr - labeled k1735 targets ( 5 10 cells / well ) at a 50:1 effector / target ratio in a final volume of 200 l calcium - free medium . after 1618 h incubation at 37c in a 5% co2 atmosphere , 100-l aliquots of cell - free supernatant were harvested and radioactivity was measured using an lkb gamma counter ( amersham pharmacia biotech , piscataway , nj ) . maximum lysis was determined by incubating cr - labeled targets in 2 n hcl . percentage specific target cell lysis was calculated as follows : 100 [ ( experimental lysis spontaneous lysis)/(maximum lysis spontaneous lysis ) ] . lytic activity of activated 3a9 cells was blocked by the inclusion of fas - fc ( data not shown ) ; unactivated 3a9 cells demonstrated no lytic activity . anti - fas killing was carried out by plating tumor cells at 5 10/well in triplicate and adding 100 ng / ml biotinylated anti - fas ( jo-2 clone ; pharmingen ) or biotinylated isotype - matched control antibody ( pharmingen ) for 30 min at 37c then adding 200 ng / ml streptavidin ( jackson immunoresearch labs . , west grove , pa ) . after a 48-h culture cell viability was determined using a tetrazolium reduction ( mtt ) assay as described previously ( 8) . values shown represent the percentage of specific cytotoxicity in anti - fas treated wells after subtracting that of the biotinylated , isotype - matched controls [ ( 1 mean anti - fas)/(mean isotype - matched control ) ] 100 . lung conditioned medium ( lcm ) was prepared according to the method of szaniaswska et al . , murine lungs were surgically excised , washed in cold pbs , cut into 12 mm pieces , washed in cold hbss , and suspended in serum - free mem with 1% bsa and rotated at 37c for 6 h. initial medium was discarded and replaced with mem with 0.01% bsa , and the lung fragments were rotated at 37c for an additional 24 h. after the final rotation , lung fragments were removed and discarded and the remaining medium was spun at 15,000 g for 15 min . the cell - free supernatants were collected ( lcm ) and their protein concentration was determined . for the determination of bioactivity , k1735 cell lines and clones were plated in media supplemented with 0.3% fetal bovine serum and 65 g / ml lcm at 10 cells / ml and cultured at 37c , 5% co2 for 48 h. after culture , relative cellular viability was determined using the tetrazolium reduction ( mtt ) assay as previously described by our laboratory ( 21 ) . background cell death was determined by culturing cells in media supplemented with 0.3% fetal bovine serum alone . specificity of lcm killing was shown by inclusion of either fas - fc ( 25 ) or isotype control fc ( ltr - fc ) at 200 ng / ml for the 48-h culture period . the presence of fasl protein in lcm was verified by western blotting . for this procedure , 10-g aliquots of lcm fasl was visualized by hybridization with a monoclonal anti - fasl ( clone 33 ; transduction laboratories , lexington , ky ) and anti mouse horseradish peroxidase was detected using the ecl system ( amersham pharmacia biotech , little chalfont , buckinghamshire , uk ) . frozen sections of murine lung were fixed with 2% formaldehyde in pbs for 10 min and washed with pbs . endogenous peroxidase was blocked with 3% h2o2 in methanol for 12 min and the sections were incubated with blocking solution ( pbs supplemented with 5% horse serum and 1% goat serum ) for 20 min . sections were then incubated with fasl - specific antibody , n-20 ( santa cruz biotechnologies , santa cruz , ca ) , at a 1:800 dilution in blocking solution overnight at 4c . after washing , antibody staining was visualized using peroxidase - conjugated goat anti rabbit igg , f(ab)2 ( jackson immunoresearch labs . ) at 1:500 in blocking solution and stable diaminobenzidine ( research genetics , inc . , c3h / gld and c3h / wt mice were purchased from the jackson laboratory ( bar harbor , me ) . single cell suspensions of k1735 melanoma cells were prepared by brief incubation of monolayer cultures in 0.02% edta and 0.25% trypsin . before injection 10 cells were injected intravenously into the lateral tail vein and metastatic lung tumor growth was enumerated by visual inspection using light microscopy 35 wk after injection . for the determination of spontaneous metastases , metastatic lung growth was determined by visual inspection using light microscopy 46 wk later . animal experimentation was performed in accordance with institutional guidelines and the regulations and standards of the usda . statistical analyses of metastatic incidence in c3h and c3h / gld mice were carried out using a 2 2 contingency table of the number of mice with metastases versus the number of mice without metastases using a one - sided analysis . statistical analyses of the number of metastases in c3h versus c3h / gld mice were carried out using a mann - whitney test . hamster anti murine fas ( jo2 ) was purchased from pharmingen ( san diego , ca ) . indirect staining was carried out as previously described ( 21 ) using 10 cells and 0.5 g anti - fas or isotype - matched control and a 1:50 dilution secondary antibody . fas staining was determined on cells that had been fixed overnight in 1% paraformaldehyde in pbs using a facscan ( becton dickinson , mountain view , ca ) with the windows set to exclude dead cells and debris . fas sensitivity was determined using the cd4 t hybridoma line , 3a9 ( 22 ) . 3a9 cells were activated by exposure to 10 ng / ml pma ( sigma chemical co. , st . louis , mo ) and 3 g / ml ionomycin ( sigma chemical co. ) for 4 h to upregulate fasl . after activation , 3a9 cells were washed three times and plated in quadruplicate in 96-well microtiter plates with cr - labeled k1735 targets ( 5 10 cells / well ) at a 50:1 effector / target ratio in a final volume of 200 l calcium - free medium . after 1618 h incubation at 37c in a 5% co2 atmosphere , 100-l aliquots of cell - free supernatant were harvested and radioactivity was measured using an lkb gamma counter ( amersham pharmacia biotech , piscataway , nj ) . maximum lysis was determined by incubating cr - labeled targets in 2 n hcl . percentage specific target cell lysis was calculated as follows : 100 [ ( experimental lysis spontaneous lysis)/(maximum lysis spontaneous lysis ) ] . lytic activity of activated 3a9 cells was blocked by the inclusion of fas - fc ( data not shown ) ; unactivated 3a9 cells demonstrated no lytic activity . anti - fas killing was carried out by plating tumor cells at 5 10/well in triplicate and adding 100 ng / ml biotinylated anti - fas ( jo-2 clone ; pharmingen ) or biotinylated isotype - matched control antibody ( pharmingen ) for 30 min at 37c then adding 200 ng / ml streptavidin ( jackson immunoresearch labs . was determined using a tetrazolium reduction ( mtt ) assay as described previously ( 8) . values shown represent the percentage of specific cytotoxicity in anti - fas treated wells after subtracting that of the biotinylated , isotype - matched controls [ ( 1 mean anti - fas)/(mean isotype - matched control ) ] 100 . lung conditioned medium ( lcm ) was prepared according to the method of szaniaswska et al . ( 24 ) with modifications . in brief , murine lungs were surgically excised , washed in cold pbs , cut into 12 mm pieces , washed in cold hbss , and suspended in serum - free mem with 1% bsa and rotated at 37c for 6 h. initial medium was discarded and replaced with mem with 0.01% bsa , and the lung fragments were rotated at 37c for an additional 24 h. after the final rotation , lung fragments were removed and discarded and the remaining medium was spun at 15,000 g for 15 min . the cell - free supernatants were collected ( lcm ) and their protein concentration was determined . for the determination of bioactivity , k1735 cell lines and clones were plated in media supplemented with 0.3% fetal bovine serum and 65 g / ml lcm at 10 cells / ml and cultured at 37c , 5% co2 for 48 h. after culture , relative cellular viability was determined using the tetrazolium reduction ( mtt ) assay as previously described by our laboratory ( 21 ) . background cell death was determined by culturing cells in media supplemented with 0.3% fetal bovine serum alone . specificity of lcm killing was shown by inclusion of either fas - fc ( 25 ) or isotype control fc ( ltr - fc ) at 200 ng / ml for the 48-h culture period . the presence of fasl protein in lcm was verified by western blotting . for this procedure , 10-g aliquots of lcm fasl was visualized by hybridization with a monoclonal anti - fasl ( clone 33 ; transduction laboratories , lexington , ky ) and anti mouse horseradish peroxidase was detected using the ecl system ( amersham pharmacia biotech , little chalfont , buckinghamshire , uk ) . frozen sections of murine lung were fixed with 2% formaldehyde in pbs for 10 min and washed with pbs . endogenous peroxidase was blocked with 3% h2o2 in methanol for 12 min and the sections were incubated with blocking solution ( pbs supplemented with 5% horse serum and 1% goat serum ) for 20 min . sections were then incubated with fasl - specific antibody , n-20 ( santa cruz biotechnologies , santa cruz , ca ) , at a 1:800 dilution in blocking solution overnight at 4c . after washing , antibody staining was visualized using peroxidase - conjugated goat anti rabbit igg , f(ab)2 ( jackson immunoresearch labs . ) at 1:500 in blocking solution and stable diaminobenzidine ( research genetics , inc . , c3h / gld and c3h / wt mice were purchased from the jackson laboratory ( bar harbor , me ) . single cell suspensions of k1735 melanoma cells were prepared by brief incubation of monolayer cultures in 0.02% edta and 0.25% trypsin . before injection 10 cells were injected intravenously into the lateral tail vein and metastatic lung tumor growth was enumerated by visual inspection using light microscopy 35 wk after injection . for the determination of spontaneous metastases , metastatic lung growth was determined by visual inspection using light microscopy 46 wk later . animal experimentation was performed in accordance with institutional guidelines and the regulations and standards of the usda . statistical analyses of metastatic incidence in c3h and c3h / gld mice were carried out using a 2 2 contingency table of the number of mice with metastases versus the number of mice without metastases using a one - sided analysis . statistical analyses of the number of metastases in c3h versus c3h / gld mice were carried out using a mann - whitney test . if lung - derived fasl elicits the apoptosis of nonmetastatic k1735 tumors in vivo , then metastatic k1735 tumors should lack fas expression or demonstrate a resistance toward fas - mediated apoptosis while their nonmetastatic counterparts should retain fas sensitivity . to test this premise , we examined fas expression and the ability to induce fasl - mediated cell death in vitro in the parental k1735 tumor ( k1735p ) and five clonally derived tumor variants with well - defined metastatic properties . k1735p ( 13 ) and the k1735 c-23 variant ( 14 ) rarely metastasize from a subcutaneously growing tumor site and are considered to have low metastatic properties . in contrast , the k1735 clones designated sw-1 and m-2 ( 15 ) are highly metastatic to the lung , whereas their counterparts k1735 c-10 and c-19 are essentially nonmetastatic ( 14 ) . as shown in fig . 1 a , fas is highly expressed on both the low and nonmetastatic k1735 tumors ( k1735p , c-23 , c-10 and c-19 ) , whereas the highly metastatic clones sw-1 and m-2 lack fas expression . although a striking correlation existed between metastatic behavior and loss of fas for the highly metastatic k1735 clones , fas was equivalently expressed on both the low and nonmetastatic k1735 tumors . however , fas expression does not necessarily predict cellular sensitivity to fasl - induced apoptosis ( 8 , 9 ) . to determine whether metastatic behavior in the low and nonmetastatic k1735 clones correlated with fas sensitivity , tumor targets were cr - labeled and incubated with a fasl - expressing effector cell ( 22 ) , and cytotoxicity was determined as described in materials and methods . as shown in fig . the nonmetastatic clones c-19 and c-10 were most sensitive to fasl - induced death , whereas the poorly metastatic tumors k1735p and c-23 were significantly more resistant to such cytotoxicity . in three out of three experiments , the low metastatic k1735 tumors were three- to fivefold more resistant to fasl - induced cytotoxicity than were their metastatic counterparts . as expected , the highly metastatic m-2 and sw-1 clones lacking fas expression were insensitive to fasl - mediated killing . an identical sensitivity profile was observed when anti fas - mediated killing was determined . c. therefore , fas sensitivity rather than fas expression appears to uniquely differentiate among those tumors that are highly metastatic , poorly metastatic , and nonmetastatic . the observation that fas sensitivity and metastatic tumor behavior are inversely correlated suggested a causal effect of fas loss - of - function on tumor progression . although fasl mrna is expressed in the lung ( 2 ) , there is scant knowledge about the distribution of fasl in this organ . to this end , we carried out immunohistochemical staining of normal murine lung using a fasl - specific antibody . 2 , a and b , the majority of fasl is expressed on lung endothelial cells , with rare staining observed in type ii pneumocytes as previously reported for human lung tissue ( 26 ) . to determine the bioactivity of lung - expressed fasl , lcm was prepared as previously described ( 24 ) . we have previously reported that lcm contains a cytotoxic factor that differentially induces cell death in nonmetastatic k1735 tumors while sparing highly metastatic tumor variants ( 20 ) . to ascertain whether such cytotoxicity was attributable to fasl , we examined lcm bioactivity against the nonmetastatic k1735 variant c-19 ( 14 ) . as shown in fig . 3 , potent , specific fasl - mediated cytotoxicity was observed in bioactive lcm preparations . approximately 50% of control k1735 c-19 cells were nonviable after a 48-h culture in lcm . fas - fc , a competitive inhibitor of fasl binding ( 25 ) , almost completely inhibited lcm - mediated killing of the k1735 c-19 cells . however , in the presence of a non - fasl binding control such as the lymphotoxin receptor fc ( ltr - fc ) , no such reversal of lcm cytotoxicity was observed . an intense band of 40 kd and an additional 32-kd mol mass form representing the nonglycosylated form of fasl ( 27 ) were consistently observed in bioactive lcm preparations ( fig . other immunoreactive lower molecular weight bands , possibly fasl degradation products , were observed in some preparations ( fig . 2 c , lane 3 ) . in nonbioactive lcm preparations subjected to multiple freeze . 2 c , lane 2 ) , substantiating a direct relationship between lcm bioactivity and fasl protein . if tumor fas loss - of - function contributes to the development of the metastatic phenotype , then uncoupling fas fasl interactions should permit the metastatic outgrowth of fas - sensitive tumors in the lung . to this end , we evaluated the metastatic behavior of the fas - sensitive k1735 tumor and clones k1735p , c-23 , and c-19 ( fig . 1 ) in normal ( c3h ) and fasl - deficient mice ( c3h / gld ) . k1735 tumors were initially injected into the subcutis of both c3h or c3h / gld mice and regional growth was monitored . although palpable tumors were usually detected earlier in c3h / gld mice than in c3h control mice , measurable tumor growth rates were nearly identical between the two strains . , the mean tumor weights at the time of surgical resection 28 d after injection were not statistically different between c3h and c3h / gld mice for any of the tumors ( table 1 ) . however , the incidence and number of spontaneous lung metastases were increased for all three tumors in mice lacking host - derived fasl . the most striking enhancement of metastatic incidence and number of metastases were observed using the k1735p tumor ( fig . this tumor showed macroscopic lung metastases in 9 out of 10 c3h / gld mice ( median 18 , range 049 ) , whereas only 3 out of 10 wild - type c3h mice showed evidence of metastatic deposits ( median 0 , range 025 ) . the increased incidence and number of metastases using the k1735p tumor was highly significant in c3h / gld mice compared to c3h controls ( difference in incidence between c3h and c3h / gld p = 0.0003 using a one - sided test ; difference in number of lung metastases p < 0.0001 using a mann - whitney test ) . we next examined the metastatic growth of the clonally derived c-23 variant that , like k1735p , is considered to have low metastatic properties in c3h mice . as shown in table 1 , c-23 was metastatic in five out of seven c3h / gld mice ( median 2 , range 010 ) , but only one out of five c3h mice ( median 0 , range 020 ) , reflecting a significantly increased incidence of metastasis in c3h / gld mice compared with wild - type controls ( difference in incidence between c3h and c3h/ gld mice p = 0.039 , a one - sided test ) . to address the effects of fasl loss - of - function on a nonmetastatic tumor , we determined lung metastasis of the c-19 clone . as shown in table 1 , the c-19 clone was spontaneously metastatic in two out of five c3h / gld mice ( range 016 ) , whereas one out of five c3h mice showed evidence of lung deposits ( range 01 ) . although not statistically significant with the number of animals used for this experiment , these findings again support a trend for increased incidence and number of metastases in fasl - deficient animals . moreover , these data suggest that the loss of fasl is sufficient for acquisition of the metastatic phenotype in a nonmetastatic tumor . a potential criticism of such experiments is that the gld host microenvironment is more permissive for metastatic tumor growth irrespective of the loss of fasl . if this were the case , then fas - insensitive , metastatic tumors such as m-2 might also demonstrate increased metastatic capacity in c3h / gld animals . to test this premise , lung metastatic growth of m-2 was evaluated in c3h and c3h / gld mice . as shown in table 1 , lung metastases were not increased in fasl - deficient mice compared with wild - type mice ( difference in lung metastases p = 0.421 , mann - whitney test ) documenting that the gld lung environment per se is not inherently more permissive for metastatic tumor outgrowth . taken together , these results demonstrate that uncoupling of fas fasl interactions can enhance tumor metastasis to organ sites constitutively expressing fasl ( k1735p , c-23 ) and may be sufficient to confer metastatic phenotype in a nonmetastatic clone ( c-19 ) . a large body of evidence supports the concept that tumor cell growth and metastasis are dependent upon the interactions of the malignant cell and the host organ environment ( 17 , 18 ) . our studies are commensurate with the interpretation that the interactions of fas and fasl on the tumor and host environment , respectively , are critical determinants of tumor cell survival and growth in the metastatic lung site . although the successful establishment of metastatic lesions has been largely correlated with tumor responsiveness to paracrine growth factors released in specific organ sites ( 17 , 18 ) , our data indicate an additional role for apoptosis - inducing factors present in the specific microenvironment . as fas - sensitive tumor cells would undergo apoptosis in organ sites constitutively expressing high levels of fasl , we expect a priori that metastatic k1735 tumors preferentially colonizing the lung would be resistant to fas - mediated apoptosis . in this report , we document that four out of four metastatic k1735 melanoma clones show a loss of fas expression and/or function compared with two nonmetastatic clones . we have observed a similar loss of fas expression and function in the highly metastatic k1735 variants c-4 and c-2 ( reference 14 and data not shown ) . the highly metastatic lung - derived clones sw-1 and m-2 demonstrated a complete loss of both fas expression and function , whereas the intermediate metastatic clones k1735p and c-23 showed a significantly decreased fas sensitivity ( fig . 1 ) . that fas sensitivity was diminished but not entirely absent in k1735p and c-23 clones with low metastatic capacity implies that even a partial loss of fas function may result in significant biologic consequences , as has been reported for tumor development in lpr mice where fas expression is diminished ( 5 ) . when fas fasl interactions are completely disrupted , such tumors may become significantly more metastatic ( table 1 ) . initiation of fas - mediated apoptosis is complex and involves , at a minimum , levels of fas sufficient to insure optimal receptor cross - linking , the assembly of critical intracellular fas - associated proteins necessary to initiate the apoptotic cascade , and the presence or absence of resistance proteins such as bcl - xl , fas - associated phosphatase 1 ( fap-1 ) , and flice inhibitory protein ( flip ) ( 9 , 2830 ) . in this respect , a balance between functional tumor - expressed fas and host - derived fasl may be critical parameters regulating the capacity of a particular tumor to grow at a specified site . for example , fas expression is absent on the highly metastatic tumors k1735 sw-1 and m-2 , precluding initiation of the apoptotic cascade by fasl cross - linking . although fas is highly expressed on low metastatic clones k1735p and c-23 , postreceptor alterations in fas signaling may prevent optimal initiation of the apoptotic cascade in response to fasl ( fig . 1 , a and b ) . similarly , the relative abundance of fasl in the microenvironment may influence the capacity of a given tumor to survive or undergo apoptosis in disparate organ sites . in support of this premise , constitutive fasl expression has been reported to be considerably higher in the murine lung than in the skin ( 3 ) , providing a cogent explanation for the apparent conundrum that the fas - sensitive k1735 tumors grew comparably in the subcutis of c3h and c3h / gld mice but were significantly more metastatic in the lung microenvironment . although we favor the interpretation that an absence of fasl in the lung microenvironment permitted the selective outgrowth of the fas - sensitive k1735 tumors in c3h/ gld mice , it is conceptually possible that an absence of fasl - positive immune effector cells encountered in the blood and lymphatics was also contributory ( 3133 ) . although the latter possibility seems unlikely in view of the fact that viable , nonmetastatic tumor cells reach the lung microenvironment in normal mice ( 16 ) , we are formally testing this premise using bone marrow chimeras . in summary , our studies provide the first evidence that host - derived fasl can inhibit the malignant potential of fas - sensitive tumor cells and suppress organ - specific metastasis . the critical involvement of fas fasl in regulation of the metastatic cascade further underscores the importance of these proteins in homeostasis and prompts consideration of strategies aimed toward modification of the tumor fas expression and sensitivity of k1735 melanoma clones . ( a ) flow cytometric analyses were carried out as described in materials and methods using a fas - specific antibody ( stippled line ) and an isotype - matched control ( solid line ) . ( b ) fasl - mediated lysis of k1735 clones in overnight cr - release assay as described in materials and methods . values represent mean sem of quadruplicate determinations within a single experiment . c10 and c19 killing were shown to be statistically different from that of k1735p and c-23 using student 's t test ( c-10 versus k1735p , p = 0.017 ; c-10 versus k1735p , p = 0.003 ; c-10 versus c-23 , p = 0.010 ; c-19 versus c-23 , p = 0.016 ) for the data shown . ( c ) anti - fas mediated killing of k1735 clones as described in materials and methods . data are representative of two to five independent determinations for fasl and anti - fas mediated killing of k1735 clones . are staining of murine lung using isotype - matched control ( a ) and fasl - specific antibody ( b ) visualized by peroxidase - labeled secondary antibodies as described in materials and methods . lcm was subjected to western blotting using a fasl - specific antibody as described in materials and methods . lane 1 , control lysate from endothelial cells ( santa cruz biotechnologies , santa cruz , ca ) ; lane 2 , lcm preparation subjected to multiple freeze thaw cycles lacking fasl bioactivity ; lane 3 , lcm preparation used for the bioactivity assay shown in fig . loss of k1735 c-19 viability after lcm culture is reversed by fas - fc . cells were plated in media supplemented with 0.3% fetal bovine serum and 65 g / ml lcm . fc ( ltr - fc ) were added at 200 ng / ml and relative cellular viability was determined at 48 h using the tetrazolium reduction ( mtt ) assay . values shown were normalized using values of cells cultured in media supplemented with 0.3% fetal bovine serum alone as background . enhanced metastasis of k1735p melanoma cells in fasl - deficient ( c3h / gld ) mice . ( a ) 5 10 k1735p cells were injected subcutaneously into the flank of c3h and c3h / gld mice . at indicated times ( b ) gross metastatic tumor growth in two representative lungs from c3h / gld ( left ) and c3h control mice ( right ) subcutaneously injected with k1735p cells . metastasis of k1735 clones in fasl - deficient ( c3h / gld ) mice cells were injected subcutaneously into mice and subcutaneous tumors were removed at 1.5-cm diameter and weighed . all mice injected had local tumor growth ; lung metastatic growth was determined by light microscopy at 5 wk after removal of subcutaneous tumor or when mice were moribund . significant difference in incidence of metastases between c3h and c3h / gld mice by test ( p = 0.003 for k1735p ; p = 0.039 for c23 ) . significant difference in number of metastases between c3h and c3h / gld , p < 0.0001 , mann - whitney test .

apoptosis induced by fas ( cd95 ) ligation is frequently lost during tumor progression ; however , there is no direct evidence to support an association of fas loss - of - function with metastatic tumor behavior . to determine whether fas loss - of - function is critical for acquisition of the metastatic phenotype , we have compared the ability of fas - sensitive k1735 murine melanomas to form spontaneous lung metastases in wild - type and fas ligand deficient mice . fas - sensitive melanoma clones are highly tumorigenic but rarely metastatic in wild - type syngeneic mice . however , in fas ligand deficient mice , both the incidence and number of metastases are increased . these findings provide the first evidence that fas fas ligand interactions can suppress metastasis and that tumor fas loss - of - function may be causally linked to metastatic progression .

Materials and Methods Fas Expression on Murine Melanoma Clones. Fas Sensitivity of Murine Melanoma Clones. Bioassay for FasL in Lung-conditioned Medium and FasL Detection by Western Blotting. Detection of FasL in Murine Lung Sections by Immunohistochemistry. Tumor Growth and Metastasis In Vivo. Results and Discussion Figures and Tables

hamster anti murine fas ( jo2 ) was purchased from pharmingen ( san diego , ca ) . after 1618 h incubation at 37c in a 5% co2 atmosphere , 100-l aliquots of cell - free supernatant were harvested and radioactivity was measured using an lkb gamma counter ( amersham pharmacia biotech , piscataway , nj ) . lytic activity of activated 3a9 cells was blocked by the inclusion of fas - fc ( data not shown ) ; unactivated 3a9 cells demonstrated no lytic activity . anti - fas killing was carried out by plating tumor cells at 5 10/well in triplicate and adding 100 ng / ml biotinylated anti - fas ( jo-2 clone ; pharmingen ) or biotinylated isotype - matched control antibody ( pharmingen ) for 30 min at 37c then adding 200 ng / ml streptavidin ( jackson immunoresearch labs . values shown represent the percentage of specific cytotoxicity in anti - fas treated wells after subtracting that of the biotinylated , isotype - matched controls [ ( 1 mean anti - fas)/(mean isotype - matched control ) ] 100 . lung conditioned medium ( lcm ) was prepared according to the method of szaniaswska et al . background cell death was determined by culturing cells in media supplemented with 0.3% fetal bovine serum alone . specificity of lcm killing was shown by inclusion of either fas - fc ( 25 ) or isotype control fc ( ltr - fc ) at 200 ng / ml for the 48-h culture period . for this procedure , 10-g aliquots of lcm fasl was visualized by hybridization with a monoclonal anti - fasl ( clone 33 ; transduction laboratories , lexington , ky ) and anti mouse horseradish peroxidase was detected using the ecl system ( amersham pharmacia biotech , little chalfont , buckinghamshire , uk ) . sections were then incubated with fasl - specific antibody , n-20 ( santa cruz biotechnologies , santa cruz , ca ) , at a 1:800 dilution in blocking solution overnight at 4c . at 1:500 in blocking solution and stable diaminobenzidine ( research genetics , inc . for the determination of spontaneous metastases , metastatic lung growth was determined by visual inspection using light microscopy 46 wk later . animal experimentation was performed in accordance with institutional guidelines and the regulations and standards of the usda . statistical analyses of metastatic incidence in c3h and c3h / gld mice were carried out using a 2 2 contingency table of the number of mice with metastases versus the number of mice without metastases using a one - sided analysis . statistical analyses of the number of metastases in c3h versus c3h / gld mice were carried out using a mann - whitney test . hamster anti murine fas ( jo2 ) was purchased from pharmingen ( san diego , ca ) . 3a9 cells were activated by exposure to 10 ng / ml pma ( sigma chemical co. , st . lytic activity of activated 3a9 cells was blocked by the inclusion of fas - fc ( data not shown ) ; unactivated 3a9 cells demonstrated no lytic activity . anti - fas killing was carried out by plating tumor cells at 5 10/well in triplicate and adding 100 ng / ml biotinylated anti - fas ( jo-2 clone ; pharmingen ) or biotinylated isotype - matched control antibody ( pharmingen ) for 30 min at 37c then adding 200 ng / ml streptavidin ( jackson immunoresearch labs . values shown represent the percentage of specific cytotoxicity in anti - fas treated wells after subtracting that of the biotinylated , isotype - matched controls [ ( 1 mean anti - fas)/(mean isotype - matched control ) ] 100 . specificity of lcm killing was shown by inclusion of either fas - fc ( 25 ) or isotype control fc ( ltr - fc ) at 200 ng / ml for the 48-h culture period . sections were then incubated with fasl - specific antibody , n-20 ( santa cruz biotechnologies , santa cruz , ca ) , at a 1:800 dilution in blocking solution overnight at 4c . , c3h / gld and c3h / wt mice were purchased from the jackson laboratory ( bar harbor , me ) . before injection 10 cells were injected intravenously into the lateral tail vein and metastatic lung tumor growth was enumerated by visual inspection using light microscopy 35 wk after injection . animal experimentation was performed in accordance with institutional guidelines and the regulations and standards of the usda . statistical analyses of metastatic incidence in c3h and c3h / gld mice were carried out using a 2 2 contingency table of the number of mice with metastases versus the number of mice without metastases using a one - sided analysis . statistical analyses of the number of metastases in c3h versus c3h / gld mice were carried out using a mann - whitney test . if lung - derived fasl elicits the apoptosis of nonmetastatic k1735 tumors in vivo , then metastatic k1735 tumors should lack fas expression or demonstrate a resistance toward fas - mediated apoptosis while their nonmetastatic counterparts should retain fas sensitivity . to test this premise , we examined fas expression and the ability to induce fasl - mediated cell death in vitro in the parental k1735 tumor ( k1735p ) and five clonally derived tumor variants with well - defined metastatic properties . in contrast , the k1735 clones designated sw-1 and m-2 ( 15 ) are highly metastatic to the lung , whereas their counterparts k1735 c-10 and c-19 are essentially nonmetastatic ( 14 ) . 1 a , fas is highly expressed on both the low and nonmetastatic k1735 tumors ( k1735p , c-23 , c-10 and c-19 ) , whereas the highly metastatic clones sw-1 and m-2 lack fas expression . although a striking correlation existed between metastatic behavior and loss of fas for the highly metastatic k1735 clones , fas was equivalently expressed on both the low and nonmetastatic k1735 tumors . however , fas expression does not necessarily predict cellular sensitivity to fasl - induced apoptosis ( 8 , 9 ) . to determine whether metastatic behavior in the low and nonmetastatic k1735 clones correlated with fas sensitivity , tumor targets were cr - labeled and incubated with a fasl - expressing effector cell ( 22 ) , and cytotoxicity was determined as described in materials and methods . as shown in fig . an identical sensitivity profile was observed when anti fas - mediated killing was determined . c. therefore , fas sensitivity rather than fas expression appears to uniquely differentiate among those tumors that are highly metastatic , poorly metastatic , and nonmetastatic . the observation that fas sensitivity and metastatic tumor behavior are inversely correlated suggested a causal effect of fas loss - of - function on tumor progression . although fasl mrna is expressed in the lung ( 2 ) , there is scant knowledge about the distribution of fasl in this organ . to this end , we carried out immunohistochemical staining of normal murine lung using a fasl - specific antibody . 2 , a and b , the majority of fasl is expressed on lung endothelial cells , with rare staining observed in type ii pneumocytes as previously reported for human lung tissue ( 26 ) . to determine the bioactivity of lung - expressed fasl , lcm was prepared as previously described ( 24 ) . we have previously reported that lcm contains a cytotoxic factor that differentially induces cell death in nonmetastatic k1735 tumors while sparing highly metastatic tumor variants ( 20 ) . to ascertain whether such cytotoxicity was attributable to fasl , we examined lcm bioactivity against the nonmetastatic k1735 variant c-19 ( 14 ) . fas - fc , a competitive inhibitor of fasl binding ( 25 ) , almost completely inhibited lcm - mediated killing of the k1735 c-19 cells . however , in the presence of a non - fasl binding control such as the lymphotoxin receptor fc ( ltr - fc ) , no such reversal of lcm cytotoxicity was observed . in nonbioactive lcm preparations subjected to multiple freeze . if tumor fas loss - of - function contributes to the development of the metastatic phenotype , then uncoupling fas fasl interactions should permit the metastatic outgrowth of fas - sensitive tumors in the lung . to this end , we evaluated the metastatic behavior of the fas - sensitive k1735 tumor and clones k1735p , c-23 , and c-19 ( fig . 1 ) in normal ( c3h ) and fasl - deficient mice ( c3h / gld ) . although palpable tumors were usually detected earlier in c3h / gld mice than in c3h control mice , measurable tumor growth rates were nearly identical between the two strains . , the mean tumor weights at the time of surgical resection 28 d after injection were not statistically different between c3h and c3h / gld mice for any of the tumors ( table 1 ) . however , the incidence and number of spontaneous lung metastases were increased for all three tumors in mice lacking host - derived fasl . the most striking enhancement of metastatic incidence and number of metastases were observed using the k1735p tumor ( fig . this tumor showed macroscopic lung metastases in 9 out of 10 c3h / gld mice ( median 18 , range 049 ) , whereas only 3 out of 10 wild - type c3h mice showed evidence of metastatic deposits ( median 0 , range 025 ) . the increased incidence and number of metastases using the k1735p tumor was highly significant in c3h / gld mice compared to c3h controls ( difference in incidence between c3h and c3h / gld p = 0.0003 using a one - sided test ; difference in number of lung metastases p < 0.0001 using a mann - whitney test ) . we next examined the metastatic growth of the clonally derived c-23 variant that , like k1735p , is considered to have low metastatic properties in c3h mice . as shown in table 1 , c-23 was metastatic in five out of seven c3h / gld mice ( median 2 , range 010 ) , but only one out of five c3h mice ( median 0 , range 020 ) , reflecting a significantly increased incidence of metastasis in c3h / gld mice compared with wild - type controls ( difference in incidence between c3h and c3h/ gld mice p = 0.039 , a one - sided test ) . to address the effects of fasl loss - of - function on a nonmetastatic tumor , we determined lung metastasis of the c-19 clone . as shown in table 1 , the c-19 clone was spontaneously metastatic in two out of five c3h / gld mice ( range 016 ) , whereas one out of five c3h mice showed evidence of lung deposits ( range 01 ) . although not statistically significant with the number of animals used for this experiment , these findings again support a trend for increased incidence and number of metastases in fasl - deficient animals . moreover , these data suggest that the loss of fasl is sufficient for acquisition of the metastatic phenotype in a nonmetastatic tumor . a potential criticism of such experiments is that the gld host microenvironment is more permissive for metastatic tumor growth irrespective of the loss of fasl . if this were the case , then fas - insensitive , metastatic tumors such as m-2 might also demonstrate increased metastatic capacity in c3h / gld animals . as shown in table 1 , lung metastases were not increased in fasl - deficient mice compared with wild - type mice ( difference in lung metastases p = 0.421 , mann - whitney test ) documenting that the gld lung environment per se is not inherently more permissive for metastatic tumor outgrowth . taken together , these results demonstrate that uncoupling of fas fasl interactions can enhance tumor metastasis to organ sites constitutively expressing fasl ( k1735p , c-23 ) and may be sufficient to confer metastatic phenotype in a nonmetastatic clone ( c-19 ) . a large body of evidence supports the concept that tumor cell growth and metastasis are dependent upon the interactions of the malignant cell and the host organ environment ( 17 , 18 ) . our studies are commensurate with the interpretation that the interactions of fas and fasl on the tumor and host environment , respectively , are critical determinants of tumor cell survival and growth in the metastatic lung site . as fas - sensitive tumor cells would undergo apoptosis in organ sites constitutively expressing high levels of fasl , we expect a priori that metastatic k1735 tumors preferentially colonizing the lung would be resistant to fas - mediated apoptosis . in this report , we document that four out of four metastatic k1735 melanoma clones show a loss of fas expression and/or function compared with two nonmetastatic clones . we have observed a similar loss of fas expression and function in the highly metastatic k1735 variants c-4 and c-2 ( reference 14 and data not shown ) . that fas sensitivity was diminished but not entirely absent in k1735p and c-23 clones with low metastatic capacity implies that even a partial loss of fas function may result in significant biologic consequences , as has been reported for tumor development in lpr mice where fas expression is diminished ( 5 ) . initiation of fas - mediated apoptosis is complex and involves , at a minimum , levels of fas sufficient to insure optimal receptor cross - linking , the assembly of critical intracellular fas - associated proteins necessary to initiate the apoptotic cascade , and the presence or absence of resistance proteins such as bcl - xl , fas - associated phosphatase 1 ( fap-1 ) , and flice inhibitory protein ( flip ) ( 9 , 2830 ) . in this respect , a balance between functional tumor - expressed fas and host - derived fasl may be critical parameters regulating the capacity of a particular tumor to grow at a specified site . for example , fas expression is absent on the highly metastatic tumors k1735 sw-1 and m-2 , precluding initiation of the apoptotic cascade by fasl cross - linking . although fas is highly expressed on low metastatic clones k1735p and c-23 , postreceptor alterations in fas signaling may prevent optimal initiation of the apoptotic cascade in response to fasl ( fig . in support of this premise , constitutive fasl expression has been reported to be considerably higher in the murine lung than in the skin ( 3 ) , providing a cogent explanation for the apparent conundrum that the fas - sensitive k1735 tumors grew comparably in the subcutis of c3h and c3h / gld mice but were significantly more metastatic in the lung microenvironment . although we favor the interpretation that an absence of fasl in the lung microenvironment permitted the selective outgrowth of the fas - sensitive k1735 tumors in c3h/ gld mice , it is conceptually possible that an absence of fasl - positive immune effector cells encountered in the blood and lymphatics was also contributory ( 3133 ) . although the latter possibility seems unlikely in view of the fact that viable , nonmetastatic tumor cells reach the lung microenvironment in normal mice ( 16 ) , we are formally testing this premise using bone marrow chimeras . in summary , our studies provide the first evidence that host - derived fasl can inhibit the malignant potential of fas - sensitive tumor cells and suppress organ - specific metastasis . the critical involvement of fas fasl in regulation of the metastatic cascade further underscores the importance of these proteins in homeostasis and prompts consideration of strategies aimed toward modification of the tumor fas expression and sensitivity of k1735 melanoma clones . ( a ) flow cytometric analyses were carried out as described in materials and methods using a fas - specific antibody ( stippled line ) and an isotype - matched control ( solid line ) . loss of k1735 c-19 viability after lcm culture is reversed by fas - fc . at indicated times ( b ) gross metastatic tumor growth in two representative lungs from c3h / gld ( left ) and c3h control mice ( right ) subcutaneously injected with k1735p cells . metastasis of k1735 clones in fasl - deficient ( c3h / gld ) mice cells were injected subcutaneously into mice and subcutaneous tumors were removed at 1.5-cm diameter and weighed . significant difference in incidence of metastases between c3h and c3h / gld mice by test ( p = 0.003 for k1735p ; p = 0.039 for c23 ) . significant difference in number of metastases between c3h and c3h / gld , p < 0.0001 , mann - whitney test .

soybean is one of the most important crops in the world , and brazil is its second largest producer . due to its use in animal feed , vegetable oil and biodiesel nevertheless , abiotic stresses , such as water deficit , cold , wind , heat or waterlogging , reduce productivity and present a major challenge in the quest for sustainable soybean production . in order to meet the ever - increasing demand , crop yield is highly affected by water deficit stress , particularly when it occurs during flowering and early pod expansion ( pedersen et al . , 2005 ) . however , soybean plants can acclimate to such conditions by triggering protective mechanisms that enable these plants to survive subsequent events of drought ( bray , 2004 ) . while studying soybean plants exposed to stress at different developmental stages , kron et al . ( 2008 ) observed that plant responses were more uniform in the v4 stage ( fehr et al . , 1971 ) . the genetic background of the cultivars br 16 ( drought sensitive ) and embrapa 48 ( drought tolerant ) under drought conditions was studied by oya et al . ( 2004 ) , and they found that at the vegetative stage , tolerant cultivars present higher growth rate and a larger leaf area compared to sensitive plants . however , to better understand plant physiology under stress , productivity analyses are hardly enough to distinguish significant tolerance differences among cultivars . the data indicate how cultivars behave under stress and , therefore , suggest which ones are suitable for further biochemical and molecular studies . a stress response is initiated when plants recognize the stress at the cellular level , it triggers a complex signal transduction network which is modulated by molecular events . gene products involved in water - deficit responses can be classified into two groups : functional or regulatory genes . functional genes include the following : proteins , osmolytes , chaperones , heat - shock proteins ( hsps ) , water - channel proteins ( multifunctional proteins involved in transport facilitation of solutes and water in leaves and roots ) , and other compounds probably conferring direct tolerance to abiotic stresses . under water stress conditions , a chemical imbalance in the cell leads to production of reactive oxygen species ( ros ) , which are damaging to lipids and proteins ( carvalho , 2008 ; molina et al . , enzymes involved in detoxification metabolism are found to protect against the stress in the tolerant common bean cultivar ( phaseolus acutifolius ) ( trkan et al . , 2005 ) . in drought - stressed tissues , the water movement within and between cells can be facilitated by aquaporins ( cocozza et al . , 2010 ) . the second group of genes involved in the response to water deficit includes molecules involved in further regulation of signal transduction and stress - responsive gene expression . these molecules are regulatory proteins , represented by various transcription factors such as dreb1 , aba - responsive element , nitrogen assimilation control protein , ethylene - response factors , kinases , phosphatases , enzymes involved in phospholipid metabolism , and other signaling molecules , such as calmodulin - binding protein ( shinozaki and yamaguchi - shinozaki , 2007 ) . in the peanut , a predicted drought tolerant legume , a set of functional and regulatory genes were detected in enriched subtractive libraries probably acting in a gradual process of drought acclimation ( govind et al . , 2009 ) . as discussed , the ability to tolerate water deficit is a complex trait controlled by many genes ( molina et al . , 2008 ; ergen and budak , 2009 ) . collections of cdnas expressed under stressed conditions have been a useful resource to study gene expression and regulation . the cdna libraries were associated to normalization steps and pcr amplification to obtain just the transcripts presented in a target situation ( diatchenko et al . , 1996 ) . a suppressive subtractive library approach makes it possible to search for genes expressed in treated samples but not in the control samples , if the forward subtraction is done . it can be used to assess changes in metabolism and in quantitative gene expression studies , such as those based on arrays . the method has been employed successfully in different research areas , such as the monitoring of citrus responses to fungus infection ( gonzlez - candelas et al . , 2010 ) and in fish infected by iridovirus ( xu et al . , 2010 ) . using the subtracted library of pigeonpea , priyanka et al . ( 2010 ) isolated a gene encoding a hybrid - rich - proline protein , which affords tolerance to water deficit and other abiotic stresses such as salt , heat , cold and abscisic acid treatment . in addition , cdna subtractive libraries have also allowed the identification of genes involved in the flower development of orange plants ( zhang et al . , 2008 ) and of genes involved in the varroa destructor mite tolerance to honey bees , drought has resulted in severe losses in soybean productivity worldwide . in order to expand previous knowledge about the water stress - sensitive br 16 and water stress - tolerant embrapa 48 soybean cultivars , the present work aimed to study early responses in gene expression of leaves and roots under water stress . seeds of the soybean cultivars embrapa 48 and br 16 , classified as tolerant and sensitive to water deficit , respectively , were germinated on filter paper during four days in a growth chamber at 25 1 c of temperature and 100% relative humidity . seedlings were placed in 36 l boxes containing 50% hoagland s solution ( hoagland and arnon , 1950 ) , which was continuously aerated and replaced on a weekly basis . these boxes were then transferred to a greenhouse under a natural photoperiod of approximately 12/12 h light / dark cycle , temperature of 30 5 c and 60 10% relative humidity ( rh ) , where the plants were allowed to grow until the v4 stage ( fehr et al . , 1971 ) . the factors were two cultivars ( br 16 and embrapa 48 ) and seven times of water deficit ( 0 , 25 , 50 , 75 , 100 , 125 and 150 min ) . the stress was applied by removing the plants out of the hydroponic solution and leaving them in boxes without nutrient solution for up to 150 min under ambient - air exposure . for each time of water deficit , roots from 10 plants were collected , pooled and frozen in liquid nitrogen before storage at 80 c . photosynthetic rate ( a ) , stomatal conductance ( gs ) , intercellular co2 concentration ( ci ) , transpiration rate ( e ) and leaf temperature ( tl ) were evaluated using a li-6400 portable photosynthesis system ( licor , inc . ) . measurements were taken on the middle leaflet of the youngest soybean leaf , fully expanded , under photon flux density of 1.000 mol m s. the water use efficiency ( wue ) of plants was determined through the ratio between photosynthetic ( a ) and transpiration ( e ) rate . data was submitted to anova analysis through the programs sanest and sas ( statistical analysis system version 8.0 ) , and the treatment means were compared by the tukey test ( p < 0.05 ) . total rna was extracted from roots and leaves using trizol reagent ( invitrogen ) according to the manufacturer s instructions . samples from each biological replicate were extracted separately , and equimolar amounts of total rna were pooled before the mrna purification . the fasttrack mag mrna isolation ( invitrogen ) was applied to isolate the mrna poly a molecules by using oligo - dt conjugated magnetic beads . the mrna samples were pooled as illustrated in figure 1 to perform the cdna reverse transcription . libraries of roots and leaves from embrapa 48 and br 16 plants were constructed based on the method of diatchenko et al . first strand cdna was synthesized from 2 g of mrna poly a pooled samples ( tester and driver ) . after the second strand synthesis , cdnas ( tester and driver ) were digested with the restriction enzyme rsa i to produce blunt end fragments . once digested , the cdnas ( only tester samples ) were divided into two samples and each sample was ligated with two different adapters ( adaptor 1 and 2r , respectively ) . as our aim was to detect transcripts expressed only in water - deficit stressed plants , the sample t0 ( control plant ) was used as the driver in the hybridization step . the cdna driver ( with no adaptor ) was added to tester samples ( 1 and 2r ) and they were submitted to the first hybridization step to eliminate sequences common in both samples or present in the control ( driver ) . for the second hybridization , tester samples 1 and 2r from the first hybridization were mixed together , and the denatured cdna driver was added again to enrich the expressed sequences in the tester . the pcr amplification was performed using the double - strand cdnas with different adapters as template . the second pcr was carried out using the nested primers to enrich the differentially expressed sequences . the six libraries from br 16 cultivar ( three from roots and three from leaves ) were sequenced through 36 bp ( base pairs ) single reads in a genome analyzer lle machine . the embrapa 48 libraries were sequenced in a genome analyzer llz using single reads of 76 bp . after base - calling , reads were mapped on a reference genome ( schmutz et al . , 2010 ) using the soap ( li et al . , 2008 ) , and non - mapped reads were assembled into contigs through the edena program following the default parameters ( hernandez et al . , 2008 ) . contig sequences were submitted to the non - redundant protein database ncbi through blast x ( altschul et al . , 1997 ) , to search for similarity to known proteins . in addition , sequences were analyzed by the software autofact ( koski et al . , 2005 ) , which is an automated annotation tool that assigns biological information for a given sequence by comparing different databases . we used the uniref90 e uniref100 , kegg ( kanehisa and goto , 2000 ) , pfam ( finn et al . , 2010 ) , smart ( schultz et al . , 1998 ) databases . in order to establish the go ( gene ontology ) terms ( ashburner et al . , 2000 ) we employed the blast2go program to classify the sequences according to the molecular function and biological process described to the respective protein ( gtz et al . seeds of the soybean cultivars embrapa 48 and br 16 , classified as tolerant and sensitive to water deficit , respectively , were germinated on filter paper during four days in a growth chamber at 25 1 c of temperature and 100% relative humidity . seedlings were placed in 36 l boxes containing 50% hoagland s solution ( hoagland and arnon , 1950 ) , which was continuously aerated and replaced on a weekly basis . these boxes were then transferred to a greenhouse under a natural photoperiod of approximately 12/12 h light / dark cycle , temperature of 30 5 c and 60 10% relative humidity ( rh ) , where the plants were allowed to grow until the v4 stage ( fehr et al . , 1971 ) . the factors were two cultivars ( br 16 and embrapa 48 ) and seven times of water deficit ( 0 , 25 , 50 , 75 , 100 , 125 and 150 min ) . the stress was applied by removing the plants out of the hydroponic solution and leaving them in boxes without nutrient solution for up to 150 min under ambient - air exposure . for each time of water deficit , roots from 10 plants were collected , pooled and frozen in liquid nitrogen before storage at 80 c . photosynthetic rate ( a ) , stomatal conductance ( gs ) , intercellular co2 concentration ( ci ) , transpiration rate ( e ) and leaf temperature ( tl ) were evaluated using a li-6400 portable photosynthesis system ( licor , inc . ) . measurements were taken on the middle leaflet of the youngest soybean leaf , fully expanded , under photon flux density of 1.000 mol m s. the water use efficiency ( wue ) of plants was determined through the ratio between photosynthetic ( a ) and transpiration ( e ) rate . data was submitted to anova analysis through the programs sanest and sas ( statistical analysis system version 8.0 ) , and the treatment means were compared by the tukey test ( p < 0.05 ) . total rna was extracted from roots and leaves using trizol reagent ( invitrogen ) according to the manufacturer s instructions . samples from each biological replicate were extracted separately , and equimolar amounts of total rna were pooled before the mrna purification . the fasttrack mag mrna isolation ( invitrogen ) was applied to isolate the mrna poly a molecules by using oligo - dt conjugated magnetic beads . the mrna samples were pooled as illustrated in figure 1 to perform the cdna reverse transcription . libraries of roots and leaves from embrapa 48 and br 16 plants were constructed based on the method of diatchenko et al . first strand cdna was synthesized from 2 g of mrna poly a pooled samples ( tester and driver ) . after the second strand synthesis , cdnas ( tester and driver ) were digested with the restriction enzyme rsa i to produce blunt end fragments . once digested , the cdnas ( only tester samples ) were divided into two samples and each sample was ligated with two different adapters ( adaptor 1 and 2r , respectively ) . as our aim was to detect transcripts expressed only in water - deficit stressed plants , the sample t0 ( control plant ) was used as the driver in the hybridization step . the cdna driver ( with no adaptor ) was added to tester samples ( 1 and 2r ) and they were submitted to the first hybridization step to eliminate sequences common in both samples or present in the control ( driver ) . for the second hybridization , tester samples 1 and 2r from the first hybridization were mixed together , and the denatured cdna driver was added again to enrich the expressed sequences in the tester . the pcr amplification was performed using the double - strand cdnas with different adapters as template . the second pcr was carried out using the nested primers to enrich the differentially expressed sequences . the six libraries from br 16 cultivar ( three from roots and three from leaves ) were sequenced through 36 bp ( base pairs ) single reads in a genome analyzer lle machine . the embrapa 48 libraries were sequenced in a genome analyzer llz using single reads of 76 bp . after base - calling , reads were mapped on a reference genome ( schmutz et al . , 2010 ) using the soap ( li et al . , 2008 ) , and non - mapped reads were assembled into contigs through the edena program following the default parameters ( hernandez et al . , 2008 ) . contig sequences were submitted to the non - redundant protein database ncbi through blast x ( altschul et al . , 1997 ) , to search for similarity to known proteins . in addition , sequences were analyzed by the software autofact ( koski et al . , 2005 ) , which is an automated annotation tool that assigns biological information for a given sequence by comparing different databases . we used the uniref90 e uniref100 , kegg ( kanehisa and goto , 2000 ) , pfam ( finn et al . , 2010 ) , smart ( schultz et al . , 1998 ) databases . in order to establish the go ( gene ontology ) terms ( ashburner et al . , 2000 ) we employed the blast2go program to classify the sequences according to the molecular function and biological process described to the respective protein ( gtz et al . plants grown hydroponically were exposed to a short - term water deficit and the physiological parameters a , gs , ci , e , tl , troom and wue are presented in figure 2 . we employed the subtractive suppressive hybridization ( ssh ) technique to obtain an enriched collection of ests expressed early in response to water deficit stress . cdna libraries were constructed to detect genes up - regulated when compared to control plants . after sequencing and base calling , approximately 65% of the total reads produced for each library was mapped on the genome . contigs assembled were searched in the ncbi database in order to access the information about proteins . biochemical roles of the proteins were determined by using the biological process level 3 from gene ontology ( figure 3 ) . approximately 71% of the sequences in br 16 and 61% of embrapa 48 showed an associated putative biological or molecular function . consequently , the number of no match and unknown genes was larger in embrapa 48 than in br 16 plants . in br 16 , we observed 121 and 452 different roles for the genes expressed in the roots and leaves , respectively . in embrapa 48 categories employed in functional classification include the following : biological regulation ( gene expression regulation , protein modification regulation ) ; signaling ( kinases , phosphatases , secondary messengers ) ; response to stress ( water deficit , osmotic and salt stress , heat , cold ) ; response to stimulus ( chemical stimulus , endogenous stimulus ) ; response to biotic stimulus ( pathogen , fungus , insect ) ; cell organization ( cytoskeleton components , cell wall organization , cellular component assembly ) ; cell cycle ( actin filament - based process , cell division ) ; senescence ( aging , cell death ) ; other biological process ( cellular component organization , cell recognition , developmental process ) ; biosynthetic process ( carbohydrate , fatty acid , hormone , small and macromolecules , pigment , nucleotide ) ; catabolic process ( protein , fatty acid , cell wall , polysaccharide ) ; nitrogen compound metabolism ( glutamate and glutamine biosynthetic process , cellular nitrogen compound metabolic process ) ; oxidation reduction ( electron transport , lipid oxidation ) and secondary metabolism ( terpenoid , phytochelatin , phytoalexin and phenylpropanoid metabolic process ) . each category was normalized by the total number of sequences observed in the respective library , which enables comparison of the results during the time course of the water deficit ( figure 3 ) . applying the venn - master diagram , we also searched for genes expressed in more than one library as described in figure 4 . br 16 root tissue genes were categorized as involved in the electron transport process ( r1 and r2 libraries ) or as encoding proteins related to the stress response class ( r2-r3 library ) ( figure 4a ) . in br 16 leaves ( l1-l2 libraries ) , we also detected genes involved in signaling reactions or involved in the transport of metabolites and electrons ( oxidation reduction process ) ( figure 4b ) . in addition , the genes in the l2-l3 libraries were also related to stress response ( figure 4b ) . a general view of l1-l2-l3 libraries confirmed that genes functionally related to transport and stress responses were also expressed in leaves over the water deficit period ( figure 4b ) . it is important to emphasize that transcripts observed in r1-r2 , for example , were different from the transcripts found in r1-r2-r3 , although similar biological processes could be detected in both libraries . interestingly , in the embrapa 48 cultivar , transcription factors and genes involved in signal transduction were the most abundant transcripts in both r1 and r2 root tissues ( figure 4c ) . genes encoding transcription factors were also predominant in the r2-r3 libraries in addition to the ones related to transport process ( electrons and molecules ) . the same pattern was found in r1-r2-r3 libraries ( figure 4c ) . processes related to protein metabolism ( assembly , folding and modification ) were very active in l2-l3 libraries as well as genes involved in transport process , response to stress and signaling . stress response genes stand for 22% of the genes identified in l1-l2-l3 libraries ( figure 4d ) . we performed a comparison between genes expressed in roots and leaves in either cultivar using the venn - master diagram tool and looking for the biological processes observed in each group . when the same contig or glyma was detected in more than one library then , considering only the non - repeated genes from each group ( tissue or cultivar ) , we were able to observe genes expressed in root and leaves as well as genes expressed in embrapa 48 and br 16 plants ( figure 5a ) . to assess biological processes in roots and leaves of both cultivars , we highlighted the more abundant putative proteins and cellular components in each water - deficit stressed tissue ( figure 5b ) . according to our data , transcripts expressed by br 16 plants in roots and leaves showed that the most frequent biological processes were similar in both tissues ( figure 5b ) . however , in embrapa 48 , the process of response to stress was predominant among transcripts from leaves and less common in roots ( figure 5b ) . among genes expressed by plants in response to water deficit , we observed that transcripts classified in the response to stress category presented similarity to proteins with responsiveness to biotic as well as to abiotic stress . these transcripts represent proteins with antimicrobial activity or proteins involved in defense and immune responses . genes related to the response to virus , pathogenic bacterium , nematodes and fungi were also detected . under abiotic stress , we identified transcripts in response to osmotic and oxidative stress , desiccation , heat , cold , water deprivation , hyperosmotic salinity and high light intensity . to assess which transcripts are expressed in response to other adverse conditions , the genes placed in the response to stress category ( n = 458 ) were compared to transcripts from plants infected by phakopsora pachyrhizi , uromyces appendiculatus , virus and treated with bradirhyzobium japonicum ( figure 6 ) . plant water deficit responses and tolerance are complex biological processes that need to be analyzed at system - level using genomics and physiological approaches ( harb et al . , 2010 ) . studies aiming to detect differences among plants in response to stress using contrasting cultivars have been performed in some legumes ( trkan et al . , 2005 ) . empiric observations during growing seasons in the field are the first step to explore plant variability based on its yield under water deficit conditions . ( 2004 ) examined the physiological characteristics of the embrapa 48 and br 16 cultivars and determined that they were moderately tolerant and highly sensitive to drought , respectively . here , we studied these cultivars at the molecular level , and our data demonstrated that the water deficit applied was effective as it triggered changes in the plant physiological behavior ( figure 2 ) . as the stress increased in the plants , photosynthesis of both cultivars was affected and was strongly inhibited after 100 min . this inhibition was related to a drastic decrease in the stomatal conductance ( gs ) and transpiration rate ( e ) of both cultivars . we also observed an increase in the leaf temperature ( from 100 min ) and internal concentration of co2 ( from 125 min ) . the water deficit did not produce significant physiological differences between embrapa 48 and br 16 cultivars . this was expected as the short - term procedure to induce stress used here is usually not enough to produce effects on plant morphological and physiological characteristics needed to distinguish the two cultivars . the experimental treatment described here aimed to study the effects of the stress on the early plant responses at a molecular level . additionally , the hydroponic system allowed the obtainment of a clean root sample , as demonstrated by martins et al . as described in the literature , subtractive libraries have been employed in a wide range of studies such as gene expression studies ( kojima et al . , 2009 ; gonzlez - candelas et al . , 2010 ; jain and chattopadhyay , 2010 ; soria - guerra et al . , 2010 ) . most of these studies were based on the construction of a physical cdna collection by cloning fragments into a vector and on the traditional big dye sequencing technology . however , in vivo cloning can be considered a laborious and expensive procedure and also has technical limitations . in order to overcome these limitations , both cultivar cdnas were sequenced using two different chemistries based on sequencing by synthesis technology ( illumina ) . as shown in table 1 , we identified many genes being regulated under water deficit , more than is often detected through subtractive libraries . larger sets of genes were also detected in the characterization of stress - responsive genes to heat in wheat ( chauhan et al . , 2011 ) and by studying salt - induced genes of grapevine ( daldoul et al . , 2010 ) . both suppressive subtractive libraries were done through in vivo cloning and by the capillary sequencing method . ( 2010 ) found 12442 contigs in the transcriptome by combining subtractive libraries and massively parallel sequencing ( pyrosequencing ) . one of the advantages of the next generation sequencing ( ngs ) over the traditional sanger methodology is a more precise measurement of the transcript levels and isoforms , in addition to other revolutionary benefits ( marioni et al . , 2009 ; wang et al . , 2009 ) the advantages and limitations found in the ngs and capillary sequencing have led to comparative studies of both methods ( cheung et al . , 2008 ; hert et al . , 2008 ; tedersoo et al . , lower costs and quickness have made the ngs a powerful tool for functional genomics studies ( shendure and ji , 2008 ; harismendy et al . , 2009 ) . under water deficit different biochemical roles have been suggested for these responsive genes , and their expression patterns make an intricate network that can be related to either water deficit acclimation or the cellular damage response ( shinozaki and yamaguchi - shinozaki , 2007 ) . the expressed genes have been shown to belong to many classes of genes ( figure 3 ) . however , the largest class of genes expressed under stress remains unknown , partly because there is no similarity in databases available to date , and partly due to the gene functions are not understood yet ( reddy et al . many genes expressed under water deficit conditions are present in the contrasting cultivars embrapa 48 and br 16 ( figure 5a ) . among others , common processes affected by the stress are those related to transport , protein folding and assembling as well as protein degradation and ubiquitination . some proteins are synthesized in requirement of defense pathways triggered by the stress whereas other proteins are degraded because its aggregation or denaturation events are predominant in water stress conditions . the transport category involves the translocation of ions , lipids , sugar and others molecules . also included in this category are the genes for aquaporins , which are proteins that might be directly involved in water deficit acclimation by facilitating water movement across the membranes ( zhao et al . , 2008 ) . we have identified several transcripts encoding the tonoplast and plasma membrane intrinsic proteins that were more frequently found in roots rather than in leaves , especially in the br 16 plants ( figure 5b ) . roles played by aquaporins in plants can be regulated in different complex ways , but the expression of aquaporins throughout the plant would most likely be regulated by water potential gradient ( zhao et al . , 2008 ) . however , there are differences between the mrna changes and its respective protein level . the changes expressed here by soybean plants in response to water stress are only at the transcriptional level . br 16 plants did not show an expressive quantitative difference in the biological processes affected by water deficit between roots and leaves ( figure 5b ) . nonetheless , embrapa 48 did show a significant contrast between both tissues ( figure 5b ) regarding the genes included in the response to stress class ( water deficit , osmotic and salt stress , heat and cold ) . in abiotic stress responses , genes are induced to protect the plants against a disturbance in cell homeostasis . under water stress , plants can become susceptible to other abiotic or biotic stresses because their normal metabolism is directed towards the acclimation to the first stress . several studies have tried to make a connection between plant responses to water deficit and heat ( sakuma et al . , 2006 ; montero - barrientos et al . , 2010 ) , cold ( li et al . , 2010 ) , or reactive oxygen species enhanced due to drought ( kocsy et al . , 2005 ; khanna - chopra and selote , 2007 ) . in the response to stress category , we observed that various transcripts were similarly involved in several abiotic stresses such as osmotic stress , cold , heat and salinity as well as those involved in response to biotic stimulus ( bacterium , fungus , nematode , virus ) ( figure 6 ) . from subtracted libraries of pigeonpea , 2010 ) identified a hybrid - proline - rich encoding gene whose multiple stress - responsive characteristics have been demonstrated under peg , mannitol , salt and heat stress , cold and aba - treatment . another gene from pigeonpea , a cyclophilin gene , presented a stress - responsive nature in arabidopsis thaliana ( sekhar et al . , 2010 ) . a bzip transcription factor gene found in soybean plants was also associated with multiple stress responses , being its expression induced by aba - treatment , drought , high salt and low temperature ( gao et al . a hydroponic system was applied successfully to grow soybean plants and to produce clean roots in a short - term water deficit experiment . we described a large collection of cdnas expressed by soybean plants under water deficit and their respective biochemical processes . although the analyses presented here represent a first step in gene expression investigation , the results indicate changes which are triggered in roots and leaves of tolerant and sensitive soybean plants .

soybean has a wide range of applications in the industry and , due to its crop potential , its improvement is widely desirable . during drought conditions , soybean crops suffer significant losses in productivity . therefore , understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques . in this study , we employed the suppressive subtractive hybridization ( ssh ) technique to investigate differentially expressed genes under water deficit conditions . embrapa 48 and br 16 soybean lines , known as drought - tolerant and -sensitive , respectively , were grown hydroponically and subjected to different short - term periods of stress by withholding the nutrient solution . using this approach , we have identified genes expressed during the early response to water deficit in roots and leaves . these genes were compared among the lines to assess probable differences in the plant transcriptomes . in general , similar biochemical processes were predominant in both cultivars ; however , there were more considerable differences between roots and leaves of embrapa 48 . moreover , we present here a fast , clean and straightforward method to obtain drought - stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress .

Introduction Material and Methods Plant growth and water stress treatment Physiological parameters measurements cDNA subtracted libraries synthesis Data analysis Results Discussion

soybean is one of the most important crops in the world , and brazil is its second largest producer . due to its use in animal feed , vegetable oil and biodiesel nevertheless , abiotic stresses , such as water deficit , cold , wind , heat or waterlogging , reduce productivity and present a major challenge in the quest for sustainable soybean production . in order to meet the ever - increasing demand , crop yield is highly affected by water deficit stress , particularly when it occurs during flowering and early pod expansion ( pedersen et al . however , soybean plants can acclimate to such conditions by triggering protective mechanisms that enable these plants to survive subsequent events of drought ( bray , 2004 ) . while studying soybean plants exposed to stress at different developmental stages , kron et al . ( 2008 ) observed that plant responses were more uniform in the v4 stage ( fehr et al . the genetic background of the cultivars br 16 ( drought sensitive ) and embrapa 48 ( drought tolerant ) under drought conditions was studied by oya et al . ( 2004 ) , and they found that at the vegetative stage , tolerant cultivars present higher growth rate and a larger leaf area compared to sensitive plants . however , to better understand plant physiology under stress , productivity analyses are hardly enough to distinguish significant tolerance differences among cultivars . the data indicate how cultivars behave under stress and , therefore , suggest which ones are suitable for further biochemical and molecular studies . gene products involved in water - deficit responses can be classified into two groups : functional or regulatory genes . under water stress conditions , a chemical imbalance in the cell leads to production of reactive oxygen species ( ros ) , which are damaging to lipids and proteins ( carvalho , 2008 ; molina et al . , enzymes involved in detoxification metabolism are found to protect against the stress in the tolerant common bean cultivar ( phaseolus acutifolius ) ( trkan et al . in drought - stressed tissues , the water movement within and between cells can be facilitated by aquaporins ( cocozza et al . the second group of genes involved in the response to water deficit includes molecules involved in further regulation of signal transduction and stress - responsive gene expression . in the peanut , a predicted drought tolerant legume , a set of functional and regulatory genes were detected in enriched subtractive libraries probably acting in a gradual process of drought acclimation ( govind et al . the cdna libraries were associated to normalization steps and pcr amplification to obtain just the transcripts presented in a target situation ( diatchenko et al . a suppressive subtractive library approach makes it possible to search for genes expressed in treated samples but not in the control samples , if the forward subtraction is done . it can be used to assess changes in metabolism and in quantitative gene expression studies , such as those based on arrays . the method has been employed successfully in different research areas , such as the monitoring of citrus responses to fungus infection ( gonzlez - candelas et al . ( 2010 ) isolated a gene encoding a hybrid - rich - proline protein , which affords tolerance to water deficit and other abiotic stresses such as salt , heat , cold and abscisic acid treatment . in addition , cdna subtractive libraries have also allowed the identification of genes involved in the flower development of orange plants ( zhang et al . , 2008 ) and of genes involved in the varroa destructor mite tolerance to honey bees , drought has resulted in severe losses in soybean productivity worldwide . in order to expand previous knowledge about the water stress - sensitive br 16 and water stress - tolerant embrapa 48 soybean cultivars , the present work aimed to study early responses in gene expression of leaves and roots under water stress . seeds of the soybean cultivars embrapa 48 and br 16 , classified as tolerant and sensitive to water deficit , respectively , were germinated on filter paper during four days in a growth chamber at 25 1 c of temperature and 100% relative humidity . the factors were two cultivars ( br 16 and embrapa 48 ) and seven times of water deficit ( 0 , 25 , 50 , 75 , 100 , 125 and 150 min ) . the stress was applied by removing the plants out of the hydroponic solution and leaving them in boxes without nutrient solution for up to 150 min under ambient - air exposure . for each time of water deficit , roots from 10 plants were collected , pooled and frozen in liquid nitrogen before storage at 80 c . measurements were taken on the middle leaflet of the youngest soybean leaf , fully expanded , under photon flux density of 1.000 mol m s. the water use efficiency ( wue ) of plants was determined through the ratio between photosynthetic ( a ) and transpiration ( e ) rate . data was submitted to anova analysis through the programs sanest and sas ( statistical analysis system version 8.0 ) , and the treatment means were compared by the tukey test ( p < 0.05 ) . total rna was extracted from roots and leaves using trizol reagent ( invitrogen ) according to the manufacturer s instructions . libraries of roots and leaves from embrapa 48 and br 16 plants were constructed based on the method of diatchenko et al . once digested , the cdnas ( only tester samples ) were divided into two samples and each sample was ligated with two different adapters ( adaptor 1 and 2r , respectively ) . as our aim was to detect transcripts expressed only in water - deficit stressed plants , the sample t0 ( control plant ) was used as the driver in the hybridization step . the cdna driver ( with no adaptor ) was added to tester samples ( 1 and 2r ) and they were submitted to the first hybridization step to eliminate sequences common in both samples or present in the control ( driver ) . for the second hybridization , tester samples 1 and 2r from the first hybridization were mixed together , and the denatured cdna driver was added again to enrich the expressed sequences in the tester . the second pcr was carried out using the nested primers to enrich the differentially expressed sequences . the six libraries from br 16 cultivar ( three from roots and three from leaves ) were sequenced through 36 bp ( base pairs ) single reads in a genome analyzer lle machine . the embrapa 48 libraries were sequenced in a genome analyzer llz using single reads of 76 bp . , 2005 ) , which is an automated annotation tool that assigns biological information for a given sequence by comparing different databases . , 2000 ) we employed the blast2go program to classify the sequences according to the molecular function and biological process described to the respective protein ( gtz et al . seeds of the soybean cultivars embrapa 48 and br 16 , classified as tolerant and sensitive to water deficit , respectively , were germinated on filter paper during four days in a growth chamber at 25 1 c of temperature and 100% relative humidity . the factors were two cultivars ( br 16 and embrapa 48 ) and seven times of water deficit ( 0 , 25 , 50 , 75 , 100 , 125 and 150 min ) . the stress was applied by removing the plants out of the hydroponic solution and leaving them in boxes without nutrient solution for up to 150 min under ambient - air exposure . for each time of water deficit , roots from 10 plants were collected , pooled and frozen in liquid nitrogen before storage at 80 c . total rna was extracted from roots and leaves using trizol reagent ( invitrogen ) according to the manufacturer s instructions . libraries of roots and leaves from embrapa 48 and br 16 plants were constructed based on the method of diatchenko et al . once digested , the cdnas ( only tester samples ) were divided into two samples and each sample was ligated with two different adapters ( adaptor 1 and 2r , respectively ) . as our aim was to detect transcripts expressed only in water - deficit stressed plants , the sample t0 ( control plant ) was used as the driver in the hybridization step . the cdna driver ( with no adaptor ) was added to tester samples ( 1 and 2r ) and they were submitted to the first hybridization step to eliminate sequences common in both samples or present in the control ( driver ) . the second pcr was carried out using the nested primers to enrich the differentially expressed sequences . the six libraries from br 16 cultivar ( three from roots and three from leaves ) were sequenced through 36 bp ( base pairs ) single reads in a genome analyzer lle machine . the embrapa 48 libraries were sequenced in a genome analyzer llz using single reads of 76 bp . , 2000 ) we employed the blast2go program to classify the sequences according to the molecular function and biological process described to the respective protein ( gtz et al . plants grown hydroponically were exposed to a short - term water deficit and the physiological parameters a , gs , ci , e , tl , troom and wue are presented in figure 2 . we employed the subtractive suppressive hybridization ( ssh ) technique to obtain an enriched collection of ests expressed early in response to water deficit stress . contigs assembled were searched in the ncbi database in order to access the information about proteins . approximately 71% of the sequences in br 16 and 61% of embrapa 48 showed an associated putative biological or molecular function . consequently , the number of no match and unknown genes was larger in embrapa 48 than in br 16 plants . in br 16 , we observed 121 and 452 different roles for the genes expressed in the roots and leaves , respectively . in embrapa 48 categories employed in functional classification include the following : biological regulation ( gene expression regulation , protein modification regulation ) ; signaling ( kinases , phosphatases , secondary messengers ) ; response to stress ( water deficit , osmotic and salt stress , heat , cold ) ; response to stimulus ( chemical stimulus , endogenous stimulus ) ; response to biotic stimulus ( pathogen , fungus , insect ) ; cell organization ( cytoskeleton components , cell wall organization , cellular component assembly ) ; cell cycle ( actin filament - based process , cell division ) ; senescence ( aging , cell death ) ; other biological process ( cellular component organization , cell recognition , developmental process ) ; biosynthetic process ( carbohydrate , fatty acid , hormone , small and macromolecules , pigment , nucleotide ) ; catabolic process ( protein , fatty acid , cell wall , polysaccharide ) ; nitrogen compound metabolism ( glutamate and glutamine biosynthetic process , cellular nitrogen compound metabolic process ) ; oxidation reduction ( electron transport , lipid oxidation ) and secondary metabolism ( terpenoid , phytochelatin , phytoalexin and phenylpropanoid metabolic process ) . each category was normalized by the total number of sequences observed in the respective library , which enables comparison of the results during the time course of the water deficit ( figure 3 ) . applying the venn - master diagram , we also searched for genes expressed in more than one library as described in figure 4 . br 16 root tissue genes were categorized as involved in the electron transport process ( r1 and r2 libraries ) or as encoding proteins related to the stress response class ( r2-r3 library ) ( figure 4a ) . in br 16 leaves ( l1-l2 libraries ) , we also detected genes involved in signaling reactions or involved in the transport of metabolites and electrons ( oxidation reduction process ) ( figure 4b ) . in addition , the genes in the l2-l3 libraries were also related to stress response ( figure 4b ) . a general view of l1-l2-l3 libraries confirmed that genes functionally related to transport and stress responses were also expressed in leaves over the water deficit period ( figure 4b ) . it is important to emphasize that transcripts observed in r1-r2 , for example , were different from the transcripts found in r1-r2-r3 , although similar biological processes could be detected in both libraries . interestingly , in the embrapa 48 cultivar , transcription factors and genes involved in signal transduction were the most abundant transcripts in both r1 and r2 root tissues ( figure 4c ) . genes encoding transcription factors were also predominant in the r2-r3 libraries in addition to the ones related to transport process ( electrons and molecules ) . processes related to protein metabolism ( assembly , folding and modification ) were very active in l2-l3 libraries as well as genes involved in transport process , response to stress and signaling . stress response genes stand for 22% of the genes identified in l1-l2-l3 libraries ( figure 4d ) . we performed a comparison between genes expressed in roots and leaves in either cultivar using the venn - master diagram tool and looking for the biological processes observed in each group . when the same contig or glyma was detected in more than one library then , considering only the non - repeated genes from each group ( tissue or cultivar ) , we were able to observe genes expressed in root and leaves as well as genes expressed in embrapa 48 and br 16 plants ( figure 5a ) . to assess biological processes in roots and leaves of both cultivars , we highlighted the more abundant putative proteins and cellular components in each water - deficit stressed tissue ( figure 5b ) . according to our data , transcripts expressed by br 16 plants in roots and leaves showed that the most frequent biological processes were similar in both tissues ( figure 5b ) . however , in embrapa 48 , the process of response to stress was predominant among transcripts from leaves and less common in roots ( figure 5b ) . among genes expressed by plants in response to water deficit , we observed that transcripts classified in the response to stress category presented similarity to proteins with responsiveness to biotic as well as to abiotic stress . under abiotic stress , we identified transcripts in response to osmotic and oxidative stress , desiccation , heat , cold , water deprivation , hyperosmotic salinity and high light intensity . to assess which transcripts are expressed in response to other adverse conditions , the genes placed in the response to stress category ( n = 458 ) were compared to transcripts from plants infected by phakopsora pachyrhizi , uromyces appendiculatus , virus and treated with bradirhyzobium japonicum ( figure 6 ) . plant water deficit responses and tolerance are complex biological processes that need to be analyzed at system - level using genomics and physiological approaches ( harb et al . studies aiming to detect differences among plants in response to stress using contrasting cultivars have been performed in some legumes ( trkan et al . empiric observations during growing seasons in the field are the first step to explore plant variability based on its yield under water deficit conditions . ( 2004 ) examined the physiological characteristics of the embrapa 48 and br 16 cultivars and determined that they were moderately tolerant and highly sensitive to drought , respectively . here , we studied these cultivars at the molecular level , and our data demonstrated that the water deficit applied was effective as it triggered changes in the plant physiological behavior ( figure 2 ) . as the stress increased in the plants , photosynthesis of both cultivars was affected and was strongly inhibited after 100 min . this inhibition was related to a drastic decrease in the stomatal conductance ( gs ) and transpiration rate ( e ) of both cultivars . the water deficit did not produce significant physiological differences between embrapa 48 and br 16 cultivars . this was expected as the short - term procedure to induce stress used here is usually not enough to produce effects on plant morphological and physiological characteristics needed to distinguish the two cultivars . the experimental treatment described here aimed to study the effects of the stress on the early plant responses at a molecular level . as described in the literature , subtractive libraries have been employed in a wide range of studies such as gene expression studies ( kojima et al . however , in vivo cloning can be considered a laborious and expensive procedure and also has technical limitations . as shown in table 1 , we identified many genes being regulated under water deficit , more than is often detected through subtractive libraries . larger sets of genes were also detected in the characterization of stress - responsive genes to heat in wheat ( chauhan et al . both suppressive subtractive libraries were done through in vivo cloning and by the capillary sequencing method . ( 2010 ) found 12442 contigs in the transcriptome by combining subtractive libraries and massively parallel sequencing ( pyrosequencing ) . one of the advantages of the next generation sequencing ( ngs ) over the traditional sanger methodology is a more precise measurement of the transcript levels and isoforms , in addition to other revolutionary benefits ( marioni et al . under water deficit different biochemical roles have been suggested for these responsive genes , and their expression patterns make an intricate network that can be related to either water deficit acclimation or the cellular damage response ( shinozaki and yamaguchi - shinozaki , 2007 ) . the expressed genes have been shown to belong to many classes of genes ( figure 3 ) . however , the largest class of genes expressed under stress remains unknown , partly because there is no similarity in databases available to date , and partly due to the gene functions are not understood yet ( reddy et al . many genes expressed under water deficit conditions are present in the contrasting cultivars embrapa 48 and br 16 ( figure 5a ) . some proteins are synthesized in requirement of defense pathways triggered by the stress whereas other proteins are degraded because its aggregation or denaturation events are predominant in water stress conditions . also included in this category are the genes for aquaporins , which are proteins that might be directly involved in water deficit acclimation by facilitating water movement across the membranes ( zhao et al . we have identified several transcripts encoding the tonoplast and plasma membrane intrinsic proteins that were more frequently found in roots rather than in leaves , especially in the br 16 plants ( figure 5b ) . roles played by aquaporins in plants can be regulated in different complex ways , but the expression of aquaporins throughout the plant would most likely be regulated by water potential gradient ( zhao et al . however , there are differences between the mrna changes and its respective protein level . the changes expressed here by soybean plants in response to water stress are only at the transcriptional level . br 16 plants did not show an expressive quantitative difference in the biological processes affected by water deficit between roots and leaves ( figure 5b ) . nonetheless , embrapa 48 did show a significant contrast between both tissues ( figure 5b ) regarding the genes included in the response to stress class ( water deficit , osmotic and salt stress , heat and cold ) . under water stress , plants can become susceptible to other abiotic or biotic stresses because their normal metabolism is directed towards the acclimation to the first stress . several studies have tried to make a connection between plant responses to water deficit and heat ( sakuma et al . , 2010 ) , or reactive oxygen species enhanced due to drought ( kocsy et al . in the response to stress category , we observed that various transcripts were similarly involved in several abiotic stresses such as osmotic stress , cold , heat and salinity as well as those involved in response to biotic stimulus ( bacterium , fungus , nematode , virus ) ( figure 6 ) . a hydroponic system was applied successfully to grow soybean plants and to produce clean roots in a short - term water deficit experiment . we described a large collection of cdnas expressed by soybean plants under water deficit and their respective biochemical processes . although the analyses presented here represent a first step in gene expression investigation , the results indicate changes which are triggered in roots and leaves of tolerant and sensitive soybean plants .

deep vein thrombosis , ischemic stroke , and pulmonary embolism are manifestations of the same disease process , summed up over 100 years ago by rudolph virchow.1 his hypothesis that thrombosis was the result of the interaction of the three factors stasis of blood flow , hypercoagulability of the blood , and damage to the vascular endothelium has become the basis of risk - association diagnosis in patients who have developed venous thrombosis embolism . atrial fibrillation ( af ) is the most common tachyarrhythmia with prevalence of over 10% in older patients ( > 70 years ) . af is the leading cause of ischemic stroke , and stroke due to af is one of the leading causes of death and adult disability.2 besides rate and rhythm control , stroke prevention is the key management strategy for patients with nonvalvular atrial fibrillation and one or more additional risk factors for stroke.3 thrombosis risk can be quantified using the chads2 or recently quantified cha2ds2-vasc scores ( documenting risk factors for stroke : history of congestive heart failure , hypertension history ; age 75 [ or age 65 years associated with one of the following : diabetes mellitus , coronary artery disease , or hypertension ] ; diabetes mellitus ; stroke or transient ischemic attack or thromboembolism history ; vascular disease history ; sex ) ( see also table 1).46 by considering these additional risk factors the score is calculated to determine whether antithrombotic therapy is required or not . anticoagulation with vitamin k antagonists ( vka ) , ever since their introduction in the 1950s , has been an enduring gold standard for stroke prevention in af as well as for the prophylaxis and long - term treatment of venous thromboembolism.7,8 vkas such as phenprocoumon ( marcumar ; meda pharma gmbh & co. kgaa , bad homburg , germany ) or warfarin ( coumadin ; bristol - myers squibb gmbh & co. kgaa , munich , germany ) prevent hepatic synthesis of coagulation factors ii , vii , ix , and x by inhibiting vitamin k - dependent -carboxylation . due to the wide spectrum of food and drug interactions of vkas , several pathological conditions , and the unpredictability of genetically determined interindividual differences in drug metabolism , treatment with vka requires more or less frequent monitoring of the anticoagulant effect with dose adjustment.9 regarding the problems and disadvantages of these drugs with respect to efficacy , safety , and quality of life , many efforts have been undertaken to develop new anticoagulants targeting only single factors of the coagulation cascade . the licensed drugs rivaroxaban ( xarelto ; bayer pharma ag , leverkusen , germany ) , dabigatran ( pradaxa ; boehringer ingelheim gmbh , ingelheim , germany ) , and apixaban ( eliquis ; bristol - myers squibb gmbh & co. kgaa ; pfizer pharma gmbh ; munich , germany ) are already available for clinical use in many countries for stroke prevention in af . other new substances targeting factor xa such as edoxaban ( lixiana ; daiichi sankyo company , limited , tokyo , japan ) are in final stages of clinical studies . the predictability of these new oral direct anticoagulants is based on their pharmacodynamic and pharmacokinetic profiles . unlike vkas , multiple food and drug interactions are not seen with noac and , thus , routine monitoring with laboratory tests is not recommended . rivaroxaban as the first direct oral factor xa inhibitor is a small molecule ( molecular weight 436 g / mol ) that is almost insoluble in water and exhibits high plasma protein binding ( 92%95% ) in humans , with serum albumin being the main binding component . the absolute bioavailability of rivaroxaban is high ( 80%100% ) and is not affected by food intake . in patients with nonvalvular atrial fibrillation receiving xarelto 20 mg once daily , median maximal concentration ( cmax ) at steady state reaches approximately 290 g / l ( 5th95th percentile : 195420 ng / ml ) and a trough concentration ( ctrough ) of approximately 32 g / l ( 5th95th percentile : 587 ng / ml ) . rivaroxaban has a dual mode of excretion with the renal route accounting for one third of the overall elimination of unchanged active drug.1012 apixaban as second direct oral factor xa inhibitor with good bioavailability and a half - life of approximately 12 hours has high affinity for factor xa similar to rivaroxaban , and inhibits free factor xa , factor xa in the prothrombinase complex , and factor xa bound to platelets . following oral administration , peak plasma concentrations are observed at about 34 hours post dosing . apixaban is eliminated predominantly via the fecal route ( 56% ) , with 25%29% of the recovered dose eliminated via urinary excretion . in patients receiving eliquis 5 mg twice daily , mean ctrough and cmax reaches approximately 19162 ng / ml.12 edoxaban is another oral reversible direct factor xa inhibitor with 62% oral bioavailability . it achieves maximum concentrations within 12 hours , its mean terminal elimination half - life is 8.7510.4 hours . edoxaban is primarily eliminated unchanged through multiple pathways , with approximately 50% of systemically absorbed drug eliminated via renal excretion . the most abundant metabolites are formed through hydrolysis with minor contribution from cytochrome p450 - 3a.13 dabigatran etexilate is a potent synthetic nonpeptide competitive rapidly acting oral direct thrombin inhibitor . dabigatran is taken orally as a prodrug in its inactive precursor form , dabigatran etexilate , which is converted after absorption by nonspecific esterases to the active substance that inhibits thrombin directly . in the treatment of atrial fibrillation , for which dabigatran was approved by the food and drug administration and the european medicine agency ( in 2011 ) pradaxa is prescribed as having a cmax at steady state of 25470.5 ng / ml ( mean standard deviation ) and a ctrough after 12 hours of 80.318.7 ng / ml in elderly subjects.14,15 as seen in table 2 , the three noac , dabigatran , rivaroxaban , and apixaban , differ in mode of action ( factor iia and factor xa inhibition ) , pharmacology , pharmacokinetic and pharmacodynamic parameters , drug interactions , and side effects . dabigatran ( pradaxa ) was the first new oral anticoagulant to be approved based on the results of the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.16 in the re - ly study , 18,113 patients with nonvalvular atrial fibrillation and at least one additional risk factor for stroke or systemic thromboembolism were randomized into two different treatment arms : dabigatran 150 mg or 110 mg twice daily or conventional therapy with the vka warfarin in therapeutic international normalized ratio ( inr)-targets of 2.03.0 . both dabigatran treatment regimes were statistically noninferior to the vka in terms of the rate of primary efficacy endpoints ( stroke and systemic embolism ) as well as with respect to the primary safety endpoint ( severe bleeding complications ) . the higher dabigatran dosing ( 150 mg twice daily ) was significantly superior to the tested vka in reducing the primary efficacy endpoints , without increasing the risk of bleeding . the lower dosing ( 110 mg twice daily ) showed a comparable effectiveness in terms of preventing stroke and thromboembolism , while the risk of bleeding complications was significantly lower . interestingly , both dabigatran dosage regimes were associated with a significantly lower risk of intracranial bleeding compared to the vka . clinically relevant side effects in gastrointestinal complaints ( dyspepsia ) and gastrointestinal bleeding occurred more frequently among dabigatran treated patients . rivaroxaban ( xarelto ) was the first oral factor xa inhibitor to have gained approval for the prophylaxis of thromboembolism in nonvalvular atrial fibrillation . in the double - blind randomized pivotal trial rivaroxaban once - daily , oral , direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation ( rocket - af ) , a high risk population of approximately 14,000 patients with nonvalvular atrial fibrillation was included ; patients had at least two risk factors or had history of stroke , transitory ischemic attack , or systemic embolism ( average chads2 score : 3.5).17 rivaroxaban 20 mg once daily was compared with the vka warfarin at inr target of 2.03.0 . patients with impaired renal function ( creatinine clearance of 3049 ml / minute ) were treated with a reduced rivaroxaban dose of 15 mg once daily . in the statistical analyses , a noninferiority of rivaroxaban compared to the conventional treatment with warfarin could be demonstrated for preventing thromboembolic complications and safety . in the on - treatment analysis , rivaroxaban resulted in a significant reduction in the primary efficacy endpoint consisting of stroke and systemic embolism compared to with the vka . the primary safety endpoint of the study consisted of all bleeding complications ( severe and nonsevere clinically significant bleeding ) and was not significantly different in both study treatment arms . rivaroxaban was associated with a significantly lower risk for intracranial bleeding and fatal bleeding complications . apixaban ( eliquis ) as second oral factor xa inhibitor was examined in two randomized , multicenter studies in patients with atrial fibrillation . in the apixaban versus acetylsalicylic acid to prevent strokes ( averroes ) trial , 5,599 patients with atrial fibrillation and at least one additional risk factor were double - blind randomized.18 patients who were not suitable for treatment with vka and , were either treated with acetylsalicylic acid ( asa ) at doses of 81324 mg once daily or apixaban at a dosing of 5 mg twice daily . it has long been known that asa has only a limited antithrombotic effect in atrial fibrillation ; therefore , apixaban could demonstrate its superior antithrombotic activity compared to asa . remarkably , the bleeding rates among apixaban were not significantly increased in comparison to asa . due to the superior benefit it was finally investigated in the apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation ( aristotle)-trial whether apixaban in the dosing of 5 mg twice daily is non - inferior to warfarin at target - inr of 23.19 the study design was double - blind and randomized , and included patients with nonvalvular af and one additional risk factor ( average chads2 score : 2.1 ) . patients with increased risk of bleeding and the presence of two risk markers ( age > 80 years , body weight less than 60 kg , creatinine > 1.5 mg / dl ) were treated with the lower dose of apixaban 2.5 mg twice daily . both the risk of stroke and systemic embolism and the risk of major bleeding complications were statistically significantly lower with apixaban than with warfarin . moreover , apixaban had an impact on the mortality of the patients that was significantly reduced in the statistical analyzes in comparison to warfarin . edoxaban ( lixiana ) , another oral factor xa inhibitor , was studied in a large multicenter phase iii trial versus warfarin in subjects with af : engage af - timi 48.20 with more than 20,000 patients , this study was the largest and longest single comparative clinical trial performed for prevention of embolic events in nonvalvular af . two edoxaban regimens ( 30 or 60 mg once daily ) were tested for noninferiority in comparison to warfarin during the treatment period of 2.8 years . the primary efficacy endpoint was stroke or systemic embolism . both once - daily regimens of edoxaban were noninferior to warfarin with respect to the prevention of stroke or systemic embolism and were associated with significantly lower rates of bleeding and death from cardiovascular causes . all studies , which compared the efficacy and safety of the new direct oral anticoagulants , used dose - adjusted warfarin as standard of control and showed noninferiority or superiority for the efficacy outcome of ischemic stroke and systemic embolism . furthermore , the rates of major bleeding complications were similar or even reduced for the new drugs in comparison with warfarin.21 table 3 summarizes the results for efficacy and safety data of dabigatran , rivaroxaban , apixaban , and edoxaban based on the re - ly , rocket af , aristotle , and engage af - timi 48 trials . no clinical comparisons regarding efficacy and safety outcomes between the new direct oral drugs have been performed with patient cohorts , and it is highly unlikely that such a comparison would be performed in the near future due to the expense and risks to the manufacturer of such an undertaking . the vka such as warfarin , acenocoumarol , and phenprocoumon require drug monitoring mainly due to their narrow therapeutic window and numerous food and drug interactions . the relationship between monitoring vkas and their efficacy / safety balance is proven.22,23 the exponential increase in studies evaluating health - related quality of life as an important outcome in anticoagulated patients has shown that monitoring these patients leads to more anticoagulation stability , lower incidence of bleeding , and less ischemic events.2430 however , active changes in lifestyle can be potentially troublesome for many patients . qualitative studies have confirmed that frequent monitoring of blood tests and visits to the clinic , anxiety related to adverse events , patient autonomy , dietary restrictions , and the impact of anticoagulant medication on physical activities have a negative impact on the health - related quality of life.24 moreover , it is often intended that the oral anticoagulation should be maintained over the long term , sometimes for the remainder of patient s life . advantages of noac include the wide therapeutic window , low inter- and intraindividual variability of the dose effect relation , and only few known drug interactions or genetic determinations of the metabolism ; thus , their clinical use is easy to handle , and frequent monitoring and dose adjustments have not been considered necessary . however , there are several situations in which it may be of assistance to assess the anticoagulant effects , even of noac such as rivaroxaban , apixaban , or dabigatran.31,32 in patients with severe renal impairment ( creatinine clearance < 30 ml / minute ) plasma levels may be significantly increased , which may lead to an increased bleeding risk when treated with rivaroxaban or apixaban . the use of these two oral factor xa inhibitors is not recommended in patients with creatinine clearance < 15 ml / minute and are used with caution in patients with creatinine clearance between 15 and 29 ml / minute , as suggested in the summary characteristics of the products.33,34 clinical case reports suggested that lower exposure of rivaroxaban or apixaban in patients with high body weight ( > 120 kg ) did not result in loss of drug efficacy.35 dose adjustment based on low body weight may also not be warranted ; however , combination of additional risk factors for bleeding ( age 80 years , body weight 60 kg , and serum creatinine 1.5 mg / dl ) might lead to dose adjustment . as substrates of cyp3a4 and p - glycoprotein , rivaroxaban and apixaban are not recommended for concomitant use with strong inhibitors of both pathways , eg , most azole antimycotics and protease inhibitors . since dabigatran is eliminated primarily by renal excretion , the use of low dosage is recommended at a reduced kidney function and also at an increased risk of bleeding in patients , as well as in the elderly ( 75 years).3639 a corresponding control value of the renal function before starting treatment and subsequently is required at regular intervals . in severe renal insufficiency ( creatinine clearance < 30 ml / minute ) , there is a contraindication for dabigatran due to accumulation . intravenous vitamin k can lower the inr more quickly than oral vitamin k , but at 24 hours , intravenous and oral vitamin k produce similar degrees of inr correction.40 due to the long half - life of vka , the antidote vitamin k for bleeding takes at least 12 hours to affect inr . fresh frozen plasma , prothrombin - complex concentrate , and recombinant factor viia have been used as general hemostatic agents in patients treated with anticoagulants who experience severe hemorrhage.41,42 for the noac , the management of bleeding can also be performed with these hemostatic agents . however , the effectiveness of these nonspecific agents for reversing the effect anticoagulation has not been established ; there are only case reports of potential reversal agents , which showed dose dependent reversal of the anticoagulant effect of the noac . due to the short half - lives and dose omission of noac therefore , less serious bleedings with noac might be more easily managed than bleedings with vka . however , there has been intensive effort to develop more specific antidotes for the new oral anticoagulants . the modified recombinant protein ( r - antidote , prt064445 ) is a specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor xa , already prooved in animal models of bleeding.43 an antidote specific for dabigatran ( adabi - fab ) has been shown to effectively reverse the anticoagulant activity of dabigatran in vivo in a rat model of anticoagulation.44 moreover , it is reported that a synthetic small molecule ( per977 ) reduces blood loss by reversing the anticoagulant activity of apixaban , rivaroxaban , and dabigatran in a rat tail transection assay.45 over- or under - anticoagulation may lead to serious bleeding or increased risk of embolic and thrombotic events.46 regarding vka , the time expressed in percentages that patients spend in therapeutic inr range is less than half in routine practice than in clinical trial settings.47,48 for noac , no routine monitoring is mandatory . therefore , the level of treatment satisfaction and efficacy will be unknown till an event appears . health - related parameters such as physical function , vitality , and general health can be easily measured . however , the major concern of doctors that oral anticoagulation , whether new or old , will lead to hemorrhage or thrombosis will mostly be solved by regular blood testing and contact with medical practitioners.2230 as mentioned , in patients over 70 years of age , the risk of a bleeding events is twice as high as in patients younger than 70 years of age . moreover , when considering the elderly , adherence , and quality of life , it must be remembered that not all noac ( ie , dabigatran ) are suitable for dosette boxes . dabigatran must remain in its original packing up until it is taken to protect it from moisture . the oral bioavailability may be decreased if the drug remains open for days in the dosette boxes , and increased by 75% compared to the reference capsule formulation when the pellets are taken without the capsule shell.36 incorrect handling may , therefore , lead to higher risk for bleeding or thrombotic events , especially in elderly patients . therefore , regular blood testing and visits to the doctors reassures effective treatment and health outcome , especially in the elderly . to improve the treatment with coumarin derivatives a standard method for reporting the prothrombin time has been assessed to ensure the proper dosage intensity that decreases the risk of bleeding while maintaining the therapeutic efficacy of the vitamin k antagonists . the target specific inhibitors dabigatran , rivaroxaban and apixaban represent a new class of anticoagulants that can not be monitored with global coagulation tests due there is not a predictable linear relationship between laboratory value and drug concentration.4956 chromogenic anti - factor xa assays have been demonstrated to have the potential for accurate measurement of plasma concentrations of the direct factor xa inhibitors.5760 however , the threshold values of drug concentrations and the degree of prolongation that might still be assumed as safe must be defined . moreover , an exact cutoff that defines a clearly increased hemorrhagic risk at trough levels of direct factor xa inhibitors has to be defined ; such threshold values are only available for dabigatran . the thrombin time ( tt ) is reported to be very sensitive to low concentrations of dabigatran.61,62 a normal tt indicates that dabigatran is either absent or at a negligible low concentration . in cases of acute emergency operations , when the detection of low dabigatran concentrations might be necessary , the tt assay provides qualitative information regarding the presence of dabigatran . a diluted tt assay can be used to accurately measure dabigatran concentrations . due to the linear dose - response curves over a wide concentration range , a diluted tt assay can be used to accurately monitor both trough and peak dabigatran levels . however , if oral anticoagulation stability could be more easily achieved over time , patients would suffer less adverse events and need less frequent blood controls . a basic need in improving the satisfaction of a patient is to first know what individual habits and social surroundings the patient has . for future practice , the physicians need to consider which patients with atrial fibrillation are best suited for treatment with the new direct oral anticoagulants , and whether it makes sense to switch patients with preexisting and well - adjusted vka therapy to noac . vka represent the gold standard therapy for the prevention of thromboembolic complications in atrial fibrillation.63 moreover , vka have now reached the lowest mean cost for the defined daily dose ; in germany in 2013 , the daily cost was 0.20 for phenprocoumon , 3.28 for dabigatran and rivaroxaban , and 3.43 for apixaban . even when event costs , inr - testing , physician visits , or prescription drug costs in a long - term 30-year model are invoiced , as performed by harrington et al,64 warfarin had the lowest mean cost of $ 77,813 , followed by rivaroxaban 20 mg ( $ 78,738 ) , dabigatran 150 mg ( $ 82,719 ) , and apixaban 5 mg ( $ 85,326 ) for a baseline patient ( defined as a 70-year - old patient with af with an increased risk for stroke [ chads2 > 1 or equivalent ] , a renal creatinine clearance of > 50 ml / minutes , and no previous contraindications to anticoagulant therapy ) . for a baseline 65-year - old patient with af and chads2 > 1 or equivalent in a time range over 20 years , the cost - utility analysis for warfarin ranged from 7,622 to 9,069 and for noac from 19,537 to 20,048.65 self - management entails the measurement of the inr by the patient using point - of - care devices and , when necessary , self - adjustment of the vka dosage.2730 in the review of regier et al,66 start - up cost in canada for self - managing treated patients was estimated to be $ 1,567 per patient , and the annual cost of physician management and self - management was estimated to be $ 357 and $ 352 , respectively , per patient . self - management for noac - treated patients has not been considered because the novel drugs are established without drug monitoring , and no point - of - care device is now available for monitoring noac - therapy . after a major event , patients with mild disability underwent 1 year of rehabilitation at a cost of $ 2,176 . for those with permanent disability , however , data of efficacy from large randomized trials exists for noac,1620 and they showed the reduction of major adverse bleeding events with noac . this is likely to lower the overall health care costs in comparison to following the vka strategy , though it is hard to get a real - world cost estimate for this . dabigatran , rivaroxaban , and apixaban are approved for stroke prevention in patients with nonvalvular af . among the factors of oral anticoagulation that induce dissatisfaction , vka as current standard therapy used for prolonged stroke management have a narrow therapeutic window , interindividual variability in dose response , and numerous drug drug and drug food interactions . however , special care is indicated in patients with increased risk of bleeding or with renal or hepatic insufficiency . older patients represent a particularly difficult cohort of patients in which both the thromboembolic risk and the risk of bleeding are increased . in these patient cohorts monitoring anticoagulation therapy methods for accurate and effective monitoring of direct oral anticoagulants are available . in order to accurately estimate the cost , the cost differences of the various anticoagulant drugs have to be calculated , but also the costs of monitoring vka versus measuring noac - concentrations in special clinical situations , the cost of bleeding events , and the costs of stroke have to be considered . uncertainties in clinical practice , that may be solved in the near future , arise in particular from : the lack of availability of specific antidotes for rapid antagonism of the anticoagulant effect of direct thrombin or factor xa inhibitors ; the lack of validated point - of - care tests to monitor and review the quality of anticoagulation in certain emergency situations ( for example , in acute bleeding and chronic renal insufficiency ) ; and from the absence of clinical data on the management of bleeding under direct oral anticoagulants or perioperative switching and bridging ( timely discontinuation and resumption in operations ) . there are also no data on possible interactions with other drugs in the presence of much comorbidity . in figure 1 therefore , it has to be carefully considered if using fixed doses without laboratory - guided dose adjustment is a real benefit for patients quality of life and adherence . to conclude , individual patients satisfaction profiles are influenced by many factors , and have to be adjusted with physicians satisfaction profiles .

atrial fibrillation ( af ) continues to be a leading cause of cerebrovascular morbidity and mortality resulting from cardioembolic stroke . oral anticoagulation therapy has been shown to decrease the incidence of cardioembolic stroke in patients with af by more than 50% . appropriate use of anticoagulation with vitamin k antagonists requires precise adherence and monitoring . a number of factors that potentially induce patients dissatisfaction reduce quality of patient life . new direct oral anticoagulants , such as the direct factor xa inhibitors rivaroxaban , apixaban , edoxaban , and the thrombin inhibitor dabigatran , were developed to overcome the limitations of the conventional anticoagulant drugs . however , models to optimize the benefit of therapy and to ensure that therapy can be safely continued are missing for the new oral anticoagulants . this review will briefly describe the new oral anticoagulants dabigatran , rivaroxaban , apixaban , and edoxaban with focus on their use for prevention of embolic events in af . moreover , it will discuss the safety , efficacy , cost data , and benefit for patients quality of life and adherence .

Introduction to thrombosis prophylaxis with new oral anticoagulants (NOAC) The pharmacokinetic profiles of NOAC Efficacy and safety studies for prophylaxis of thromboembolism in nonvalvular atrial fibrillation of NOAC Health outcomes and patients quality of life Cost analyses Conclusion

deep vein thrombosis , ischemic stroke , and pulmonary embolism are manifestations of the same disease process , summed up over 100 years ago by rudolph virchow.1 his hypothesis that thrombosis was the result of the interaction of the three factors stasis of blood flow , hypercoagulability of the blood , and damage to the vascular endothelium has become the basis of risk - association diagnosis in patients who have developed venous thrombosis embolism . atrial fibrillation ( af ) is the most common tachyarrhythmia with prevalence of over 10% in older patients ( > 70 years ) . af is the leading cause of ischemic stroke , and stroke due to af is one of the leading causes of death and adult disability.2 besides rate and rhythm control , stroke prevention is the key management strategy for patients with nonvalvular atrial fibrillation and one or more additional risk factors for stroke.3 thrombosis risk can be quantified using the chads2 or recently quantified cha2ds2-vasc scores ( documenting risk factors for stroke : history of congestive heart failure , hypertension history ; age 75 [ or age 65 years associated with one of the following : diabetes mellitus , coronary artery disease , or hypertension ] ; diabetes mellitus ; stroke or transient ischemic attack or thromboembolism history ; vascular disease history ; sex ) ( see also table 1).46 by considering these additional risk factors the score is calculated to determine whether antithrombotic therapy is required or not . anticoagulation with vitamin k antagonists ( vka ) , ever since their introduction in the 1950s , has been an enduring gold standard for stroke prevention in af as well as for the prophylaxis and long - term treatment of venous thromboembolism.7,8 vkas such as phenprocoumon ( marcumar ; meda pharma gmbh & co. kgaa , bad homburg , germany ) or warfarin ( coumadin ; bristol - myers squibb gmbh & co. kgaa , munich , germany ) prevent hepatic synthesis of coagulation factors ii , vii , ix , and x by inhibiting vitamin k - dependent -carboxylation . due to the wide spectrum of food and drug interactions of vkas , several pathological conditions , and the unpredictability of genetically determined interindividual differences in drug metabolism , treatment with vka requires more or less frequent monitoring of the anticoagulant effect with dose adjustment.9 regarding the problems and disadvantages of these drugs with respect to efficacy , safety , and quality of life , many efforts have been undertaken to develop new anticoagulants targeting only single factors of the coagulation cascade . the licensed drugs rivaroxaban ( xarelto ; bayer pharma ag , leverkusen , germany ) , dabigatran ( pradaxa ; boehringer ingelheim gmbh , ingelheim , germany ) , and apixaban ( eliquis ; bristol - myers squibb gmbh & co. kgaa ; pfizer pharma gmbh ; munich , germany ) are already available for clinical use in many countries for stroke prevention in af . other new substances targeting factor xa such as edoxaban ( lixiana ; daiichi sankyo company , limited , tokyo , japan ) are in final stages of clinical studies . rivaroxaban as the first direct oral factor xa inhibitor is a small molecule ( molecular weight 436 g / mol ) that is almost insoluble in water and exhibits high plasma protein binding ( 92%95% ) in humans , with serum albumin being the main binding component . in patients with nonvalvular atrial fibrillation receiving xarelto 20 mg once daily , median maximal concentration ( cmax ) at steady state reaches approximately 290 g / l ( 5th95th percentile : 195420 ng / ml ) and a trough concentration ( ctrough ) of approximately 32 g / l ( 5th95th percentile : 587 ng / ml ) . rivaroxaban has a dual mode of excretion with the renal route accounting for one third of the overall elimination of unchanged active drug.1012 apixaban as second direct oral factor xa inhibitor with good bioavailability and a half - life of approximately 12 hours has high affinity for factor xa similar to rivaroxaban , and inhibits free factor xa , factor xa in the prothrombinase complex , and factor xa bound to platelets . in patients receiving eliquis 5 mg twice daily , mean ctrough and cmax reaches approximately 19162 ng / ml.12 edoxaban is another oral reversible direct factor xa inhibitor with 62% oral bioavailability . in the treatment of atrial fibrillation , for which dabigatran was approved by the food and drug administration and the european medicine agency ( in 2011 ) pradaxa is prescribed as having a cmax at steady state of 25470.5 ng / ml ( mean standard deviation ) and a ctrough after 12 hours of 80.318.7 ng / ml in elderly subjects.14,15 as seen in table 2 , the three noac , dabigatran , rivaroxaban , and apixaban , differ in mode of action ( factor iia and factor xa inhibition ) , pharmacology , pharmacokinetic and pharmacodynamic parameters , drug interactions , and side effects . dabigatran ( pradaxa ) was the first new oral anticoagulant to be approved based on the results of the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.16 in the re - ly study , 18,113 patients with nonvalvular atrial fibrillation and at least one additional risk factor for stroke or systemic thromboembolism were randomized into two different treatment arms : dabigatran 150 mg or 110 mg twice daily or conventional therapy with the vka warfarin in therapeutic international normalized ratio ( inr)-targets of 2.03.0 . rivaroxaban ( xarelto ) was the first oral factor xa inhibitor to have gained approval for the prophylaxis of thromboembolism in nonvalvular atrial fibrillation . in the double - blind randomized pivotal trial rivaroxaban once - daily , oral , direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation ( rocket - af ) , a high risk population of approximately 14,000 patients with nonvalvular atrial fibrillation was included ; patients had at least two risk factors or had history of stroke , transitory ischemic attack , or systemic embolism ( average chads2 score : 3.5).17 rivaroxaban 20 mg once daily was compared with the vka warfarin at inr target of 2.03.0 . apixaban ( eliquis ) as second oral factor xa inhibitor was examined in two randomized , multicenter studies in patients with atrial fibrillation . in the apixaban versus acetylsalicylic acid to prevent strokes ( averroes ) trial , 5,599 patients with atrial fibrillation and at least one additional risk factor were double - blind randomized.18 patients who were not suitable for treatment with vka and , were either treated with acetylsalicylic acid ( asa ) at doses of 81324 mg once daily or apixaban at a dosing of 5 mg twice daily . due to the superior benefit it was finally investigated in the apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation ( aristotle)-trial whether apixaban in the dosing of 5 mg twice daily is non - inferior to warfarin at target - inr of 23.19 the study design was double - blind and randomized , and included patients with nonvalvular af and one additional risk factor ( average chads2 score : 2.1 ) . patients with increased risk of bleeding and the presence of two risk markers ( age > 80 years , body weight less than 60 kg , creatinine > 1.5 mg / dl ) were treated with the lower dose of apixaban 2.5 mg twice daily . moreover , apixaban had an impact on the mortality of the patients that was significantly reduced in the statistical analyzes in comparison to warfarin . edoxaban ( lixiana ) , another oral factor xa inhibitor , was studied in a large multicenter phase iii trial versus warfarin in subjects with af : engage af - timi 48.20 with more than 20,000 patients , this study was the largest and longest single comparative clinical trial performed for prevention of embolic events in nonvalvular af . all studies , which compared the efficacy and safety of the new direct oral anticoagulants , used dose - adjusted warfarin as standard of control and showed noninferiority or superiority for the efficacy outcome of ischemic stroke and systemic embolism . furthermore , the rates of major bleeding complications were similar or even reduced for the new drugs in comparison with warfarin.21 table 3 summarizes the results for efficacy and safety data of dabigatran , rivaroxaban , apixaban , and edoxaban based on the re - ly , rocket af , aristotle , and engage af - timi 48 trials . no clinical comparisons regarding efficacy and safety outcomes between the new direct oral drugs have been performed with patient cohorts , and it is highly unlikely that such a comparison would be performed in the near future due to the expense and risks to the manufacturer of such an undertaking . the relationship between monitoring vkas and their efficacy / safety balance is proven.22,23 the exponential increase in studies evaluating health - related quality of life as an important outcome in anticoagulated patients has shown that monitoring these patients leads to more anticoagulation stability , lower incidence of bleeding , and less ischemic events.2430 however , active changes in lifestyle can be potentially troublesome for many patients . qualitative studies have confirmed that frequent monitoring of blood tests and visits to the clinic , anxiety related to adverse events , patient autonomy , dietary restrictions , and the impact of anticoagulant medication on physical activities have a negative impact on the health - related quality of life.24 moreover , it is often intended that the oral anticoagulation should be maintained over the long term , sometimes for the remainder of patient s life . advantages of noac include the wide therapeutic window , low inter- and intraindividual variability of the dose effect relation , and only few known drug interactions or genetic determinations of the metabolism ; thus , their clinical use is easy to handle , and frequent monitoring and dose adjustments have not been considered necessary . however , there are several situations in which it may be of assistance to assess the anticoagulant effects , even of noac such as rivaroxaban , apixaban , or dabigatran.31,32 in patients with severe renal impairment ( creatinine clearance < 30 ml / minute ) plasma levels may be significantly increased , which may lead to an increased bleeding risk when treated with rivaroxaban or apixaban . the use of these two oral factor xa inhibitors is not recommended in patients with creatinine clearance < 15 ml / minute and are used with caution in patients with creatinine clearance between 15 and 29 ml / minute , as suggested in the summary characteristics of the products.33,34 clinical case reports suggested that lower exposure of rivaroxaban or apixaban in patients with high body weight ( > 120 kg ) did not result in loss of drug efficacy.35 dose adjustment based on low body weight may also not be warranted ; however , combination of additional risk factors for bleeding ( age 80 years , body weight 60 kg , and serum creatinine 1.5 mg / dl ) might lead to dose adjustment . since dabigatran is eliminated primarily by renal excretion , the use of low dosage is recommended at a reduced kidney function and also at an increased risk of bleeding in patients , as well as in the elderly ( 75 years).3639 a corresponding control value of the renal function before starting treatment and subsequently is required at regular intervals . fresh frozen plasma , prothrombin - complex concentrate , and recombinant factor viia have been used as general hemostatic agents in patients treated with anticoagulants who experience severe hemorrhage.41,42 for the noac , the management of bleeding can also be performed with these hemostatic agents . however , there has been intensive effort to develop more specific antidotes for the new oral anticoagulants . the modified recombinant protein ( r - antidote , prt064445 ) is a specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor xa , already prooved in animal models of bleeding.43 an antidote specific for dabigatran ( adabi - fab ) has been shown to effectively reverse the anticoagulant activity of dabigatran in vivo in a rat model of anticoagulation.44 moreover , it is reported that a synthetic small molecule ( per977 ) reduces blood loss by reversing the anticoagulant activity of apixaban , rivaroxaban , and dabigatran in a rat tail transection assay.45 over- or under - anticoagulation may lead to serious bleeding or increased risk of embolic and thrombotic events.46 regarding vka , the time expressed in percentages that patients spend in therapeutic inr range is less than half in routine practice than in clinical trial settings.47,48 for noac , no routine monitoring is mandatory . health - related parameters such as physical function , vitality , and general health can be easily measured . however , the major concern of doctors that oral anticoagulation , whether new or old , will lead to hemorrhage or thrombosis will mostly be solved by regular blood testing and contact with medical practitioners.2230 as mentioned , in patients over 70 years of age , the risk of a bleeding events is twice as high as in patients younger than 70 years of age . moreover , when considering the elderly , adherence , and quality of life , it must be remembered that not all noac ( ie , dabigatran ) are suitable for dosette boxes . to improve the treatment with coumarin derivatives a standard method for reporting the prothrombin time has been assessed to ensure the proper dosage intensity that decreases the risk of bleeding while maintaining the therapeutic efficacy of the vitamin k antagonists . the target specific inhibitors dabigatran , rivaroxaban and apixaban represent a new class of anticoagulants that can not be monitored with global coagulation tests due there is not a predictable linear relationship between laboratory value and drug concentration.4956 chromogenic anti - factor xa assays have been demonstrated to have the potential for accurate measurement of plasma concentrations of the direct factor xa inhibitors.5760 however , the threshold values of drug concentrations and the degree of prolongation that might still be assumed as safe must be defined . moreover , an exact cutoff that defines a clearly increased hemorrhagic risk at trough levels of direct factor xa inhibitors has to be defined ; such threshold values are only available for dabigatran . for future practice , the physicians need to consider which patients with atrial fibrillation are best suited for treatment with the new direct oral anticoagulants , and whether it makes sense to switch patients with preexisting and well - adjusted vka therapy to noac . vka represent the gold standard therapy for the prevention of thromboembolic complications in atrial fibrillation.63 moreover , vka have now reached the lowest mean cost for the defined daily dose ; in germany in 2013 , the daily cost was 0.20 for phenprocoumon , 3.28 for dabigatran and rivaroxaban , and 3.43 for apixaban . even when event costs , inr - testing , physician visits , or prescription drug costs in a long - term 30-year model are invoiced , as performed by harrington et al,64 warfarin had the lowest mean cost of $ 77,813 , followed by rivaroxaban 20 mg ( $ 78,738 ) , dabigatran 150 mg ( $ 82,719 ) , and apixaban 5 mg ( $ 85,326 ) for a baseline patient ( defined as a 70-year - old patient with af with an increased risk for stroke [ chads2 > 1 or equivalent ] , a renal creatinine clearance of > 50 ml / minutes , and no previous contraindications to anticoagulant therapy ) . for a baseline 65-year - old patient with af and chads2 > 1 or equivalent in a time range over 20 years , the cost - utility analysis for warfarin ranged from 7,622 to 9,069 and for noac from 19,537 to 20,048.65 self - management entails the measurement of the inr by the patient using point - of - care devices and , when necessary , self - adjustment of the vka dosage.2730 in the review of regier et al,66 start - up cost in canada for self - managing treated patients was estimated to be $ 1,567 per patient , and the annual cost of physician management and self - management was estimated to be $ 357 and $ 352 , respectively , per patient . dabigatran , rivaroxaban , and apixaban are approved for stroke prevention in patients with nonvalvular af . among the factors of oral anticoagulation that induce dissatisfaction , vka as current standard therapy used for prolonged stroke management have a narrow therapeutic window , interindividual variability in dose response , and numerous drug drug and drug food interactions . however , special care is indicated in patients with increased risk of bleeding or with renal or hepatic insufficiency . in these patient cohorts monitoring anticoagulation therapy methods for accurate and effective monitoring of direct oral anticoagulants are available . in order to accurately estimate the cost , the cost differences of the various anticoagulant drugs have to be calculated , but also the costs of monitoring vka versus measuring noac - concentrations in special clinical situations , the cost of bleeding events , and the costs of stroke have to be considered . uncertainties in clinical practice , that may be solved in the near future , arise in particular from : the lack of availability of specific antidotes for rapid antagonism of the anticoagulant effect of direct thrombin or factor xa inhibitors ; the lack of validated point - of - care tests to monitor and review the quality of anticoagulation in certain emergency situations ( for example , in acute bleeding and chronic renal insufficiency ) ; and from the absence of clinical data on the management of bleeding under direct oral anticoagulants or perioperative switching and bridging ( timely discontinuation and resumption in operations ) . in figure 1 therefore , it has to be carefully considered if using fixed doses without laboratory - guided dose adjustment is a real benefit for patients quality of life and adherence .

in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health . they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . individuals do not see themselves as multi - ill , but as needing support for their needs as they know them . it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health . they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . individuals do not see themselves as multi - ill , but as needing support for their needs as they know them . it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? lindberg observed examples of meaningful co - operation at the local level , with colleagues from different organizations meeting and pooling their knowledge of local conditions with the patient or user as the focal point . the phenomenon has been variously called the chain of care , integrated care , seamless care or shared care . this co - operation aims at creating a continuing relationship with the patient / user regardless of who at a given moment is the responsible provider . edgren and stenberg found these practical attributes of co - operation in cass : a common task is shared among the co - working agents.collaboration is in people s minds , it is instinctive behaviour.each actor s capabilities are known and respected.a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . collaboration is in people s minds , it is instinctive behaviour . each actor s capabilities are known and respected . a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . according to brommels in his presentation to the ehma - conference 2006 , the cas approach means , among other things : identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts.avoiding strict definitions of roles and concentrating on agreed actions.giving agents their freedom to organize current activities . identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts . avoiding strict definitions of roles and concentrating on agreed actions . giving agents their freedom to organize current activities . if we are to improve our understanding of how a health or social service provider functions as an integrated part of a locally driven health and social service system , we need an innovative , in terms of changing practice , model to guide our thinking . traditional models view systems as machines . it helps us understand what happens in dynamic living systems , where many agents are interconnected . in order to make clear its significance let us first recapitulate the machine model . for a long time effective organizations the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] . rationality , objectivity , stability and predictability are the terms we associate with this approach . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action . the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . davis coined the expression any time , any place when he describes how time and space restrictions this has given us enormous possibilities to communicate , to become connected , to network . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old . so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al . they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool . it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care . complexity can be expressed as the amount of information needed to describe or understand something . and the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . . they may either compete or co - operate according to their sense of their interests and what will bring them an advantage . self - organization is about creating order or increasing the regularity of the system without help from the outside . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien . according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . the agents see the point , because they create order out of many local interactions . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . emergency treatment is a clear example of independent agents interacting locally with other independent agents . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly . a common vision , easy to understand and to communicate , there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . complexity science emphasizes the inherent power of development and self - organizing nature of the system . attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage . for example in general terms , there is a positive feedback loop between income and consumption in an economy . the bigger the income of the individuals , negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science positive and negative feedback there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives . it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs , they are so much better placed to act than any centrally located management ever could be . they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them . but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . for a long time effective organizations have been looked upon like well - oiled machines . the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] . rationality , objectivity , stability and predictability are the terms we associate with this approach . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action . the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . davis coined the expression any time , any place when he describes how time and space restrictions this has given us enormous possibilities to communicate , to become connected , to network . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old . so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al . they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool . it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care . complexity can be expressed as the amount of information needed to describe or understand something . and an important part of complexity science is the complex adaptive system ( cas ) . the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . . they may either compete or co - operate according to their sense of their interests and what will bring them an advantage . self - organization is about creating order or increasing the regularity of the system without help from the outside . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien . according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . the agents see the point , because they create order out of many local interactions . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . emergency treatment is a clear example of independent agents interacting locally with other independent agents . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly . a common vision , easy to understand and to communicate , was created to connect the two groups . there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . complexity science emphasizes the inherent power of development and self - organizing nature of the system . attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . working in isolation the burden of coordination passes to the patient . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage . for example in general terms , there is a positive feedback loop between income and consumption in an economy . the bigger the income of the individuals , the more the whole population consume , which further increasing their income as individuals . negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs , they are so much better placed to act than any centrally located management ever could be . they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . some theorists would claim that cas is nothing but the emperor s new clothes . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them . but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . when the competence necessary for carrying out a given task does not lie within one individual provider organization , co - operation between agents within the system comes into play to discharge that task . we are no longer talking about the individual organization / agent but shaping overall workable solutions taking a patient / user perspective . this applies to matching care with the needs of different patient groups and of individual patients . the cas approach helps the management to understand why the traditional top down way of managing may meet with problems in organizations with complex tasks . an important discussion is about how the top management in fact executes its steering function . leaders may consider accepting complexity instead of trying to reduce it , formulate few simple and concrete goals , communicate and give feedback and measure performance . when we perceive health and social service organizations as cass we should gain more insight into the processes that go on within and between organizations . are we willing to face the interdependence between health and social services , the dependence on collaboration to deliver appropriate integrated care ? if we do , complexity science could be an important step towards fresh thinking in order to fulfil our patients and users presently unfulfilled needs . edgar borgenhammar , phd ( economics ) , mba ( berkeley ) , dsi ( lund ) , professor emeritus , from the nordic school of public health in gteborg , former hospital chief executive , sweden peter carswell , dr . , centre for health services research and policy , faculty of medical and health sciences , university of auckland , auckland , new zealand alene hokenstad , project director , united hospital fund , new york , usa

introductionorganizations can be regarded as systems . the traditional model of systems views them as machines . this seems to be insufficient when it comes to understanding and organizing complex tasks . to better understand integrated care we should approach organizations as constantly changing living organisms , where many agents are interconnected in so - called complex adaptive systems ( cas).theory and discussionthe term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . adaptive means that system change occurs through successive adaptations . a cas consists of several subsystems called agents , which act in dependence of one another . examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . studying a cas , the focus is on the interaction and communication between agents . although these thoughts are not new , the cas - approach has not yet been widely applied to the management of integrated care . this helps the management to understand why the traditional top down way of managing , following the machine model thinking , may meet with problems in interdependent organizations with complex tasks.conclusionwhen we perceive health and social services as cass we should gain more insight into the processes that go on within and between organizations and how top management , for example within a hospital , in fact executes its steering function .

Introduction Background Problem statement Theory and discussion The machine model thinking Complex adaptive systems (CASs) Characteristics of a CAS Management and control in a CAS Criticism of the CAS approach Conclusion Reviewers

in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . individuals do not see themselves as multi - ill , but as needing support for their needs as they know them . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? lindberg observed examples of meaningful co - operation at the local level , with colleagues from different organizations meeting and pooling their knowledge of local conditions with the patient or user as the focal point . the phenomenon has been variously called the chain of care , integrated care , seamless care or shared care . this co - operation aims at creating a continuing relationship with the patient / user regardless of who at a given moment is the responsible provider . edgren and stenberg found these practical attributes of co - operation in cass : a common task is shared among the co - working agents.collaboration is in people s minds , it is instinctive behaviour.each actor s capabilities are known and respected.a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . according to brommels in his presentation to the ehma - conference 2006 , the cas approach means , among other things : identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts.avoiding strict definitions of roles and concentrating on agreed actions.giving agents their freedom to organize current activities . identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts . if we are to improve our understanding of how a health or social service provider functions as an integrated part of a locally driven health and social service system , we need an innovative , in terms of changing practice , model to guide our thinking . traditional models view systems as machines . it helps us understand what happens in dynamic living systems , where many agents are interconnected . in order to make clear its significance let us first recapitulate the machine model . for a long time effective organizations the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . so why turn to complexity science and to complex adaptive systems ( cas ) ? they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care . complexity can be expressed as the amount of information needed to describe or understand something . and the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . self - organization is about creating order or increasing the regularity of the system without help from the outside . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . a common vision , easy to understand and to communicate , there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . complexity science emphasizes the inherent power of development and self - organizing nature of the system . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . for example in general terms , there is a positive feedback loop between income and consumption in an economy . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science positive and negative feedback there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . so why turn to complexity science and to complex adaptive systems ( cas ) ? they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool . complexity can be expressed as the amount of information needed to describe or understand something . the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . they may either compete or co - operate according to their sense of their interests and what will bring them an advantage . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . a common vision , easy to understand and to communicate , was created to connect the two groups . there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . working in isolation the burden of coordination passes to the patient . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . the bigger the income of the individuals , the more the whole population consume , which further increasing their income as individuals . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . when the competence necessary for carrying out a given task does not lie within one individual provider organization , co - operation between agents within the system comes into play to discharge that task . the cas approach helps the management to understand why the traditional top down way of managing may meet with problems in organizations with complex tasks . an important discussion is about how the top management in fact executes its steering function . when we perceive health and social service organizations as cass we should gain more insight into the processes that go on within and between organizations . are we willing to face the interdependence between health and social services , the dependence on collaboration to deliver appropriate integrated care ?

with the aggravation of environmental pollution , lung cancer is almost the most malignant tumor in the world with its high morbidity and mortality.1 the most common histologic subtype is non - small cell lung cancer ( nsclc ) , which accounts for 80% of all lung cancers.2 although much progress has been made in the treatment of lung cancer , early diagnosis is difficult and the majority of patients has progressed to an advanced stage when diagnosed . the median survival rate for these patients is only 811 months.3 in 2004 , a landmark discovery had been made in that somatic mutations in the epidermal growth factor receptor ( egfr ) were associated with sensitivity to egfr tyrosine kinase ( tk ) inhibitors ( tkis ) ( egfr - tki).4,5 in subsequent large - scale randomized clinical trials , the relationship between egfr mutation status and efficacy of the egfr - tki drug was clearly explained.68 based on these findings , egfr mutation status in the tk domain can determine the treatment of advanced nsclc . mutations associated with enhanced sensitivity to egfr - tkis are found in exons 1821 of the tk domain of egfr ; in particular , del e746-a750 in exon 19 and the l858r point mutation in exon 21 account for nearly 90% of all the mutations in egfr in lung cancer.7,9,10 nowadays , the detection of egfr mutation status in nsclc patients has become an expert consensus.11 different methodologies have been developed for molecular testing , such as the amplified refractory mutation system ( arms ) , high - resolution melting , dna direct sequencing , and next - generation sequencing ( ngs ) . dna direct sequencing is considered the gold standard for the assessment of egfr mutation status in nsclc ; however , it is time consuming and laborious . the arms method is widely used in the clinical testing ; however , the commercial assay kit for egfr is very expensive , and the experiment needs to be done under good experimental conditions with sophisticated real - time polymerase chain reaction ( pcr ) instruments.12 immunohistochemistry ( ihc ) is a well - established method that is widely applied in conventional pathological diagnosis . egfr mutation - specific rabbit monoclonal antibodies against e746-a750 deletion and l858r ( cell signaling technology , inc . , danvers , ma , usa ) have been applied in ihc application . this provides a simple and rapid screening method for assessing egfr mutation status.1315 a nanofluorescent material , fluorescent semiconductor nanocrystal quantum dots ( qds ) , have been widely used in labeling some molecules , such as streptavidin and antibodies , through carbodiimide chemistry , optionally using edac ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) . qd ( 605 nm)-labeled streptavidin emits bright red fluorescence with 605 nm as the maximum emission wavelength while being stimulated by an excitation light source < 580 nm , which is different from green background autofluorescence . those labeled materials can be successfully applied to biological imaging and ihc detection of gene mutations , such as with human epidermal growth factor receptor 2 ( her 2 ) amplification in breast cancer.16,17 moreover , multiple markers can be visualized on one cell for in vitro multiplexed imaging ; for example , clinically significant tumor biomarkers including her2 , egfr , progesterone receptor , estrogen receptor , and mammalian target of rapamycin can be detected quantitatively and simultaneously in breast cancer cells using multicolor qds.18 qd - based immunofluorescence histochemistry ( qds - ihc ) is an established method ; it has been validated in many published papers.17,19,20 the staining signal detected by qds - ihc is much stronger with a lower background when compared with ihc.20 based on the application of egfr mutation - specific antibodies , this study was designed to develop a qd - based immunofluorescent approach for egfr mutation detection , which is a simple , quick , and highly sensitive molecular method for diagnosing egfr mutation status in nsclc samples . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) . this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture . nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series . qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . , antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections . after that , the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . , finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes . after rinsing for three times , sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer . after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen . after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) . the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) . the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown yellow or brown . immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs . the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded . staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) . accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen , people s republic of china ) has been widely used in the clinical laboratory . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion , the results were analyzed according to the criteria defined by the manufacturer s instructions . statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) . cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement . all tests were two - sided , and a p - value of < 0.05 was considered statistically significant . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) . this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture . nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series . qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . , antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections . specimens were incubated at 4c overnight with those three primary antibodies , respectively . after that , the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . , finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes . after rinsing for three times , sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer . resected tumor specimens were stained simultaneously using these three antibodies according to the manufacturer s instructions . after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen . after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) . following three washes in tbs the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) . the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown yellow or brown . immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs . the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded . staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) . accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen , people s republic of china ) has been widely used in the clinical laboratory . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion , the results were analyzed according to the criteria defined by the manufacturer s instructions . statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) . cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement . all tests were two - sided , and a p - value of < 0.05 was considered statistically significant . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) . thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) . we then compared the mutation status between adx - arms and immunostaining - based egfr . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) . thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) . we then compared the mutation status between adx - arms and immunostaining - based egfr . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . the superior optical and electronic properties of qds over conventional organic dyes , such as high brightness , high photostability , continuous absorption , narrow emission bandwidth , and the ability to simultaneously excite multiple fluorescent colors , make them attractive labels for the development of qds - ihc imaging for multiplexing cancer biomarker detection on ffpe tissues.24,25 in our study , we confirmed that qds - ihc is a simple and standardized method for detecting egfr mutations , and it has high sensitivity and specificity when compared with real - time pcr . in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer . in general , patients with advanced nsclc ( stage iiib ) do not benefit from surgery alone and are best managed by initial chemotherapy , chemotherapy plus radiation therapy , or radiation therapy alone . egfr mutation status plays a critical role in the therapeutic decision making for these patients . nowadays , egfr - tkis have been recommended as a first - line therapy in nsclc patients with activating mutations of egfr , including gefitinib , erlotinib , afatinib , and so on . several clinical studies have recently shown that egfr - tkis are superior to chemotherapy in nsclc patients with an egfr - activating mutation.26,27 therefore , it is highly important to evaluate the egfr mutation status in advanced nsclc patients , especially before any clinical therapy decision is undertaken . exon 19 del e746-a750 and exon 21 l858r point mutations represent the majority of egfr mutations.27,28 analysis of egfr mutations has become an important tool for targeted therapy in lung cancer , and recently , many efforts have been made to find a more specific and sensitive method ( including arms and ngs ) to detect them.29,30 when compared with conventional dna direct sequencing , targeted ngs provides a more accurate and clinically useful molecular classification method for lung adenocarcinoma.30 however , these techniques are relatively expensive for routine use in clinical laboratories , and they depend on the quality of the samples . the goal of our study was to evaluate the accuracy and sensitivity of qds - ihc in detecting egfr mutations in nsclc when compared with traditional ihc and arms . our results showed that egfr mutations in 40.0% ( 26/65 ) of nsclc patients were detected by qds - ihc , 12 ( 46.2% ) cases showed e746-a750-specific staining , and 14 ( 53.8% ) patients were l858r mutant - specific . we observed nearly perfect consistency between the positive immunostaining results of qds - ihc and adx - arms when detecting egfr mutations status . the sensitivity of qds - ihc when detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; both demonstrated antibody specificity of 100.0% ( 26/26 ) . however , ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . despite the small number of cases in our study , we also identified that qds - ihc combined with egfr mutation - specific antibodies to detect an egfr mutation has a high specificity and sensitivity in nsclc patients . furthermore , when compared with adx - arms and traditional ihc , qds - ihc for egfr mutation detection can be performed on very small biopsy specimens and pleural effusion cell blocks . this is especially the case in instances when the number of tumor cells of these precious biopsy samples could not reach dna or rna extraction requirements . according to our knowledge , this was the first report on the detection of egfr mutations using qds - ihc in nsclc patients . our study also showed that these methods could precisely detect egfr exon 19 deletion or exon 21 l858r mutation status ; adx - arms had the best sensitivity , which could also detect exon 18 point mutation and exon 20 insertion . nevertheless , adx - arms was more expensive and required strict conditions for routine use in clinical laboratories , which is difficult to carry out and popularize in a basic hospital setting . lung adenocarcinoma showed higher egfr mutations than squamous cell carcinoma ; this mutation was identified by three methods , and it was found that adenocarcinoma exhibits different biological behaviors and requires a different therapeutic strategy . in the present study , we found that qds exhibit excellent photostability , a broad excitation spectrum , and a long fluorescence lifetime.20,31 qds - ihc could accurately detect egfr mutation protein localization in nsclc . taken together , the qds - ihc technique achieves levels of sensitivity and specificity that are sufficient for detecting egfr mutation signals in ffpe surgery , biopsy , and cell block specimens , while minimizing costs and optimizing therapeutic options . combining this method with adx - arms

backgroundepidermal growth factor receptor ( egfr ) mutation status plays an important role in therapeutic decision making for non - small cell lung cancer ( nsclc ) patients . since egfr mutation - specific antibodies ( e746-a750del and l858r ) have been developed , egfr mutation detection by immunohistochemistry ( ihc ) is a suitable screening test . on this basis , we want to establish a new screening test , quantum dots immunofluorescence histochemistry ( qds - ihc ) , to assess egfr gene mutation in nsclc tissues , and we compared it to traditional ihc and amplification refractory mutation system ( arms).materials and methodsegfr gene mutations were detected by qds - ihc , ihc , and adx - arms in 65 cases of nsclc composed of 55 formalin - fixed , paraffin - embedded specimens and ten pleural effusion cell blocks , including 13 squamous cell carcinomas , two adenosquamous carcinomas , and 50 adenocarcinomas.resultspositive rates of egfr gene mutations detected by qds - ihc , ihc , and adx - arms were 40.0% , 36.9% , and 46.2% , respectively , in 65 cases of nsclc patients . the sensitivity of qds - ihc when detecting egfr mutations , as compared to adx - arms , was 86.7% ( 26/30 ) ; the specificity for both antibodies was 100.0% ( 26/26 ) . ihc sensitivity was 80.0% ( 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms had perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01).conclusionqds - ihc is a simple and standardized method to detect egfr mutations with its high sensitivity and specificity , as compared with real - time polymerase chain reaction . in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer .

Introduction Materials and methods Patients and samples QD immunofluorescence histochemistry staining Immunohistochemistry to detect EGFR mutation QDs-IHC and IHC scoring DNA extraction from NSCLC FFPE tissues and cell blocks Detection of EGFR mutations in exon 1821 by ARMS Statistical analysis Results EGFR mutation detected by QDs-IHC and IHC using mutation-specific antibodies in NSCLC EGFR mutation detected by ADx-ARMS Comparison of EGFR mutation detection by QDs-IHC, IHC, and ADx-ARMS Discussion Conclusion

with the aggravation of environmental pollution , lung cancer is almost the most malignant tumor in the world with its high morbidity and mortality.1 the most common histologic subtype is non - small cell lung cancer ( nsclc ) , which accounts for 80% of all lung cancers.2 although much progress has been made in the treatment of lung cancer , early diagnosis is difficult and the majority of patients has progressed to an advanced stage when diagnosed . the median survival rate for these patients is only 811 months.3 in 2004 , a landmark discovery had been made in that somatic mutations in the epidermal growth factor receptor ( egfr ) were associated with sensitivity to egfr tyrosine kinase ( tk ) inhibitors ( tkis ) ( egfr - tki).4,5 in subsequent large - scale randomized clinical trials , the relationship between egfr mutation status and efficacy of the egfr - tki drug was clearly explained.68 based on these findings , egfr mutation status in the tk domain can determine the treatment of advanced nsclc . mutations associated with enhanced sensitivity to egfr - tkis are found in exons 1821 of the tk domain of egfr ; in particular , del e746-a750 in exon 19 and the l858r point mutation in exon 21 account for nearly 90% of all the mutations in egfr in lung cancer.7,9,10 nowadays , the detection of egfr mutation status in nsclc patients has become an expert consensus.11 different methodologies have been developed for molecular testing , such as the amplified refractory mutation system ( arms ) , high - resolution melting , dna direct sequencing , and next - generation sequencing ( ngs ) . dna direct sequencing is considered the gold standard for the assessment of egfr mutation status in nsclc ; however , it is time consuming and laborious . the arms method is widely used in the clinical testing ; however , the commercial assay kit for egfr is very expensive , and the experiment needs to be done under good experimental conditions with sophisticated real - time polymerase chain reaction ( pcr ) instruments.12 immunohistochemistry ( ihc ) is a well - established method that is widely applied in conventional pathological diagnosis . egfr mutation - specific rabbit monoclonal antibodies against e746-a750 deletion and l858r ( cell signaling technology , inc . this provides a simple and rapid screening method for assessing egfr mutation status.1315 a nanofluorescent material , fluorescent semiconductor nanocrystal quantum dots ( qds ) , have been widely used in labeling some molecules , such as streptavidin and antibodies , through carbodiimide chemistry , optionally using edac ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) . those labeled materials can be successfully applied to biological imaging and ihc detection of gene mutations , such as with human epidermal growth factor receptor 2 ( her 2 ) amplification in breast cancer.16,17 moreover , multiple markers can be visualized on one cell for in vitro multiplexed imaging ; for example , clinically significant tumor biomarkers including her2 , egfr , progesterone receptor , estrogen receptor , and mammalian target of rapamycin can be detected quantitatively and simultaneously in breast cancer cells using multicolor qds.18 qd - based immunofluorescence histochemistry ( qds - ihc ) is an established method ; it has been validated in many published papers.17,19,20 the staining signal detected by qds - ihc is much stronger with a lower background when compared with ihc.20 based on the application of egfr mutation - specific antibodies , this study was designed to develop a qd - based immunofluorescent approach for egfr mutation detection , which is a simple , quick , and highly sensitive molecular method for diagnosing egfr mutation status in nsclc samples . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . we then compared the mutation status between adx - arms and immunostaining - based egfr . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . the superior optical and electronic properties of qds over conventional organic dyes , such as high brightness , high photostability , continuous absorption , narrow emission bandwidth , and the ability to simultaneously excite multiple fluorescent colors , make them attractive labels for the development of qds - ihc imaging for multiplexing cancer biomarker detection on ffpe tissues.24,25 in our study , we confirmed that qds - ihc is a simple and standardized method for detecting egfr mutations , and it has high sensitivity and specificity when compared with real - time pcr . in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer . egfr mutation status plays a critical role in the therapeutic decision making for these patients . nowadays , egfr - tkis have been recommended as a first - line therapy in nsclc patients with activating mutations of egfr , including gefitinib , erlotinib , afatinib , and so on . several clinical studies have recently shown that egfr - tkis are superior to chemotherapy in nsclc patients with an egfr - activating mutation.26,27 therefore , it is highly important to evaluate the egfr mutation status in advanced nsclc patients , especially before any clinical therapy decision is undertaken . exon 19 del e746-a750 and exon 21 l858r point mutations represent the majority of egfr mutations.27,28 analysis of egfr mutations has become an important tool for targeted therapy in lung cancer , and recently , many efforts have been made to find a more specific and sensitive method ( including arms and ngs ) to detect them.29,30 when compared with conventional dna direct sequencing , targeted ngs provides a more accurate and clinically useful molecular classification method for lung adenocarcinoma.30 however , these techniques are relatively expensive for routine use in clinical laboratories , and they depend on the quality of the samples . the goal of our study was to evaluate the accuracy and sensitivity of qds - ihc in detecting egfr mutations in nsclc when compared with traditional ihc and arms . our results showed that egfr mutations in 40.0% ( 26/65 ) of nsclc patients were detected by qds - ihc , 12 ( 46.2% ) cases showed e746-a750-specific staining , and 14 ( 53.8% ) patients were l858r mutant - specific . we observed nearly perfect consistency between the positive immunostaining results of qds - ihc and adx - arms when detecting egfr mutations status . the sensitivity of qds - ihc when detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; both demonstrated antibody specificity of 100.0% ( 26/26 ) . however , ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . despite the small number of cases in our study , we also identified that qds - ihc combined with egfr mutation - specific antibodies to detect an egfr mutation has a high specificity and sensitivity in nsclc patients . furthermore , when compared with adx - arms and traditional ihc , qds - ihc for egfr mutation detection can be performed on very small biopsy specimens and pleural effusion cell blocks . according to our knowledge , this was the first report on the detection of egfr mutations using qds - ihc in nsclc patients . our study also showed that these methods could precisely detect egfr exon 19 deletion or exon 21 l858r mutation status ; adx - arms had the best sensitivity , which could also detect exon 18 point mutation and exon 20 insertion . in the present study , we found that qds exhibit excellent photostability , a broad excitation spectrum , and a long fluorescence lifetime.20,31 qds - ihc could accurately detect egfr mutation protein localization in nsclc . taken together , the qds - ihc technique achieves levels of sensitivity and specificity that are sufficient for detecting egfr mutation signals in ffpe surgery , biopsy , and cell block specimens , while minimizing costs and optimizing therapeutic options .

malignant glaucoma is a rare but serious condition of secondary angle closure mostly seen after filtration surgery , but it may also occur spontaneously.1 it has also been described after laser iridotomy,2 laser capsulotomy,3 cataract surgery,4 topical miotic therapy,5 and blunt trauma.6 some basic mechanisms of ciliolenticular block glaucoma are poorly understood . lens disproportion and lens ciliary body apposition in small eyes and anterior hyaloid changes with increased hydraulic resistance are supposed to be major pathophysiological factors.7 besides these factors , poor vitreous flow and a tendency towards expansion of the choroidal volume in small eyes with angle closure refractory to iridotomy are further possible mechanisms of malignant glaucoma as proposed by quigley et al.8 these features lead to aqueous misdirection into the vitreous cavity restricted by the anterior hyaloid membrane and additional forward movement of the lens iris diaphragm.9,10 it is clinically characterized by an axial shallowing of the anterior chamber in the absence of a pupillary block mechanism , choroidal effusion , or hemorrhage , despite the presence of a patent iridectomy.11 intraocular pressure ( iop ) is usually dramatically increased , but may also be within the normal range.12 treatment of malignant glaucoma should be done stepwise . medical treatment includes topical antiglaucomatous medication , cycloplegic agents , aqueous suppressants , systemic carbonic anhydrase inhibitors , and hyperosmotic drugs inducing a posterior movement of the lens iris diaphragm , reducing production of aqueous humor , and dehydrating the vitreous volume.13 the performance of laser capsulotomy,14 or laser anterior hyaloidectomy,15 and argon laser of ciliary processes through a peripheral iridectomy16 to relieve the ciliolenticular block are recommended for unsuccessful medical treatment . when medical and laser treatment failed , surgery should be performed to interrupt the ciliolenticular block . pars plana vitrectomy in pseudophakic eyes or combined vitrectomy and cataract surgery in phakic eyes are effective methods to allow aqueous outflow into the anterior chamber.17,18 apart from anterior vitrectomy alone or in combination with phacoemulsification1719 or trabeculectomy20 via a posterior approach ( pars plana ) , a procedure called zonulo - hyaloido - vitrectomy through a peripheral iridectomy or iridotomy via the anterior chamber was described by lois et al in 2001.21 due to the difficulty in visualization of the anterior hyaloid membrane in phakic eyes and the risk of damaging the lens , chen et al published a new technique of videoendoscope - guided fluorescein - assisted vitrectomy in phakic eyes.22 in this study , a series of patients diagnosed with malignant glaucoma , who underwent pars plana vitrectomy to interrupt the ciliolenticular block mechanism , were reviewed . major outcome measures were the event preceding malignant glaucoma , the efficacy of vitrectomy including success rate based on iop reduction and need for medical treatment , and complications following vitrectomy in eyes with and without previous trabeculectomy . clinical records of 15 patients were reviewed retrospectively undergoing pars plana vitrectomy for treatment of malignant glaucoma at the university eye hospital of wuerzburg , germany between 1995 and 2010 . the following parameters were recorded : age , sex , lens status , primary ophthalmic diagnosis , past medical history , glaucoma medication , and the event preceding malignant glaucoma . preoperatively , all patients received a standard ophthalmic examination including best corrected visual acuity measurement , applanation tonometry , slit lamp biomicroscopy , and indirect ophthalmoscopy . clinical inclusion criteria of malignant glaucoma were as follows : shallow anterior chamber in the presence of a patent iridectomy or iridotomy after a choroidal hemorrhage or effusion have been ruled out . however , three patients were included according to the inclusion criteria despite having an iop < 20 mmhg . patients were divided into a group with ( trab group ) and one without ( nontrab group ) previous trabeculectomy to evaluate the influence of vitrectomy on success of patients who had undergone filtration surgery before . success was defined as an iop 21 mmhg and iop reduction by 20% ( definition one ) or an iop < 18 mmhg to baseline measures ( definition two ) with ( qualified success ) and without ( complete success ) iop - lowering medication . prior to surgical treatment , neodymium : yttrium - aluminium - garnet ( nd : yag ) laser iridotomy was performed in all patients of the nontrab group to rule out a pupillary block . of these , three patients required additional surgical iridectomy for inconsistent iridotomy . as intensive medical treatment ( including cycloplegic agents and topical and systemic iop - lowering medication ) remained unsuccessful and iop was persistently elevated , pars plana vitrectomy became a prerequisite . a 20-gauge port was used in six patients of the trab group and in all six patients of the nontrab group and a 23-gauge port in three patients of the trab group . anterior vitrectomy via pars plana was performed using a three - port technique to disrupt the anterior hyaloid membrane ensuring aqueous outflow from the vitreous cavity into the anterior chamber in order to interrupt the ciliolenticular block mechanism . existing adhesions and remnants of the vitreous body around the peripheral iridectomy were removed to allow patency for anterior aqueous flow . vitrectomy was continued until the anterior chamber deepened . in phakic eyes with significant cataract , combined vitrectomy and cataract extraction spss version 19.0 for windows ( ibm corporation , armonk , ny , usa ) was used for statistical analyses and to create figures . categorical variables were evaluated with pearson s chi - squared test or fisher s exact test . differences between groups in single discrete variables of nonnormal distribution were tested for significance using the mann . a maximum two - tailed p - value of < 0.05 was considered an indication of statistical significance . clinical records of 15 patients were reviewed retrospectively undergoing pars plana vitrectomy for treatment of malignant glaucoma at the university eye hospital of wuerzburg , germany between 1995 and 2010 . the following parameters were recorded : age , sex , lens status , primary ophthalmic diagnosis , past medical history , glaucoma medication , and the event preceding malignant glaucoma . preoperatively , all patients received a standard ophthalmic examination including best corrected visual acuity measurement , applanation tonometry , slit lamp biomicroscopy , and indirect ophthalmoscopy . clinical inclusion criteria of malignant glaucoma were as follows : shallow anterior chamber in the presence of a patent iridectomy or iridotomy after a choroidal hemorrhage or effusion have been ruled out . however , three patients were included according to the inclusion criteria despite having an iop < 20 mmhg . patients were divided into a group with ( trab group ) and one without ( nontrab group ) previous trabeculectomy to evaluate the influence of vitrectomy on success of patients who had undergone filtration surgery before . success was defined as an iop 21 mmhg and iop reduction by 20% ( definition one ) or an iop < 18 mmhg to baseline measures ( definition two ) with ( qualified success ) and without ( complete success ) iop - lowering medication . prior to surgical treatment , neodymium : yttrium - aluminium - garnet ( nd : yag ) laser iridotomy was performed in all patients of the nontrab group to rule out a pupillary block . of these as intensive medical treatment ( including cycloplegic agents and topical and systemic iop - lowering medication ) remained unsuccessful and iop was persistently elevated , pars plana vitrectomy became a prerequisite . a 20-gauge port was used in six patients of the trab group and in all six patients of the nontrab group and a 23-gauge port in three patients of the trab group . anterior vitrectomy via pars plana was performed using a three - port technique to disrupt the anterior hyaloid membrane ensuring aqueous outflow from the vitreous cavity into the anterior chamber in order to interrupt the ciliolenticular block mechanism . existing adhesions and remnants of the vitreous body around the peripheral iridectomy were removed to allow patency for anterior aqueous flow . vitrectomy was continued until the anterior chamber deepened . in phakic eyes with significant cataract , combined vitrectomy and cataract extraction spss version 19.0 for windows ( ibm corporation , armonk , ny , usa ) was used for statistical analyses and to create figures . categorical variables were evaluated with pearson s chi - squared test or fisher s exact test . differences between groups in single discrete variables of nonnormal distribution were tested for significance using the mann . a maximum two - tailed p - value of < 0.05 was considered an indication of statistical significance . the results of 15 patients undergoing anterior vitrectomy for treatment of malignant glaucoma during a 12-month follow - up are described . nine patients ( trab group ) developed a ciliolenticular block following filtration surgery with a patent iridectomy in the absence of a pupillary block , hypotony , choroidal effusion or hemorrhage ( table 1 ) . in six patients ( nontrab group ) , several intraocular surgeries , laser , or cyclodestructive treatments malignant glaucoma occurred in seven patients after trabeculectomy and in two patients after combined trabeculectomy and cataract extraction . the median time between filtration surgery and malignant glaucoma was 41.4 days ( range : 5205 days ) . of the patients in the nontrab group , one patient developed a ciliolenticular block after neodymium : yttrium - aluminium - garnet ( nd : yag ) laser peripheral iridotomy five patients after cataract surgery . mean time to malignant glaucoma was 14.0 days ( range : 141 days ) with one patient , who had a cataract extraction 9 years prior , excluded . the mean age of six female and three male patients in the trab group was 65 years ( range : 4188 years ) . four women and two men with a mean age of 78 years ( range : 6185 years ) were included in the nontrab group and were significantly older than patients with filtering blebs ( p = 0.046 ) . patients in both groups had a median axial length of 22.0 1.0 mm and 22.5 1.0 mm ( p = 0.383 ) with a median lens thickness of 4.8 0.9 mm and 4.6 0.7 mm ( p = 0.800 ) , respectively . five patients in the trab group and all the patients in the nontrab group were pseudophakic at the time of diagnosing malignant glaucoma . indications for combined surgery were a significant cataract and failed deepening of the anterior chamber during vitrectomy . the mean iop of patients in the trab group was 25.0 9.3 mmhg at the time of malignant glaucoma and was lower than in patients of the nontrab group ( 38.8 21.0 mmhg ) , although no statistical difference was found ( p = 0.135 ) . figure 1 illustrates iop development during follow - up for both groups . a reduction in iop iop was 20.0 3.9 mmhg in the trab group and 20.3 3.3 mmhg in the nontrab group at the 12-month visit . no statistical difference in iop was found between the two groups ( p = 0.571 ) . preoperatively , patients of the trab group received a total number of 2.0 1.3 ( range one to four ) topical agents . preoperatively , patients of the nontrab group needed 3.0 1.3 ( range two to five ) topical iop - lowering drugs . additional systemic medication ( acetazolamide , mannitol ) was necessary in all patients of both groups to lower iop . the number of patients with iop - lowering medication dropped to two patients in the trab group needing 2.0 1.4 medication after 1 month . one patient in the nontrab group was on 4.0 topical medications at the 1-month visit . however , seven patients in the trab group received a total of 2.3 0.8 medications and four patients in the nontrab group received 2.5 1.0 medications at the last available visit of each patient ( range : 7 days to 58 months ) . no statistical difference concerning pre- and postoperative iop - lowering medication was found between the two groups during follow - up ( p = 0.781 ) . according to the criteria for qualified success , two ( 40.0% ) patients in the trab group fulfilled both criteria of qualified success after 12 months ( follow - up rate 55.6% ) . none of the patients in the nontrab group had an iop 21 mmhg and 20% reduction of iop from baseline or an iop < 18 mmhg with or without iop - lowering medication ( follow - up rate 33.3% ) . complete success rate was 20.0% ( one patient ) according to both definitions ( iop 21 mmhg and 20% iop reduction ; iop < 18 mmhg ) in the trab group at the 12-month visit ( follow - up rate 55.6% ) . in the nontrab group , there was no statistical difference in complete success found between the two groups during follow - up ( table 3 ) . pars plana vitrectomy was successful in terminating malignant glaucoma and facilitated intraoperative deepening of the anterior chamber in all cases . the incidence of complications and surgical revisions are shown in table 4 . three of nine patients ( 33.3% ) in the trab group had transient hypotony ( defined as an iop < 5 mmhg ) for a mean duration of 3.3 3.2 days ( range : 17 days ) . all three patients with filtering blebs suffering from hypotony had a choroidal detachment for a duration ranging 39 days . postoperative hypotony lasting > 10 days without choroidal detachment was seen in one patient ( 16.7% ) of the nontrab group . there was no statistical difference in length of hypotony between both groups ( p = 0.500 ) . in four patients ( 44.4% ) of the trab group , anterior chamber flattened again for 17 days postoperatively and deepened spontaneously thereafter without further interventions . in contrast , in two patients ( 33.3% ) of the nontrab group , the anterior chamber was shallow for 3 days after vitrectomy , deepened spontaneously , and remained deep thereafter . further adverse events were failure of filtering blebs in four patients and progression of cataract in two patients of the trab group . neither choroidal effusion , blebitis , endophthalmitis , nor iris incarceration was seen in either group during follow - up . incidence of further surgical interventions including cyclodestructive procedures and revitrectomy is summarized in table 4 . in five patients ( 55.6% ) of the trab group and in three patients ( 50% ) of the nontrab group , one or more additional iop - lowering procedures nine patients ( trab group ) developed a ciliolenticular block following filtration surgery with a patent iridectomy in the absence of a pupillary block , hypotony , choroidal effusion or hemorrhage ( table 1 ) . in six patients ( nontrab group ) , several intraocular surgeries , laser , or cyclodestructive treatments malignant glaucoma occurred in seven patients after trabeculectomy and in two patients after combined trabeculectomy and cataract extraction . the median time between filtration surgery and malignant glaucoma was 41.4 days ( range : 5205 days ) . of the patients in the nontrab group , one patient developed a ciliolenticular block after neodymium : yttrium - aluminium - garnet ( nd : yag ) laser peripheral iridotomy five patients after cataract surgery . mean time to malignant glaucoma was 14.0 days ( range : 141 days ) with one patient , who had a cataract extraction 9 years prior , excluded . the mean age of six female and three male patients in the trab group was 65 years ( range : 4188 years ) . four women and two men with a mean age of 78 years ( range : 6185 years ) were included in the nontrab group and were significantly older than patients with filtering blebs ( p = 0.046 ) . patients in both groups had a median axial length of 22.0 1.0 mm and 22.5 1.0 mm ( p = 0.383 ) with a median lens thickness of 4.8 0.9 mm and 4.6 0.7 mm ( p = 0.800 ) , respectively . five patients in the trab group and all the patients in the nontrab group were pseudophakic at the time of diagnosing malignant glaucoma . indications for combined surgery were a significant cataract and failed deepening of the anterior chamber during vitrectomy . the mean iop of patients in the trab group was 25.0 9.3 mmhg at the time of malignant glaucoma and was lower than in patients of the nontrab group ( 38.8 21.0 mmhg ) , although no statistical difference was found ( p = 0.135 ) . figure 1 illustrates iop development during follow - up for both groups . a reduction in iop iop was 20.0 3.9 mmhg in the trab group and 20.3 3.3 mmhg in the nontrab group at the 12-month visit . no statistical difference in iop was found between the two groups ( p = 0.571 ) . preoperatively , patients of the trab group received a total number of 2.0 1.3 ( range one to four ) topical agents . preoperatively , patients of the nontrab group needed 3.0 1.3 ( range two to five ) topical iop - lowering drugs . additional systemic medication ( acetazolamide , mannitol ) was necessary in all patients of both groups to lower iop . the number of patients with iop - lowering medication dropped to two patients in the trab group needing 2.0 1.4 medication after 1 month . one patient in the nontrab group was on 4.0 topical medications at the 1-month visit . however , seven patients in the trab group received a total of 2.3 0.8 medications and four patients in the nontrab group received 2.5 1.0 medications at the last available visit of each patient ( range : 7 days to 58 months ) . no statistical difference concerning pre- and postoperative iop - lowering medication was found between the two groups during follow - up ( p = 0.781 ) . according to the criteria for qualified success , two ( 40.0% ) patients in the trab group fulfilled both criteria of qualified success after 12 months ( follow - up rate 55.6% ) . none of the patients in the nontrab group had an iop 21 mmhg and 20% reduction of iop from baseline or an iop < 18 mmhg with or without iop - lowering medication ( follow - up rate 33.3% ) . complete success rate was 20.0% ( one patient ) according to both definitions ( iop 21 mmhg and 20% iop reduction ; iop < 18 mmhg ) in the trab group at the 12-month visit ( follow - up rate 55.6% ) . in the nontrab group , there was no statistical difference in complete success found between the two groups during follow - up ( table 3 ) . pars plana vitrectomy was successful in terminating malignant glaucoma and facilitated intraoperative deepening of the anterior chamber in all cases . the incidence of complications and surgical revisions are shown in table 4 . three of nine patients ( 33.3% ) in the trab group had transient hypotony ( defined as an iop < 5 mmhg ) for a mean duration of 3.3 3.2 days ( range : 17 days ) . all three patients with filtering blebs suffering from hypotony had a choroidal detachment for a duration ranging 39 days . postoperative hypotony lasting > 10 days without choroidal detachment was seen in one patient ( 16.7% ) of the nontrab group . there was no statistical difference in length of hypotony between both groups ( p = 0.500 ) . in four patients ( 44.4% ) of the trab group , anterior chamber flattened again for 17 days postoperatively and deepened spontaneously thereafter without further interventions . in contrast , in two patients ( 33.3% ) of the nontrab group , the anterior chamber was shallow for 3 days after vitrectomy , deepened spontaneously , and remained deep thereafter . further adverse events were failure of filtering blebs in four patients and progression of cataract in two patients of the trab group . neither choroidal effusion , blebitis , endophthalmitis , nor iris incarceration was seen in either group during follow - up . incidence of further surgical interventions including cyclodestructive procedures and revitrectomy is summarized in table 4 . in five patients ( 55.6% ) of the trab group and in three patients ( 50% ) of the nontrab group , one or more additional iop - lowering procedures were necessary . in three patients of the trab group , four diode cyclophotocoagulations and cyclocryocoagulations were performed , respectively . two patients in the nontrab group needed diode cyclophotocoagulation and three patients received cyclocryocoagulation . no statistical difference regarding complications or surgical revisions ciliolenticular block glaucoma remains one of the most challenging serious adverse events necessitating immediate treatment to reduce aqueous misdirection into the vitreous and consecutive displacement of the lens iris diaphragm . in a recent review by ng and morgan , different pathophysiological features are described to play an important role for development of ciliolenticular block glaucoma , although not every part is clearly understood.7 lens disproportion and lens ciliary body apposition in at - risk eyes ( hyperopic , nanophthalmic ) and anterior hyaloid changes with increased hydraulic resistance due to a strong adhesion between vitreous , lens , and ciliary body are major pathogenic factors.7 in angle closure eyes , the lens is disproportionally large and increases in size , thereby pushing the iris and ciliary body forwards . in addition , as the lens ciliary body distance decreases , the fluid resistance increases and aqueous humor passes through the anterior vitreous restricted by the anterior hyaloid . moreover , lens apposition to the ciliary body may lead to reduced aqueous flow into the anterior chamber . both lens disproportion and lens ciliary body apposition result in forward movement of the lens iris diaphragm and a shallow anterior chamber.7 since malignant glaucoma also occurs in aphakic eyes , the lens seems not to be the only cause . flow resistance increases as the aqueous humor passes through the vitreous body and compresses the anterior hyaloid face . the pressure differential between the anterior and posterior chamber leads to an anterior displacement of iris and ciliary body . quigley et al described poor vitreous flow and a tendency towards expansion of the choroidal volume in small eyes with angle closure refractory to iridotomy as possible mechanisms of malignant glaucoma.8 anterior vitrectomy for malignant glaucoma refractory to medical and laser treatment facilitates aqueous humor outflow into the anterior chamber and reflects one recognizing pathophysiologic role of the vitreous in the mechanism of malignant glaucoma.7,23,24 results of vitrectomy in pseudophakic and aphakic eyes developing malignant glaucoma have been published in several previous studies.1822,25,26 nevertheless , vitrectomy alone in phakic eyes may be disappointing due to the difficulty in visualization and removal of the anterior hyaloid membrane without lens damaging.18,25,26 therefore , combined cataract extraction and vitrectomy even in eyes without lens opacities or mild lens opacities are strongly recommended . in this study of 15 patients diagnosed with malignant glaucoma , the efficacy and safety of pars plana vitrectomy for treatment of the ciliolenticular block was evaluated . the results were based on data of patients with and without previous filtration surgery during a median follow - up of 16.4 months . it was found that vitrectomy for malignant glaucoma was successful in iop reduction after medical treatment failed . vitrectomy seems to be effective in patients with and without previous trabeculectomy with adequate reduction of iop , although iop - lowering medication is necessary in nearly all cases to maintain desired iop . byrnes et al reported on a case series report of 20 patients with malignant glaucoma and success of vitrectomy in 50% of phakic patients and 90% of pseudophakic eyes.18 they recommended combined vitrectomy and crystalline lens extraction to allow removal of the entire anterior vitreous without damaging the lens . they defined success as immediate backward movement of the iris lens diaphragm during vitrectomy recovering the aqueous outflow , and deepening of the anterior chamber . this is in contrast to the current study in which success was based on criteria of iop reduction and medication use . although the anterior chamber deepened after vitrectomy in all cases of the current study , medication or subsequent iop - lowering procedures became necessary in the majority of patients to reach target iop . a study by harbour et al , in which surgical outcomes of vitrectomy of ten pseudophakic and 14 phakic eyes were compared , revealed a successful initial vitrectomy in 100% of seven phakic eyes undergoing concurrent lens extraction and in 71% of seven phakic eyes without cataract extraction.26 in pseudophakic patients , therefore , lensectomy during vitrectomy may be considered in cases of dense cataract or inability to deepen the anterior chamber . in the current study , more than two - thirds of patients were pseudophakic at the time of vitrectomy , whereas two patients received cataract extraction during vitrectomy . thus , two patients remained phakic and also had a successful iop reduction after deepening of the anterior chamber , but showed a progression of cataract during follow - up . in the current study , the 23-gauge transconjunctival sutureless vitrectomy was equally effective compared to the standard three - port 20-gauge system regarding reduction of iop and success rate . while the 23-gauge approach is frequently associated with a higher incidence of postoperative hypotony , no differences were found in complication rates between both systems . this is in line with previously published data on complications after vitrectomy for malignant glaucoma mostly encountering transient shallowing of the anterior chamber , hypotony , retinal or choroidal detachment , corneal decompensation , and bleb failure needing surgical revision.18,26 lois et al reported a novel technique of zonulo - hyaloido - vitrectomy using a vitreous cutter through a peripheral iridectomy or iridotomy via the anterior chamber in five patients.21 resolution of malignant glaucoma was achieved in all cases during a follow - up of 5.5 months . lois et al stated that zonulo - hyaloido - vitrectomy via an anterior approach appears to be an option in the treatment of pseudophakic malignant glaucoma and can be safely done by an anterior segment surgeon.21 due to the difficulty in visualization of the anterior hyaloid membrane in phakic eyes , chen et al introduced a novel technique of videoendoscope - guided fluorescein - assisted vitrectomy without cataract extraction in selected cases of prepresbyopic patients to avoid the need for clear lens extraction.22 however , they stated that the limited illumination field of the videoendoscope , when simultaneously used with the vitreous cutter , may contribute to an unintentional touch of the lens during vitrectomy . previous studies have addressed the importance of lensectomy to achieve desired iop reduction after the initial vitrectomy since pseudophakic eyes compared to phakic eyes show a higher success rate in terminating malignant glaucoma.18,19,25,26 the current study has some limitations . first , retrospective data commonly has more sources of error due to confounding and bias . second , the sample size was too small to detect small differences between the two groups . it is difficult to collect a large number of cases since malignant glaucoma is a very rare disease . prospective trials with greater statistical power will be needed to establish surgical methods of malignant glaucoma . in summary , pars plana vitrectomy using the 20- and 23-gauge approach is effective for treatment of malignant glaucoma in patients with and without previous filtration surgery . efficacy regarding reduction of iop , success , and complications rates was similar in eyes with and without previous trabeculectomy .

purposeto assess the outcomes of pars plana vitrectomy for the treatment of malignant glaucoma in patients with and without previous filtration surgery.patients and methodsdata of 15 patients developing malignant glaucoma after trabeculectomy ( 60% ) or following ophthalmic interventions other than filtration surgery ( 40% ) were recorded retrospectively . pars plana vitrectomy was performed in case of failed medical or laser treatment recreating the normal pathway of aqueous humor . the main outcome measures were the postoperative intraocular pressure ( iop ) , the frequency of complications , and success rate based on the following criteria : iop reduction by 20% and to 21 mmhg ( definition one ) or an iop < 18 mmhg ( definition two ) with ( qualified success ) and without ( complete success ) glaucoma medication.resultsvitrectomy reduced iop from baseline in eyes with and without previous trabeculectomy during a median follow - up of 16.4 months ( range 7 days to 58 months ) ; although the majority of patients required glaucoma medication to reach desired iop . the complete success rates were 11% ( both definitions ) for patients with filtering blebs and none of the patients without previous trabeculectomy had complete success at the 12-month visit . complications were few and included transient shallowing of the anterior chamber , choroidal detachment , corneal decompensation , filtering bleb failure , and need for further iop - lowering procedures.conclusionpars plana vitrectomy is equally effective for malignant glaucoma caused by trabeculectomy or interventions other than filtration surgery , although iop - lowering medication is necessary in nearly all cases to maintain target iop .

Introduction Material and methods Patients Surgical technique Data analysis Results Baseline data IOP Medication Success Complications and further interventional management Discussion Conclusion

malignant glaucoma is a rare but serious condition of secondary angle closure mostly seen after filtration surgery , but it may also occur spontaneously.1 it has also been described after laser iridotomy,2 laser capsulotomy,3 cataract surgery,4 topical miotic therapy,5 and blunt trauma.6 some basic mechanisms of ciliolenticular block glaucoma are poorly understood . lens disproportion and lens ciliary body apposition in small eyes and anterior hyaloid changes with increased hydraulic resistance are supposed to be major pathophysiological factors.7 besides these factors , poor vitreous flow and a tendency towards expansion of the choroidal volume in small eyes with angle closure refractory to iridotomy are further possible mechanisms of malignant glaucoma as proposed by quigley et al.8 these features lead to aqueous misdirection into the vitreous cavity restricted by the anterior hyaloid membrane and additional forward movement of the lens iris diaphragm.9,10 it is clinically characterized by an axial shallowing of the anterior chamber in the absence of a pupillary block mechanism , choroidal effusion , or hemorrhage , despite the presence of a patent iridectomy.11 intraocular pressure ( iop ) is usually dramatically increased , but may also be within the normal range.12 treatment of malignant glaucoma should be done stepwise . medical treatment includes topical antiglaucomatous medication , cycloplegic agents , aqueous suppressants , systemic carbonic anhydrase inhibitors , and hyperosmotic drugs inducing a posterior movement of the lens iris diaphragm , reducing production of aqueous humor , and dehydrating the vitreous volume.13 the performance of laser capsulotomy,14 or laser anterior hyaloidectomy,15 and argon laser of ciliary processes through a peripheral iridectomy16 to relieve the ciliolenticular block are recommended for unsuccessful medical treatment . pars plana vitrectomy in pseudophakic eyes or combined vitrectomy and cataract surgery in phakic eyes are effective methods to allow aqueous outflow into the anterior chamber.17,18 apart from anterior vitrectomy alone or in combination with phacoemulsification1719 or trabeculectomy20 via a posterior approach ( pars plana ) , a procedure called zonulo - hyaloido - vitrectomy through a peripheral iridectomy or iridotomy via the anterior chamber was described by lois et al in 2001.21 due to the difficulty in visualization of the anterior hyaloid membrane in phakic eyes and the risk of damaging the lens , chen et al published a new technique of videoendoscope - guided fluorescein - assisted vitrectomy in phakic eyes.22 in this study , a series of patients diagnosed with malignant glaucoma , who underwent pars plana vitrectomy to interrupt the ciliolenticular block mechanism , were reviewed . major outcome measures were the event preceding malignant glaucoma , the efficacy of vitrectomy including success rate based on iop reduction and need for medical treatment , and complications following vitrectomy in eyes with and without previous trabeculectomy . clinical records of 15 patients were reviewed retrospectively undergoing pars plana vitrectomy for treatment of malignant glaucoma at the university eye hospital of wuerzburg , germany between 1995 and 2010 . the following parameters were recorded : age , sex , lens status , primary ophthalmic diagnosis , past medical history , glaucoma medication , and the event preceding malignant glaucoma . patients were divided into a group with ( trab group ) and one without ( nontrab group ) previous trabeculectomy to evaluate the influence of vitrectomy on success of patients who had undergone filtration surgery before . success was defined as an iop 21 mmhg and iop reduction by 20% ( definition one ) or an iop < 18 mmhg to baseline measures ( definition two ) with ( qualified success ) and without ( complete success ) iop - lowering medication . as intensive medical treatment ( including cycloplegic agents and topical and systemic iop - lowering medication ) remained unsuccessful and iop was persistently elevated , pars plana vitrectomy became a prerequisite . anterior vitrectomy via pars plana was performed using a three - port technique to disrupt the anterior hyaloid membrane ensuring aqueous outflow from the vitreous cavity into the anterior chamber in order to interrupt the ciliolenticular block mechanism . vitrectomy was continued until the anterior chamber deepened . clinical records of 15 patients were reviewed retrospectively undergoing pars plana vitrectomy for treatment of malignant glaucoma at the university eye hospital of wuerzburg , germany between 1995 and 2010 . the following parameters were recorded : age , sex , lens status , primary ophthalmic diagnosis , past medical history , glaucoma medication , and the event preceding malignant glaucoma . clinical inclusion criteria of malignant glaucoma were as follows : shallow anterior chamber in the presence of a patent iridectomy or iridotomy after a choroidal hemorrhage or effusion have been ruled out . patients were divided into a group with ( trab group ) and one without ( nontrab group ) previous trabeculectomy to evaluate the influence of vitrectomy on success of patients who had undergone filtration surgery before . success was defined as an iop 21 mmhg and iop reduction by 20% ( definition one ) or an iop < 18 mmhg to baseline measures ( definition two ) with ( qualified success ) and without ( complete success ) iop - lowering medication . of these as intensive medical treatment ( including cycloplegic agents and topical and systemic iop - lowering medication ) remained unsuccessful and iop was persistently elevated , pars plana vitrectomy became a prerequisite . anterior vitrectomy via pars plana was performed using a three - port technique to disrupt the anterior hyaloid membrane ensuring aqueous outflow from the vitreous cavity into the anterior chamber in order to interrupt the ciliolenticular block mechanism . the results of 15 patients undergoing anterior vitrectomy for treatment of malignant glaucoma during a 12-month follow - up are described . four women and two men with a mean age of 78 years ( range : 6185 years ) were included in the nontrab group and were significantly older than patients with filtering blebs ( p = 0.046 ) . patients in both groups had a median axial length of 22.0 1.0 mm and 22.5 1.0 mm ( p = 0.383 ) with a median lens thickness of 4.8 0.9 mm and 4.6 0.7 mm ( p = 0.800 ) , respectively . indications for combined surgery were a significant cataract and failed deepening of the anterior chamber during vitrectomy . the mean iop of patients in the trab group was 25.0 9.3 mmhg at the time of malignant glaucoma and was lower than in patients of the nontrab group ( 38.8 21.0 mmhg ) , although no statistical difference was found ( p = 0.135 ) . preoperatively , patients of the nontrab group needed 3.0 1.3 ( range two to five ) topical iop - lowering drugs . the number of patients with iop - lowering medication dropped to two patients in the trab group needing 2.0 1.4 medication after 1 month . however , seven patients in the trab group received a total of 2.3 0.8 medications and four patients in the nontrab group received 2.5 1.0 medications at the last available visit of each patient ( range : 7 days to 58 months ) . no statistical difference concerning pre- and postoperative iop - lowering medication was found between the two groups during follow - up ( p = 0.781 ) . according to the criteria for qualified success , two ( 40.0% ) patients in the trab group fulfilled both criteria of qualified success after 12 months ( follow - up rate 55.6% ) . none of the patients in the nontrab group had an iop 21 mmhg and 20% reduction of iop from baseline or an iop < 18 mmhg with or without iop - lowering medication ( follow - up rate 33.3% ) . complete success rate was 20.0% ( one patient ) according to both definitions ( iop 21 mmhg and 20% iop reduction ; iop < 18 mmhg ) in the trab group at the 12-month visit ( follow - up rate 55.6% ) . in the nontrab group , there was no statistical difference in complete success found between the two groups during follow - up ( table 3 ) . pars plana vitrectomy was successful in terminating malignant glaucoma and facilitated intraoperative deepening of the anterior chamber in all cases . three of nine patients ( 33.3% ) in the trab group had transient hypotony ( defined as an iop < 5 mmhg ) for a mean duration of 3.3 3.2 days ( range : 17 days ) . all three patients with filtering blebs suffering from hypotony had a choroidal detachment for a duration ranging 39 days . in contrast , in two patients ( 33.3% ) of the nontrab group , the anterior chamber was shallow for 3 days after vitrectomy , deepened spontaneously , and remained deep thereafter . in five patients ( 55.6% ) of the trab group and in three patients ( 50% ) of the nontrab group , one or more additional iop - lowering procedures nine patients ( trab group ) developed a ciliolenticular block following filtration surgery with a patent iridectomy in the absence of a pupillary block , hypotony , choroidal effusion or hemorrhage ( table 1 ) . mean time to malignant glaucoma was 14.0 days ( range : 141 days ) with one patient , who had a cataract extraction 9 years prior , excluded . four women and two men with a mean age of 78 years ( range : 6185 years ) were included in the nontrab group and were significantly older than patients with filtering blebs ( p = 0.046 ) . patients in both groups had a median axial length of 22.0 1.0 mm and 22.5 1.0 mm ( p = 0.383 ) with a median lens thickness of 4.8 0.9 mm and 4.6 0.7 mm ( p = 0.800 ) , respectively . indications for combined surgery were a significant cataract and failed deepening of the anterior chamber during vitrectomy . the mean iop of patients in the trab group was 25.0 9.3 mmhg at the time of malignant glaucoma and was lower than in patients of the nontrab group ( 38.8 21.0 mmhg ) , although no statistical difference was found ( p = 0.135 ) . preoperatively , patients of the nontrab group needed 3.0 1.3 ( range two to five ) topical iop - lowering drugs . the number of patients with iop - lowering medication dropped to two patients in the trab group needing 2.0 1.4 medication after 1 month . however , seven patients in the trab group received a total of 2.3 0.8 medications and four patients in the nontrab group received 2.5 1.0 medications at the last available visit of each patient ( range : 7 days to 58 months ) . no statistical difference concerning pre- and postoperative iop - lowering medication was found between the two groups during follow - up ( p = 0.781 ) . according to the criteria for qualified success , two ( 40.0% ) patients in the trab group fulfilled both criteria of qualified success after 12 months ( follow - up rate 55.6% ) . none of the patients in the nontrab group had an iop 21 mmhg and 20% reduction of iop from baseline or an iop < 18 mmhg with or without iop - lowering medication ( follow - up rate 33.3% ) . complete success rate was 20.0% ( one patient ) according to both definitions ( iop 21 mmhg and 20% iop reduction ; iop < 18 mmhg ) in the trab group at the 12-month visit ( follow - up rate 55.6% ) . in the nontrab group , there was no statistical difference in complete success found between the two groups during follow - up ( table 3 ) . pars plana vitrectomy was successful in terminating malignant glaucoma and facilitated intraoperative deepening of the anterior chamber in all cases . three of nine patients ( 33.3% ) in the trab group had transient hypotony ( defined as an iop < 5 mmhg ) for a mean duration of 3.3 3.2 days ( range : 17 days ) . all three patients with filtering blebs suffering from hypotony had a choroidal detachment for a duration ranging 39 days . in contrast , in two patients ( 33.3% ) of the nontrab group , the anterior chamber was shallow for 3 days after vitrectomy , deepened spontaneously , and remained deep thereafter . in five patients ( 55.6% ) of the trab group and in three patients ( 50% ) of the nontrab group , one or more additional iop - lowering procedures were necessary . in a recent review by ng and morgan , different pathophysiological features are described to play an important role for development of ciliolenticular block glaucoma , although not every part is clearly understood.7 lens disproportion and lens ciliary body apposition in at - risk eyes ( hyperopic , nanophthalmic ) and anterior hyaloid changes with increased hydraulic resistance due to a strong adhesion between vitreous , lens , and ciliary body are major pathogenic factors.7 in angle closure eyes , the lens is disproportionally large and increases in size , thereby pushing the iris and ciliary body forwards . in addition , as the lens ciliary body distance decreases , the fluid resistance increases and aqueous humor passes through the anterior vitreous restricted by the anterior hyaloid . both lens disproportion and lens ciliary body apposition result in forward movement of the lens iris diaphragm and a shallow anterior chamber.7 since malignant glaucoma also occurs in aphakic eyes , the lens seems not to be the only cause . quigley et al described poor vitreous flow and a tendency towards expansion of the choroidal volume in small eyes with angle closure refractory to iridotomy as possible mechanisms of malignant glaucoma.8 anterior vitrectomy for malignant glaucoma refractory to medical and laser treatment facilitates aqueous humor outflow into the anterior chamber and reflects one recognizing pathophysiologic role of the vitreous in the mechanism of malignant glaucoma.7,23,24 results of vitrectomy in pseudophakic and aphakic eyes developing malignant glaucoma have been published in several previous studies.1822,25,26 nevertheless , vitrectomy alone in phakic eyes may be disappointing due to the difficulty in visualization and removal of the anterior hyaloid membrane without lens damaging.18,25,26 therefore , combined cataract extraction and vitrectomy even in eyes without lens opacities or mild lens opacities are strongly recommended . in this study of 15 patients diagnosed with malignant glaucoma , the efficacy and safety of pars plana vitrectomy for treatment of the ciliolenticular block was evaluated . the results were based on data of patients with and without previous filtration surgery during a median follow - up of 16.4 months . it was found that vitrectomy for malignant glaucoma was successful in iop reduction after medical treatment failed . vitrectomy seems to be effective in patients with and without previous trabeculectomy with adequate reduction of iop , although iop - lowering medication is necessary in nearly all cases to maintain desired iop . byrnes et al reported on a case series report of 20 patients with malignant glaucoma and success of vitrectomy in 50% of phakic patients and 90% of pseudophakic eyes.18 they recommended combined vitrectomy and crystalline lens extraction to allow removal of the entire anterior vitreous without damaging the lens . they defined success as immediate backward movement of the iris lens diaphragm during vitrectomy recovering the aqueous outflow , and deepening of the anterior chamber . although the anterior chamber deepened after vitrectomy in all cases of the current study , medication or subsequent iop - lowering procedures became necessary in the majority of patients to reach target iop . thus , two patients remained phakic and also had a successful iop reduction after deepening of the anterior chamber , but showed a progression of cataract during follow - up . in the current study , the 23-gauge transconjunctival sutureless vitrectomy was equally effective compared to the standard three - port 20-gauge system regarding reduction of iop and success rate . this is in line with previously published data on complications after vitrectomy for malignant glaucoma mostly encountering transient shallowing of the anterior chamber , hypotony , retinal or choroidal detachment , corneal decompensation , and bleb failure needing surgical revision.18,26 lois et al reported a novel technique of zonulo - hyaloido - vitrectomy using a vitreous cutter through a peripheral iridectomy or iridotomy via the anterior chamber in five patients.21 resolution of malignant glaucoma was achieved in all cases during a follow - up of 5.5 months . lois et al stated that zonulo - hyaloido - vitrectomy via an anterior approach appears to be an option in the treatment of pseudophakic malignant glaucoma and can be safely done by an anterior segment surgeon.21 due to the difficulty in visualization of the anterior hyaloid membrane in phakic eyes , chen et al introduced a novel technique of videoendoscope - guided fluorescein - assisted vitrectomy without cataract extraction in selected cases of prepresbyopic patients to avoid the need for clear lens extraction.22 however , they stated that the limited illumination field of the videoendoscope , when simultaneously used with the vitreous cutter , may contribute to an unintentional touch of the lens during vitrectomy . previous studies have addressed the importance of lensectomy to achieve desired iop reduction after the initial vitrectomy since pseudophakic eyes compared to phakic eyes show a higher success rate in terminating malignant glaucoma.18,19,25,26 the current study has some limitations . in summary , pars plana vitrectomy using the 20- and 23-gauge approach is effective for treatment of malignant glaucoma in patients with and without previous filtration surgery . efficacy regarding reduction of iop , success , and complications rates was similar in eyes with and without previous trabeculectomy .

photochemistry is among the most powerful complexity generating tools available to chemists . fundamentally groundbreaking reactions such as di--methane rearrangements and arene olefin cycloadditions have rarely been exceeded in their potential to construct molecular architecture , which would be unachievable in the absence of the reaction . however , traditional batch - mode photochemistry has a number of disadvantages that have hindered its implementation and development in traditional synthetic organic laboratories . typical batch reactor systems often suffer from poor uv penetration , long diffusion lengths , and poor temperature control . while there have been a number of examples utilizing flow chemistry to increase the effectiveness of photochemistry , we have focused our application on reaction screening and discovery of complex chemotypes . to this end , we have developed a microfluidic - based photochemistry platform capable of running pulse or continuous flow reactions , which is more suitable for high - throughput chemistry . in recent years , a number of exciting reaction discovery and development paradigms have been highlighted in the literature . examples such as multidimensional reaction screening developed in our laboratory , mcmillan s advent of enabled serendipity , and hartwig s use of mass spectrometry to analyze thousands of reaction mixtures are a testament to the potential of screening reactions in parallel . over the past several years , we have developed reaction screening techniques that take advantage of analytical reaction scale and microfluidics . herein , we report the expansion of our microfluidic platform to incorporate photo - optics for higher levels of wavelength control and its application to a multidimensional reaction screen . we have developed a photochemical microfluidic platform utilizing a 1 kw capillary mercury lamp and microreactor fabricated with schott 8337 , a uv transparent glass . in an effort to advance the utility of photochemistry through expanded wavelength control , we designed a system that also incorporates photo - optical components ( figure 1 ) . specifically , a plano - concave lens focuses and collimates the beam , followed by a heat mirror that reflects ir wavelengths . directly in front of the microfluidic device a wide range of uv filters , both long - pass and band - pass , are commercially available . one of the exciting applications of our photochemical platform is multidimensional reaction screening wherein multiple substrates are evaluated in photochemical reactions with varying wavelength , sensitizer , temperature , and solvent . with this in mind , we selected a variety of substrates ( 14 ) that were part of the compound library collection in the cmld - bu ( figure 2 ) . the absorption spectrum of each compound indicated the presence of a chromophore that could potentially be excited under photochemical conditions . along with four long - pass uv filters , we selected a series of sensitizers ( matched with appropriate uv filters ) with triplet energies ranging from 41 to 74 kcal / mol , as well as two electron - transfer sensitizers . multidimensional reaction screening parameters . two temperatures ( room temperature and 60 c ) and two solvents ( ch3cn and thf ) were also screened . each reaction was performed on 100 g scale at a concentration of 0.05 m. the sensitizers were utilized at 1.0 mol equiv . the residence time within the microfluidic device was 5 min ( 8 l / min ) . the reactions were subsequently collected into a 96 well - plate and analyzed by uplc / elsd . each reaction showing potential new products by uplc analysis was scaled up using continuous flow and the same microfluidic device and photochemical platform . the continuous flow reactions afforded appropriate amounts of material for structure elucidation . in total however , while we were able to reproduce reactions ( as shown by the identical uplc spectra ) , only four substrates afforded isolable products . the reaction screen revealed multiple conditions leading to cyclopropane 5 via an oxa - di--methane rearrangement ( eq 1).1 the reaction was facilitated by both direct excitation and triplet sensitization . further optimization of the reaction revealed that reactions with a wavelength window of 340405 nm were not productive unless sensitized with benzophenone , which has a triplet energy of 68 kcal / mol and marginal absorption in the uv filter window ( figure 3 ) . reaction screen profile . however , when the reaction was carried out in absence of a sensitizer and a blue - shifted uv filter ( 305365 nm ) , it proceeded to full conversion and was exceptionally clean . in this case , the reaction was fully quenched in the presence of sensitizers with triplet energies ranging from 41 to 53 kcal / mol . the second reaction identified was a product derived from substrate 2 ( scheme 1 ) . when heated in acetonitrile and irradiated at wavelengths > 280 nm , 2 undergoes a homolytic c n bond fragmentation leading to diradical intermediate 6 followed by a 1,4-hydride shift to form isoquinoline 7 , which rapidly isomerizes to diastereomer 8 during column chromatography . although this is a simple reaction , it was notable that the original reaction conditions from the reaction screen resulted in incomplete conversion and significant decomposition . utilizing the flexibility of the platform , we were able to optimize the transformation with a lower temperature and shorter residence time ( 10 c and 3 min ) and slightly longer uv wavelength ( > 295 nm ) . a third reaction was observed in the reaction screen utilizing tetracyclic scaffold 3 . elucidation of the purified material revealed cyclobutanone 9 , which is derived from a 1,3-acyl shift ( eq 2 ) . the reaction screen profile revealed that utilizing a uv filter > 280 nm resulted in nearly complete conversion but significant decomposition by h nmr ( figure 4a ) . at wavelengths the reaction screen also revealed that the reaction was fully quenched by both acetophenone and benzophenone ( triplet energies of 68 and 74 kcal / mol , respectively).2 ( a ) reaction screen profile . ( b ) absorption spectrum of 3 with bars indicating that reactivity is wavelength dependent . on the basis of data from the reaction screen , we carried out further optimization focused on wavelength variation . as summarized in figure 4b , reactions focused on a chromophore at 255 nm resulted in no reaction . significant loss of reactivity was observed at wavelengths > 340 nm and no reactivity at wavelengths > 370 nm . notably , decomposition could be substantially avoided at wavelengths > 320 nm . utilizing the > 300 nm uv filter , we evaluated longer residence times in order to drive the reaction to completion . however , this resulted in formation of a second product which was determined to be tetrahydrofluorene 10 which was derived from a thermal decarbonylation . because cyclobutanone 9 is extremely unstable and difficult to purify thus , microwave irradiation immediately following the photochemical transformation afforded ketone 10 as a 1:1 mixture of diastereomers in 27% yield ( scheme 2 ) . we further evaluated the scope of this reaction as a one - pot , two - step procedure utilizing compounds 11,13 , and 15 . reaction of substrate 11 afforded ketone 12 ( as a 1.5:1 mixture ) in 43% yield , whereas cyclobutanone 14 could be isolated cleanly from 13 and did not decarbonylate under microwave radiations . finally , ketol substrate 15 did not show any sign of reactivity under several photochemical conditions . a fourth reaction was identified in the multidimensional reaction screen involving glycal - derived substrate 4 ( eq 3 ) . however , upon scale - up and nmr analysis , it was apparent two products were present and , upon isolation and elucidation , we determined that indole 16 and cyclobutane 17 were the major products . indole 16 is derived from a simple photo - fries migration of the sulfonyl moiety to c3 , while cyclobutane 17 may arise from an initial [ 2 + 2 ] cycloaddition followed by migration of the sulfonate to the indole c7.3 close analysis of the reaction screening results and follow - up optimization suggested that the reaction pathways were wavelength dependent . as illustrated in figure 5 , a uv filter with a window of 260300 nm resulted in the exclusive formation of photo - fries product 16 . a slightly red - shifted uv filter ( > 275 nm ) resulted in a mixture of 16 and 17 ( favoring 16 ) , and a uv filter with a longer wavelength window of 290340 nm favored formation of cyclobutane 17 the reaction was further optimized by varying residence time and temperature . thus , indole 16 could be isolated in 50% yield when the reaction was carried out at 60 c with a 5 min residence time , and cyclobutane 17 could be isolated in 24% yield ( no indole 16 was observed ) when the reaction was carried out at room temperature with a 10 min residence time . most notably , irradiation of 16 at various wavelengths and conditions did not result in formation of 17 . it has been shown that indoles bearing n - acyl functionality can undergo [ 2 + 2 ] cycloadditions along with competing photo - fries rearrangement . however , if the fries rearrangement occurs from a high energy singlet excited state , longer wavelengths may favor [ 2 + 2]-cycloaddition , which proceeds via a 1,4-diradical in the triplet excited state . indeed , irradiation of boc - protected 18 and acetate - protected 20 at 290340 nm afforded only cyclobutanes 19 and 21 ( scheme 3 ) . however , reactions at shorter wavelengths did not result in migration to c3 but only in decomposition . indoles that were not acylated ( n h or n ch3 ) were unreactive under the photochemical reaction conditions . changing the electronics of the indole ring with 5-chloro substitution ( 22 ) resulted in only the photo - fries product ( 23 ) in low yield . in conclusion , we have successfully adapted a photochemical microfluidics platform for use in multidimensional reaction screening . by adding a photo - optics system , we are able to effectively control the wavelength of the reactions utilizing uv filters . such control allows for introduction of irradiation wavelength as a screening variable as well as downstream optimization of reactions that are highly dependent on wavelength . utilizing the platform in a multidimensional reaction screen , we were able to identify new chemotypes derived from photochemical transformations of complex scaffolds . most notably , we identified complex chemotypes derived from an oxa - di--methane rearrangement of a bicyclo[3.2.1]octanoid scaffold , a 1,3-acyl shift of bicyclic ketol , and a wavelength - dependent photo - fries/[2 + 2]-cycloaddition . the new scaffolds are currently being used to develop methodologies and access additional complex and unique molecules for use in high - throughput screening and medicinal chemistry programs . all reactions were carried out in a flame - dried apparatus under an ar atmosphere , unless otherwise noted . solvents ( methylene chloride , thf , and acetonitrile ) were dried by passage through columns of neutral alumina . flash chromatography was performed using silica gel ( siliaflash p60 , 230400 mesh ) purchased from silicycle , or a flash chromatography system utilizing silica gel columns from interchim . analytical thin - layer chromatography was performed on 0.25 mm sio2 60 f-254 plates . tlc plates were analyzed using uv illumination and staining with basic kmno4 solution . chemical shifts ( ) are reported in parts per million ( ppm ) relative to the solvent ( cdcl3 at 7.27 ( 77.0 ) ppm or c6d6 at 7.16 ( 128 ) ppm ) . coupling constants ( j ) are reported in hertz ( hz ) with multiplicities denoted as br ( broad ) , s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , m ( multiplet ) . stock solutions of substrate ( 0.1 m ) and sensitizers ( 0.1 m ) were prepared in ch3cn or thf ( anhydrous ) while maintaining dry handling . the solutions were placed in a custom - designed 96-well aluminum holding block fitted with oven - dried glass sleeves . the block was attached to the microfluidics platform , and a slow , steady stream of argon was passed through the inert gas chamber . the system was fitted with the appropriate uv filter , and the parameters were set to achieve 5 min residence time at the chosen temperature . each reaction was analyzed by uplc / ms / elsd ( 1090% ch3cn , 2 min ) . reactions showing apparent reactivity were subsequently scaled up and isolated , using the system in continuous flow , for further characterization . the system was fitted with the appropriate optical filter and set at the appropriate temperature . a solution of substrate ( 1.0 equiv ) in ch3cn was loaded into a syringe and flowed through the device via a syringe pump set at the appropriate flow rate to obtain the desired residence time . the reactions were collected in a vial , concentrated in vacuo , and purified by flash chromatography . gly - omehcl ( 0.084 g , 0.666 mmol ) was added to a solution of aldehyde 24(51 ) ( 0.122 g , 0.444 mmol ) in dcm ( 2.2 ml ) , followed by mgso4 ( 0.266 g , 2.22 mmol ) and et3n ( 0.08 ml , 0.666 mmol ) . it was then concentrated in vacuo and dissolved in etoac ( 5 ml ) , filtered , and concentrated in vacuo . the resulting solid was dissolved in dcm ( 1.5 ml ) and agotf ( 0.011 g , 0.044 mmol ) was added , followed by molecular sieves ( 0.03 g ) and tfa ( 0.04 ml , 0.489 mmol ) . the resulting mixture was stirred at room temperature for 1 h and filtered over a cotton plug . the filtrate was concentrated in vacuo to afford intermediate which was dissolved in phcf3 ( 4.4 ml ) to be used directly in the next step . 1-methyl-1h - pyrrole-2,5-dione ( 0.049 g , 0.444 mmol ) was then added followed by hunig s base ( 0.09 ml , 0.489 mmol ) . the reaction mixture was stirred at room temperature for 3 h and concentrated in vacuo . purification by flash chromatography ( 5:1 hexanes / etoac ) afforded 2 as a white amorphous solid ( 0.063 g , 31% ) over 3 steps : rf = 0.58 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2956 , 2924 , 1708 , 1435 , 1329 , 1118 ; h nmr ( cdcl3 , 400 mhz ) 7.467.50 ( m , 2h ) , 7.397.42 ( m , 3h ) , 7.31 ( bs , 1h ) , 7.04 ( d , j = 7.6 hz , 1h ) , 5.36 ( s , 1h ) , 5.20 ( d , j = 7.6 hz , 1h ) , 4.91 ( s , 1h ) , 3.86 ( s , 3h ) , 3.74 ( d , j = 7.6 hz , 1h ) , 3.48 ( t , j = 7.6 hz , 1h ) , 2.89 ( s , 3h ) ; c nmr ( cdcl3 , 100 mhz ) 176.3 , 174.7 , 170.4 , 148.7 , 136.1 , 135.9 , 132.1 , 129.6 , 129.2 , 128.8 , 127.8 ( d , jc f = 32 hz ) , 127.6 , 126.0 ( d , jc f = 4 hz ) , 125.8 , 125.0 , 124.5 ( d , jc f = 281 hz ) , 124.3 ( d , jc f = 4 hz ) , 102.0 , 67.1 , 63.4 , 53.6 , 51.5 , 46.5 ; hrms - esi ( m / z ) [ m + h ] calcd for c24h20f3n2o4 457.13785 , found 457.1362 . ( 1 ) prepared according to general continuous flow procedure using 1(21 ) and benzophenone ( 1 equiv ) in ch3cn ( 0.05 m ) with a 340405 nm band - pass filter and a 2 min residence time at room temperature . ( 2 ) prepared according to general continuous flow procedure using 1 in ch3cn ( 0.05 m ) with a 305365 nm band - pass filter and a 2 min residence time at room temperature . prepared according to the general continuous flow procedure using 2 ( 0.0143 g , 0.031 mmol ) in ch3cn ( 1.24 ml ) with a 295 nm long pass filter and a 4 min residence time at 10 c . the reaction mixture was concentrated in vacuo to afford 7 ; h nmr ( cdcl3 , 500 mhz ) 8.49 ( s , 1h ) , 8.10 ( s , 1h ) , 7.998.04 ( m , 2h ) , 7.857.89 ( m , 1h ) , 7.437.54 ( m , 4h ) , 5.35 ( d , j = 8.5 hz , 1h ) , 3.91 ( ddd , j = 11.0 , 8.5 , 4.0 hz , 1h ) , 3.23 ( s , 3h ) , 3.20 ( s , 1h ) , 2.99 ( dd , j = 18.0 , 4.0 hz , 1h ) , 2.25 ( dd , j = 17.5 , 11.0 hz , 1h ) ; c nmr ( cdcl3 , 125 mhz ) 178.4 , 176.4 , 171.8 , 156.4 , 151.7 , 139.3 , 137.8 , 129.8 , 129.7 , 129.6 , 129.5 , 129.2 , 129.2 , 127.2 , 126.9 , 126.5 ( d , jc f = 2 hz ) , 125.8 , 123.1(d , jc f = 4 hz ) , 116.1 , 51.9 , 47.6 , 42.3 , 30.4 , 25.7 . purification by flash chromatography ( 5:1 hexanes / etoac ) afforded 8 as an oil ( 0.0051 g , 36% ) : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2958 , 2924 , 2852 , 1703 , 1635 , 1436 , 1329 , 1294 , 1122 ; h nmr ( cdcl3 , 400 mhz ) 8.59 ( s , 1h ) , 8.11 ( m , 3h ) , 8.04 ( d , j = 8.4 hz , 1h ) , 7.90 ( d , j = 9.6 hz , 1h ) , 7.447.54 ( m , 4h ) , 5.01 ( d , j = 5.6 hz , 1h ) , 4.61 ( q , j = 6.0 hz , 1h ) , 3.64 ( s , 3h ) , 3.143.20 ( m , 1h ) , 3.12 ( s , 1h ) , 2.87 ( dd , j = 17.6 , 3.6 hz , 1h ) ; c nmr ( cdcl3 , 100 mhz ) 178.6 , 175.4 , 171.9 , 155.2 , 151.7 , 139.4 , 138.3 , 129.7 , 129.2 , 128.8 , 127.3 , 126.5 , 126.3 , 124.1 , 124.0 , 116.5 , 52.4 , 50.1 , 41.7 , 33.2 , 25.8 ; hrms - esi ( m / z ) [ m + h ] calcd for c24h20f3n2o4 457.1375 , found 457.1373 . prepared according to general continuous flow procedure using ketone 3(30 ) ( 0.0014 g , 0.05 mmol ) in ch3cn ( 1 ml ) with a 305365 nm band - pass filter and a 10 min residence time at 60 c . the reaction mixture was then transferred to a microwave flask and heated to 120 c for 15 min in a cem microwave . purification by flash chromatography ( 15:1:1 hexanes / dcm / etoac ) afforded a 1:1 mixture of 10a and 10b . 10a : ( 0.002 g , 14% ) as a yellow oil ; rf = 0.53 ( 15:1:1 hexanes / dcm / etoac ) ; ir ( thin film ) ( cm ) 2960 , 2931 , 1816 , 1711 , 1460 , 1362 , 755 ; h nmr ( cdcl3 , 400 mhz ) 7.137.20 ( m , 4h ) , 5.33 ( d , j = 4.4 hz , 1h ) , 3.87 ( d , j = 4.4 hz , 1h ) , 3.46 ( q , j = 7.2 hz , 1h ) , 3.07 ( dd , j = 15.2 , 8.0 hz , 1h ) , 2.943.00 ( m , 1h ) , 2.69 ( dd , j = 15.2 , 6.8 hz , 1h ) , 2.38 ( s , 3h ) , 2.34 ( dd , j = 16.8 , 7.2 hz , 1h ) , 2.172.24 ( m,1h ) , 2.10 ( dd , j = 17.2 , 6.0 hz , 1h ) , 0.94 ( d , j = 4.4 hz , 3h ) , 0.91 ( d , j = 5.2 hz , 3h ) ; c nmr ( cdcl3 , 100 mhz ) 198.4 , 149.5 , 147.9 , 142.5 , 126.8 , 126.8 , 125.0 , 123.7 , 113.3 , 45.9 , 42.2 , 38.6 , 37.7 , 35.8 , 29.3 , 25.2 , 21.3 , 21.2 ; hrms - esi ( m / z ) [ m + h ] calcd for c18h23o 255.1749 , found 255.1773 . 10b : ( 0.0018 g , 13% ) as a colorless oil ; rf = 0.5 ( 15:1 : hexanes / dcm / etoac ) ; ir ( thin film ) ( cm ) 2960 , 2927 , 1709 , 1665 , 1459 , 1353 , 757 ; h nmr ( c6d6 , 400 mhz ) 7.047.12 ( m , 4h ) , 5.29 ( d , j = 4.0 hz , 1h ) , 3.25 ( q , j = 6.8 hz , 1h ) , 2.822.92 ( m , 2h ) , 2.68 ( d , j = 4.4 hz , 1h ) , 2.57 ( dd , j = 14.4 , 4.8 hz , 1h ) , 2.19 ( dd , j = 17.2 , 7.6 hz , 1h ) , 2.05 ( sept , j = 7.2 hz , 1h ) , 1.97 ( dd , j = 16.8 , 5.2 hz , 1h ) , 1.78 ( s , 3h ) , 0.87 ( d , j = 6.4 hz , 6h ) ; c nmr ( c6d6 , 100 mhz ) 207.2 , 148.1 , 146.8 , 143.1 , 127.1 , 127.1 , 125.5 , 123.8 , 116.5 , 54.0 , 42.8 , 39.1 , 38.8 , 35.9 , 28.9 , 28.2 , 21.5 , 21.4 ; hrms - esi ( m / z ) [ m + h ] calcd for c18h22o 255.1749 , found 255.1754 . prepared according to the general continuous flow procedure using ketone 11(30 ) ( 0.0125 g , 0.053 mmol ) in ch3cn ( 1.1 ml ) with a 305365 nm band - pass filter and a 10 min residence time at 60 c . the reaction mixture was then transferred to a microwave flask and heated to 120 c for 15 min in a cem microwave . purification by flash chromatography ( 10:1 hexanes / etoac ) afforded a 1:1 mixture of 12a and 12b . 12a : ( 0.003 g , 26% ) as a yellow oil ; rf = 0.52 ( 5:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2961 , 2872 , 1707 , 1665 , 1063 ; h nmr ( c6d6 , 400 mhz ) 5.43 ( d , j = 6.4 hz , 1h ) , 4.32 ( ddd , j = 7.8 , 5.1 , 3.1 hz , 1h ) , 3.75 ( dd , j = 6.4 , 2.9 hz , 1h ) , 3.68 ( td , j = 8.4 , 3.5 hz , 1h ) , 3.313.37 ( m , 1h ) , 2.58 ( qd , j = 7.8 , 3.1 hz , 1h ) , 2.21 ( dd , j = 16.8 , 3.2 hz , 1h ) , 2.002.08 ( m , 2h ) , 1.92 ( s , 3h ) , 1.571.64 ( m , 1h ) , 1.261.26 ( m , 1h ) , 0.86 ( t , j = 6.8 hz , 6h ) ; c nmr ( c6d6 , 100 mhz ) 198.2 , 150.2 , 113.2 , 77.5 , 67.1 , 46.2 , 37.3 , 36.0 , 33.6 , 30.7 , 24.6 , 21.2 , 21.1 ; hrms - esi ( m / z ) [ m + h ] calcd for c13h21o2 209.1542 , found 209.1547 . 12b : ( 0.002 g , 17% ) as an oil : rf = 0.47 ( 5:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2962 , 2873 , 1708 , 1673 , 1355 , 1057 ; h nmr ( cd3cn , 500 mhz ) 5.55 ( d , j = 5.0 hz , 1h ) , 4.07 ( ddd , j = 6.6 , 5.7 , 3.8 hz , 1h ) , 3.78 ( td , j = 8.0 , 4.7 hz , 1h ) , 3.56 ( dt , j = 8.5 , 7.5 hz , 1h ) , 3.05 ( t , j = 4.5 hz , 1h ) , 2.53 ( quint . , j = 7.0 hz , 1h ) , 2.172.27 ( m , 2h ) , 2.13 ( s , 3h ) , 1.992.08 ( m , 2h ) , 1.57 ( dtd , j = 11.9 , 7.7 , 6.6 hz , 1h ) , 0.99 ( d , j = 3.0 hz , 3h ) , 0.98 ( d , j = 2.0 hz , 3h ) ; c nmr ( cd3cn , 125 mhz ) 209.5 , 146.9 , 116.5 , 77.3 , 67.1 , 54.2 , 38.4 , 36.2 , 33.6 , 30.9 , 29.1 , 21.6 , 21.6 ; hrms - esi ( m / z ) [ m + h ] calcd for c13h21o2 209.1542 , found 209.1545 . prepared according to general continuous flow procedure using ketone 13(33 ) ( 0.016 g , 0.055 mmol ) in ch3cn ( 2.3 ml ) , with a 290340 nm band - pass filter and a 10 min residence time at 60 c . purification by flash chromatography ( 6:1 hexanes / etoac ) afforded 14 ( 0.0044 , 27% ) as a colorless oil : rf = 0.58 ( 3:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3406 , 2958 , 2921 , 1710 , 1611 , 1512 , 1464 , 1247 , 1177 ; h nmr ( cdcl3 , 400 mhz ) 7.17 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 5.615.64 ( m , 1h ) , 3.81 ( s , 3h ) , 3.113.13 ( m , 1h ) , 2.812.88 ( m , 1h ) , 2.30 ( sept . , j = 6.4 hz , 1h ) , 2.182.26 ( m , 4h ) , 2.042.11 ( m , 2h ) , 1.73 ( ddd , j = 12.9 , 11.3 , 6.3 hz , 1h ) , 1.06 ( d , j = 4.4 hz , 3h ) , 1.04 ( d , j = 4.0 hz , 3h ) ; c nmr ( cdcl3 , 100 mhz ) 209.7 , 158.2 , 147.3 , 138.8 , 128.1 , 115.5 , 114.1 , 114.1 , 55.6 , 49.3 , 36.5 , 35.7 , 34.0 , 31.2 , 28.9 , 21.9 , 21.4 ; hrms - esi ( m / z ) [ m - h2o ] calcd for c19h22o2 283.1698 , found 283.1706 . prepared according to the general continuous flow procedure using indole 4(34 ) in ch3cn ( 0.01 m ) and a 10 min residence time at room temperature . purification using flash chromatography ( 2:1 to 1:2 hexanes / etoac ) afforded a mixture of indole 16 and cyclobutane 17 . 16 : colorless oil ; rf = 0.33 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3286 , 2925 , 1723 , 1488 , 1454 , 1433 , 1305 , 1282 , 1135 , 1111 , 1087 ; h nmr ( cdcl3 , 500 mhz ) 10.07 ( bs , 1h ) , 7.59 ( dd , j = 9.2 , 2.4 hz , 1h ) , 7.367.46 ( m , 5h ) , 7.287.29 ( m , 1h ) , 7.02 ( td , j = 9.0 , 2.4 hz , 1h ) , 6.196.26 ( m , 2h ) , 5.315.34 ( m , 2h ) , 5.16 ( d , j = 15.9 hz , 1h ) , 4.26 ( dd , j = 7.2 , 1.4 hz , 1h ) , 3.80 ( dt , j = 7.2 , 4.8 hz , 1h ) , 3.74 ( dd , j = 10.5 , 4 . hz , 1h ) , 3.62 ( dd , j = 10.5 , 5.0 hz , 1h ) , 3.51 ( s , 3h ) , 3.13 ( s , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 159.4 ( d , jc f = 238 hz ) , 143.5 , 139.2 , 131.1 , 131.0 , 128.8 , 128.6 , 128.5 , 128.3 , 126.4 ( d , jc f = 11 hz ) , 125.7 , 112.9 ( d , jc f = 10 hz ) , 112.2 ( d , jc f = 26 hz ) , 109.9 , 104.8 ( d , jc f = 25 hz ) , 74.5 , 73.3 , 73.1 , 69.8 , 63.0 , 59.7 , 45.5 ; hrms - esi ( m / z ) [ m + na ] calcd for c23h24fnnao5s 468.1257 , found 468.1269 . 17 : colorless oil ; rf = 0.16 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3387 , 2925 , 2854 , 1461 , 1300 , 1133 ; h nmr ( cdcl3 , 500 mhz ) 7.337.44 ( m , 6h ) , 6.97 ( dd , j = 8.5 , 3.0 hz , 1h ) , 5.49 ( ddd , j = 7.8 , 2.6 , 0.9 hz , 1h ) , 5.01 ( d , j = 4.0 hz , 1h ) , 4.39 ( t , j = 4.7 hz , 1h ) , 4.15 ( d , j = 7.9 hz , 1h ) , 3.87 ( d , j = 9.5 hz , 1h ) , 3.693.73 ( m , 3h ) , 3.49 ( d , j = 9.2 hz , 1h ) , 3.36 ( s , 3h ) , 3.163.20 ( m , 1h ) , 2.99 ( s , 3h ) , 2.64 ( ddd , j = 8.4 , 6.9 , 4.0 hz , 1h ) ; c nmr ( cdcl3 , 125 mhz ) 155.3 ( d , jc f = 239 hz ) , 147.5 , 140.4 , 137.5 ( d , jc f = 8 hz ) , 128.6 , 127.6 , 125.5 , 118.0 ( d , jc f = 7 hz ) , 111.4 ( d , jc f = 26 hz ) , 74.6 , 74.3 , 73.8 , 72.5 , 71.3 , 70.0 , 59.4 , 44.3 , 43.9 , 43.0 , 40.0 ; hrms - esi ( m / z ) [ m + h ] calcd for c23h25fno5s 446.1437 , found 446.1433 . tbaf ( 0.54 ml , 0 . 539 mmol , 1.0 m solution in thf ) was added dropwise to a solution of indole 4 ( 0.12 g , 0.269 mmol ) in thf ( 5 ml ) . the reaction mixture was stirred at room temperature overnight . a saturated solution ( 5 ml ) of nh4cl the mixture was extracted with etoac ( 3 5 ml ) , washed with brine ( 2 3 ml ) , dried over na2so4 , and concentrated in vacuo . purification by flash chromatography ( 2:1 hexanes / etoac ) afforded indole 25 ( 0.075 g , 75% ) as a colorless oil : rf = 0.65 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3316 , 2922 , 2853 , 1486 , 1452 , 1318 , 1169 , 1078 ; h nmr ( cdcl3 , 500 mhz ) 8.69 ( bs , 1h ) , 7.227.44 ( m , 2h ) , 7.317.39 ( m , 3h ) , 7.217.25 ( m , 2h ) , 6.92 ( td , j = 9.1 , 2.6 hz , 1h ) , 6.39 ( d , j = 1.2 hz , 1h ) , 6.11 ( m , 1h ) , 5.30 ( m , 1h ) , 4.85 ( d , j = 12.8 hz , 1h ) , 4.78 ( d , j = 13.1 hz , 1h ) , 4.224.24 ( m , 1h ) , 3.69 ( dt , j = 7.6 , 3.8 hz , 1h ) , 3.63 ( dd , j = 10.4 , 4.3 hz , 1h ) , 3.51 ( dd , j = 10.4 , 3.4 hz , 1h ) , 3.37 ( s , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 158.1(d , jc f = 232 hz ) , 139.4 , 137.5 , 133.1 , 130.2 , 128.8 ( d , jc f = 10 hz ) , 128.7 , 128.3 , 126.8 , 111.6 ( d , jc f = 10 hz ) , 110.5 ( d , jc f = 26 hz ) , 105.5 ( d , jc f = 23 hz ) , 101.6 , 101.5 , 74.4 , 72.4 , 70.9 , 70.4 , 64.5 , 59.5 ; hrms - esi ( m / z ) [ m + h ] calcd for c22h23fno3 368.1662 , found 368.1658 . boc2o ( 0.036 g , 0.163 mmol ) and dmap ( 0.001 g , 0.008 mmol ) were added to a solution of indole 25 ( 0.03 g , 0.082 mmol ) in dcm ( 2 ml ) . the mixture was extracted with dcm ( 3 5 ml ) , washed with brine ( 2 3 ml ) , dried over na2so4 , and concentrated in vacuo . purification by flash chromatography ( 3:1 hexanes / etoac ) afforded indole 18 ( 0.023 g , 61% ) as a yellow oil : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2978 , 2930 , 1731 , 1471 , 1446 , 1369 , 1258 , 1155 , 1155 , 1111 ; h nmr ( cdcl3 , 500 mhz ) 8.03 ( dd , j = 9.2 , 4.6 hz , 1h ) , 7.447.46 ( m , 2h ) , 7.307.39 ( m , 3h ) , 7.16 ( dd , j = 8.5 , 2.4 hz , 1h ) , 6.99 ( td , j = 9.0 , 2.5 hz , 1h ) , 6.63 ( s , 1h ) , 6.106.19 ( m , 2h ) , 5.315.34 ( m , 1h ) , 5.05 ( d , j = 14.3 hz , 1h ) , 4.92 ( d , j = 14.3 hz , 1h ) , 4.26 ( dd , j = 7.9 , 1.5 hz , 1h ) , 3.663.69 ( m , 1h ) , 3.60 ( dd , j = 10.4 , 4.6 hz , 1h ) , 3.52 ( dd , j = 10.2 , 2.6 hz , 1h ) , 3.353.38 ( m , 1h ) , 3.31 ( s , 3h ) , 1.69 ( s , 9h ) ; c nmr ( cdcl3 , 100 mhz ) 160.7 , 158.3 , 150.3 , 149.2 , 140.1 , 139.7 , 133.2 , 130.2 , 130.1 , 129.9 128.7 , 128.4 , 128.2 , 127.4 , 116.7 ( d , jc f = 9 hz ) , 111.8 ( d , jc f = 4 hz ) , 106.1 ( d , jc f = 23 hz ) , 84.7 , 74.5 , 72.0 , 70.6 , 70.5 , 66.1 , 59.5 , 28.5 ; hrms - esi ( m / z ) [ m + na ] calcd for c27h30fnnao5 490.2006 , found 490.2004 . ac2o ( 0.04 ml , 0.441 mmol ) and et3n ( 0.02 ml , 0.165 mmol ) were added to a solution of indole 25 ( 0.0405 g , 0.11 mmol ) and dmap ( 0.003 g , 0.022 mmol ) in dce ( 0.5 ml ) . the resulting mixture was heated to 60 c for 18 h. a saturated solution ( 1 ml ) of nh4cl was then added . the mixture was extracted with etoac ( 3 2 ml ) , washed with brine ( 2 3 ml ) , dried over na2so4 , and concentrated in vacuo . purification by flash chromatography ( 3:1 hexanes / etoac ) afforded indole 20 ( 0.03 g , 66% ) as a yellow oil : rf = 0.64 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2925 , 2892 , 1705 , 1614 , 1601 , 1470 , 1448 , 1372 , 1309 , 1205 , 1090 ; h nmr ( cdcl3 , 400 mhz ) 7.98 ( dd , j = 9.0 , 4.3 hz , 1h ) , 7.297.45 ( m , 5h ) , 7.19 ( dd , j = 8.6 , 2.7 hz , 1h ) , 7.04 ( td , j = 9.1 , 2.5 hz , 1h ) , 6.69 ( s , 1h ) , 6.106.13 ( m , 2h ) , 5.31 ( s , 1h ) , 5.03 ( d , j = 14.5 hz , 1h ) , 4.92 ( d , j = 14.1 hz , 1h ) , 4.27 ( dd , j = 7.8 , 2.0 hz , 1h ) , 3.543.67 ( m , 2h ) , 3.48 ( dd , j = 10.2 , 2.7 hz , 1h ) , 3.29 ( s , 3h ) , 2.82 ( s , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 170.2 , 159.3 ( d , jc f = 223 hz ) , 139.6 , 133.6 , 130.7 , 130.2 , 128.7 , 128.3 , 128.2 , 126.9 , 116.7(d , jc f = 11 hz ) , 112.5 ( d , jc f = 31 hz ) , 111.2 ( d , jc f = 4.5 hz ) , 106.6 ( d , jc f = 29 hz ) , 74.5 , 72.0 , 70.6 , 70.2 , 65.9 , 59.5 , 26.8 ; hrms - esi ( m / z ) [ m + na ] calcd for c24h24fnnao4 432.1587 , found 432.1574 . 4-fluoro-2-iodo - n - methylaniline ( 0.164 g , 0.652 mmol ) was added dropwise to a solution of alkyne 26(6 ) ( 0.252 g , 0.978 mmol ) , pd(pph3)2cl2 ( 0.023 g , 0.033 mmol ) , and cui ( 0.006 g , 0.033 mmol ) in et2nh ( 7 ml ) at 50 c . the resulting mixture was stirred at 50 c for 2 h. the solution was then cooled to room temperature , and the solvent was evaporated . purification by flash chromatography ( benzene then 4:1 hexanes / etoac ) afforded 27 ( 0.169 g , 68% ) as a brown oil : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2916 , 2849 , 1515 , 1488 , 1451 , 1315 , 1265 , 1166 , 1076 ; h nmr ( cdcl3 , 400 mhz ) 7.297.45 ( m , 6h ) , 7.02 ( dd , j = 8.8 , 2.9 hz , 1h ) , 6.97 ( td , j = 8.7 , 2.9 hz , 1h ) , 6.49 ( dd , j = 9.0 , 4.3 hz , 1h ) , 6.126.22 ( m , 2h ) , 5.325.34 ( m , 1h ) , 4.55 ( s , 2h ) , 4.334.36 ( m , 1h ) , 3.633.68 ( m , 2h ) , 3.523.56 ( m , 1h ) , 3.35 ( m , 3h ) , 2.87 ( s , 3h ) ; c nmr ( cdcl3 , 100 mhz ) 147.2 , 139.5 , 130.4 , 128.7 , 128.6 , 128.4 , 128.4 , 128.3 , 128.2 , 126.9 , ( d , jc f = 24 hz ) , 117.4 ( d , jc f = 22 hz ) , 110.0 ( d , jc f = 8 hz ) , 91.9 , 82.4 , 74.4 , 71.8 , 70.5 , 70.0 , 59.6 , 57.6 , 31.0 ; hrms - esi ( m / z ) [ m + h ] calcd for c23h25fno3 382.1818 , found 382.1213 . prepared according to general continuous flow procedure using indole 18 ( 0.015 g , 0.031 mmol ) in ch3cn ( 3.1 ml ) with a 290340 nm band - pass filter and a 10 min residence time at room temperature . purification by flash chromatography ( 5:1 hexanes / etoac ) afforded indole 19 ( 0.0059 g , 40% ) as a yellow oil : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3429 , 2971 , 2930 , 2881 , 1707 , 1479 , 1452 , 1369 , 1260 , 1140 , 1089 ; h nmr ( cdcl3 , 500 mhz ) 7.70 ( bs , 1h ) , 7.387.43 ( m , 4h ) , 7.337.37 ( m , 1h ) , 6.71 ( t , j = 7.8 hz , 1h ) , 5.29 ( dd , j = 8.1 , 2.6 hz , 1h ) , 5.00 ( d , j = 4.0 hz , 1h ) , 4.42 ( t , j = 5.0 hz , 1h ) , 4.064.15 ( m , 2h ) , 3.703.75 ( m , 3h ) , 3.60 ( d , j = 7.0 hz , 1h ) , 3.343.39 ( m , 4h ) , 2.58 ( td , j = 7.7 , 4.1 hz , 1h ) , 1.58 ( s , 9h ) ; c nmr ( cdcl3 , 125 mhz ) 161.0 , 159.9 , 158.1 , 151.9 , 140.3 , 128.6 , 127.6 , 125.6 , 116.1 ( d , jc f = 8 hz ) , 113.7 ( d , jc f = 22 hz ) , 111.2 ( d , jc f = 30 hz ) , 81.5 , 74.2 , 73.6 , 73.2 , 72.4 , 69.9 , 59.4 , 43.7 , 39.9 , 38.6 , 28.8 ; hrms - esi ( m / z ) [ m + na ] calcd for c27h30fnnao5 490.2006 , found 490.2013 . prepared according to the general continuous flow procedure using indole 20 ( 0.016 g , 0.038 mmol ) in ch3cn ( 0.01 m , 3.8 ml ) with a 290340 nm band - pass filter and a 10 min residence time at room temperature . purification using flash chromatography ( 2:1:1 hexanes / etoac / dcm ) afforded indole 21 ( 0.0023 g , 16% ) as a yellow oil : rf = 0.35 ( 2:1:1 hexanes / etoac / dcm ) ; ir ( thin film ) ( cm ) 3403 , 2923 , 2853 , 1737 , 1462 , 1127 ; h nmr ( c6d6 , 500 mhz ) 7.007.38 ( m , 6h ) , 6.79 ( dd , j = 10.1 , 2.6 hz , 1h ) , 5.72 ( dd , j = 8.1 , 2.4 hz , 1h ) , 4.84 ( d , j = 3.9 hz , 1h ) , 4.40 ( t , j = 4.6 hz , 1h ) , 3.78 ( d , j = 8.1 hz , 1h ) , 3.67 ( d , j = 6.8 hz , 1h ) , 3.48 ( d , j = 9.0 hz , 1h ) , 3.23 ( dd , j = 10.5 , 4.4 hz , 1h ) , 3.13 ( dd , j = 10.5 , 5.4 hz , 1h ) , 2.892.90 ( m , 4h ) , 2.63 ( t , j = 7.5 hz , 1h ) , 2.27 ( td , j = 7.6 , 3.9 hz , 1h ) , 1.99 ( s , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 198.5 , 155.1 , 153.2 , 151.0 , 140.6 , 136.4 ( d , jc f = 8 hz ) , 128.6 , 127.5 , 125.6 , 117.6 ( d , jc f = 6 hz ) , 112.2 ( d , jc f = 23 hz ) , 105.3 , 74.3 , 73.8 , 73.3 , 72.7 , 71.1 , 70.0 , 59.4 , 44.1 , 43.4 , 39.7 , 29.9 ; hrms - esi ( m / z ) [ m + h ] calcd for c24h25fno4 410.1768 , found 410.1762 . prepared according to the general continuous flow procedure using indole 22(34 ) ( 0.014 g , 0.03 mmol ) in ch3cn ( 3 ml ) with a 305 nm long - pass filter and a 10 min residence time at room temperature . purification by flash chromatography ( 3:1 hexanes / etoac ) afforded indole 23 ( 0.023 g , 16% ) as a yellow oil : rf = 0.38 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3335 , 2925 , 1722 , 1681 , 1449 , 1420 , 1302 , 1146 , 1125 ; h nmr ( cdcl3 , 500 mhz ) 10.13 ( bs , 1h ) , 7.83 ( d , j = 8.5 hz , 3h ) , 7.367.46 ( m , 6h ) , 7.34 ( dd , j = 1.8 , 0.6 hz , 1h ) , 6.196.25 ( m , 2h ) , 5.315.36 ( m , 2h ) , 5.15 ( d , j = 15.9 hz , 1h ) , 4.244.26 ( m , 1h ) , 3.77 ( d , j = 7.9 hz , 1h ) , 3.58 ( d , j = 7.0 hz , 1h ) , 3.49 ( d , j = 7.0 hz , 1h ) , 3.25 ( dd , j = 10.5 , 4.1 hz , 1h ) , 3.13 ( dd , j = 10.5 , 5.0 hz , 1h ) , 2.92 ( s , 3h ) , 2.50 ( t , j = 7.8 hz , 1h ) , 2.30 ( s , 3h ) , 2.10 ( ddd , j = 8.5 , 6.6 , 4.1 hz , 1h ) ; c nmr ( cdcl3 , 125 mhz ) 142.8 , 139.1 , 131.0 , 129.7 , 128.8 , 128.5 , 128.3 , 125.7 , 124.2 , 123.4 , 120.3 , 111.9 , 110.0 , 74.5 , 73.4 , 73.2 , 69.7 , 62.9 , 59.8 , 53.7 , 45.7 , 31.2 ; hrms - esi ( m / z ) [ m + na ] calcd for c23h24clnnao5s 484.0961 , found 484.0965 .

we have developed an automated photochemical microfluidics platform that integrates a 1 kw high - pressure hg vapor lamp and allows for analytical pulse flow or preparative continuous flow reactions . herein , we will discuss the use of this platform toward the discovery of new chemotypes through multidimensional reaction screening . we will highlight the ability to discretely control wavelengths with optical filters , allowing for control of reaction outcomes .

Introduction Results and Discussion Experimental Section

photochemistry is among the most powerful complexity generating tools available to chemists . while there have been a number of examples utilizing flow chemistry to increase the effectiveness of photochemistry , we have focused our application on reaction screening and discovery of complex chemotypes . to this end , we have developed a microfluidic - based photochemistry platform capable of running pulse or continuous flow reactions , which is more suitable for high - throughput chemistry . examples such as multidimensional reaction screening developed in our laboratory , mcmillan s advent of enabled serendipity , and hartwig s use of mass spectrometry to analyze thousands of reaction mixtures are a testament to the potential of screening reactions in parallel . over the past several years , we have developed reaction screening techniques that take advantage of analytical reaction scale and microfluidics . herein , we report the expansion of our microfluidic platform to incorporate photo - optics for higher levels of wavelength control and its application to a multidimensional reaction screen . we have developed a photochemical microfluidic platform utilizing a 1 kw capillary mercury lamp and microreactor fabricated with schott 8337 , a uv transparent glass . in an effort to advance the utility of photochemistry through expanded wavelength control , we designed a system that also incorporates photo - optical components ( figure 1 ) . directly in front of the microfluidic device a wide range of uv filters , both long - pass and band - pass , are commercially available . one of the exciting applications of our photochemical platform is multidimensional reaction screening wherein multiple substrates are evaluated in photochemical reactions with varying wavelength , sensitizer , temperature , and solvent . with this in mind , we selected a variety of substrates ( 14 ) that were part of the compound library collection in the cmld - bu ( figure 2 ) . along with four long - pass uv filters , we selected a series of sensitizers ( matched with appropriate uv filters ) with triplet energies ranging from 41 to 74 kcal / mol , as well as two electron - transfer sensitizers . multidimensional reaction screening parameters . each reaction was performed on 100 g scale at a concentration of 0.05 m. the sensitizers were utilized at 1.0 mol equiv . each reaction showing potential new products by uplc analysis was scaled up using continuous flow and the same microfluidic device and photochemical platform . the continuous flow reactions afforded appropriate amounts of material for structure elucidation . the second reaction identified was a product derived from substrate 2 ( scheme 1 ) . utilizing the flexibility of the platform , we were able to optimize the transformation with a lower temperature and shorter residence time ( 10 c and 3 min ) and slightly longer uv wavelength ( > 295 nm ) . on the basis of data from the reaction screen , we carried out further optimization focused on wavelength variation . utilizing the > 300 nm uv filter , we evaluated longer residence times in order to drive the reaction to completion . we further evaluated the scope of this reaction as a one - pot , two - step procedure utilizing compounds 11,13 , and 15 . reaction of substrate 11 afforded ketone 12 ( as a 1.5:1 mixture ) in 43% yield , whereas cyclobutanone 14 could be isolated cleanly from 13 and did not decarbonylate under microwave radiations . a fourth reaction was identified in the multidimensional reaction screen involving glycal - derived substrate 4 ( eq 3 ) . however , upon scale - up and nmr analysis , it was apparent two products were present and , upon isolation and elucidation , we determined that indole 16 and cyclobutane 17 were the major products . indole 16 is derived from a simple photo - fries migration of the sulfonyl moiety to c3 , while cyclobutane 17 may arise from an initial [ 2 + 2 ] cycloaddition followed by migration of the sulfonate to the indole c7.3 close analysis of the reaction screening results and follow - up optimization suggested that the reaction pathways were wavelength dependent . however , reactions at shorter wavelengths did not result in migration to c3 but only in decomposition . in conclusion , we have successfully adapted a photochemical microfluidics platform for use in multidimensional reaction screening . by adding a photo - optics system , we are able to effectively control the wavelength of the reactions utilizing uv filters . such control allows for introduction of irradiation wavelength as a screening variable as well as downstream optimization of reactions that are highly dependent on wavelength . utilizing the platform in a multidimensional reaction screen , we were able to identify new chemotypes derived from photochemical transformations of complex scaffolds . most notably , we identified complex chemotypes derived from an oxa - di--methane rearrangement of a bicyclo[3.2.1]octanoid scaffold , a 1,3-acyl shift of bicyclic ketol , and a wavelength - dependent photo - fries/[2 + 2]-cycloaddition . the new scaffolds are currently being used to develop methodologies and access additional complex and unique molecules for use in high - throughput screening and medicinal chemistry programs . stock solutions of substrate ( 0.1 m ) and sensitizers ( 0.1 m ) were prepared in ch3cn or thf ( anhydrous ) while maintaining dry handling . the block was attached to the microfluidics platform , and a slow , steady stream of argon was passed through the inert gas chamber . reactions showing apparent reactivity were subsequently scaled up and isolated , using the system in continuous flow , for further characterization . it was then concentrated in vacuo and dissolved in etoac ( 5 ml ) , filtered , and concentrated in vacuo . ( 1 ) prepared according to general continuous flow procedure using 1(21 ) and benzophenone ( 1 equiv ) in ch3cn ( 0.05 m ) with a 340405 nm band - pass filter and a 2 min residence time at room temperature . ( 2 ) prepared according to general continuous flow procedure using 1 in ch3cn ( 0.05 m ) with a 305365 nm band - pass filter and a 2 min residence time at room temperature . prepared according to the general continuous flow procedure using 2 ( 0.0143 g , 0.031 mmol ) in ch3cn ( 1.24 ml ) with a 295 nm long pass filter and a 4 min residence time at 10 c . prepared according to general continuous flow procedure using ketone 3(30 ) ( 0.0014 g , 0.05 mmol ) in ch3cn ( 1 ml ) with a 305365 nm band - pass filter and a 10 min residence time at 60 c . 10b : ( 0.0018 g , 13% ) as a colorless oil ; rf = 0.5 ( 15:1 : hexanes / dcm / etoac ) ; ir ( thin film ) ( cm ) 2960 , 2927 , 1709 , 1665 , 1459 , 1353 , 757 ; h nmr ( c6d6 , 400 mhz ) 7.047.12 ( m , 4h ) , 5.29 ( d , j = 4.0 hz , 1h ) , 3.25 ( q , j = 6.8 hz , 1h ) , 2.822.92 ( m , 2h ) , 2.68 ( d , j = 4.4 hz , 1h ) , 2.57 ( dd , j = 14.4 , 4.8 hz , 1h ) , 2.19 ( dd , j = 17.2 , 7.6 hz , 1h ) , 2.05 ( sept , j = 7.2 hz , 1h ) , 1.97 ( dd , j = 16.8 , 5.2 hz , 1h ) , 1.78 ( s , 3h ) , 0.87 ( d , j = 6.4 hz , 6h ) ; c nmr ( c6d6 , 100 mhz ) 207.2 , 148.1 , 146.8 , 143.1 , 127.1 , 127.1 , 125.5 , 123.8 , 116.5 , 54.0 , 42.8 , 39.1 , 38.8 , 35.9 , 28.9 , 28.2 , 21.5 , 21.4 ; hrms - esi ( m / z ) [ m + h ] calcd for c18h22o 255.1749 , found 255.1754 . prepared according to the general continuous flow procedure using ketone 11(30 ) ( 0.0125 g , 0.053 mmol ) in ch3cn ( 1.1 ml ) with a 305365 nm band - pass filter and a 10 min residence time at 60 c . prepared according to general continuous flow procedure using ketone 13(33 ) ( 0.016 g , 0.055 mmol ) in ch3cn ( 2.3 ml ) , with a 290340 nm band - pass filter and a 10 min residence time at 60 c . prepared according to the general continuous flow procedure using indole 4(34 ) in ch3cn ( 0.01 m ) and a 10 min residence time at room temperature . 16 : colorless oil ; rf = 0.33 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3286 , 2925 , 1723 , 1488 , 1454 , 1433 , 1305 , 1282 , 1135 , 1111 , 1087 ; h nmr ( cdcl3 , 500 mhz ) 10.07 ( bs , 1h ) , 7.59 ( dd , j = 9.2 , 2.4 hz , 1h ) , 7.367.46 ( m , 5h ) , 7.287.29 ( m , 1h ) , 7.02 ( td , j = 9.0 , 2.4 hz , 1h ) , 6.196.26 ( m , 2h ) , 5.315.34 ( m , 2h ) , 5.16 ( d , j = 15.9 hz , 1h ) , 4.26 ( dd , j = 7.2 , 1.4 hz , 1h ) , 3.80 ( dt , j = 7.2 , 4.8 hz , 1h ) , 3.74 ( dd , j = 10.5 , 4 . purification by flash chromatography ( 3:1 hexanes / etoac ) afforded indole 20 ( 0.03 g , 66% ) as a yellow oil : rf = 0.64 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2925 , 2892 , 1705 , 1614 , 1601 , 1470 , 1448 , 1372 , 1309 , 1205 , 1090 ; h nmr ( cdcl3 , 400 mhz ) 7.98 ( dd , j = 9.0 , 4.3 hz , 1h ) , 7.297.45 ( m , 5h ) , 7.19 ( dd , j = 8.6 , 2.7 hz , 1h ) , 7.04 ( td , j = 9.1 , 2.5 hz , 1h ) , 6.69 ( s , 1h ) , 6.106.13 ( m , 2h ) , 5.31 ( s , 1h ) , 5.03 ( d , j = 14.5 hz , 1h ) , 4.92 ( d , j = 14.1 hz , 1h ) , 4.27 ( dd , j = 7.8 , 2.0 hz , 1h ) , 3.543.67 ( m , 2h ) , 3.48 ( dd , j = 10.2 , 2.7 hz , 1h ) , 3.29 ( s , 3h ) , 2.82 ( s , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 170.2 , 159.3 ( d , jc f = 223 hz ) , 139.6 , 133.6 , 130.7 , 130.2 , 128.7 , 128.3 , 128.2 , 126.9 , 116.7(d , jc f = 11 hz ) , 112.5 ( d , jc f = 31 hz ) , 111.2 ( d , jc f = 4.5 hz ) , 106.6 ( d , jc f = 29 hz ) , 74.5 , 72.0 , 70.6 , 70.2 , 65.9 , 59.5 , 26.8 ; hrms - esi ( m / z ) [ m + na ] calcd for c24h24fnnao4 432.1587 , found 432.1574 . purification by flash chromatography ( benzene then 4:1 hexanes / etoac ) afforded 27 ( 0.169 g , 68% ) as a brown oil : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 2916 , 2849 , 1515 , 1488 , 1451 , 1315 , 1265 , 1166 , 1076 ; h nmr ( cdcl3 , 400 mhz ) 7.297.45 ( m , 6h ) , 7.02 ( dd , j = 8.8 , 2.9 hz , 1h ) , 6.97 ( td , j = 8.7 , 2.9 hz , 1h ) , 6.49 ( dd , j = 9.0 , 4.3 hz , 1h ) , 6.126.22 ( m , 2h ) , 5.325.34 ( m , 1h ) , 4.55 ( s , 2h ) , 4.334.36 ( m , 1h ) , 3.633.68 ( m , 2h ) , 3.523.56 ( m , 1h ) , 3.35 ( m , 3h ) , 2.87 ( s , 3h ) ; c nmr ( cdcl3 , 100 mhz ) 147.2 , 139.5 , 130.4 , 128.7 , 128.6 , 128.4 , 128.4 , 128.3 , 128.2 , 126.9 , ( d , jc f = 24 hz ) , 117.4 ( d , jc f = 22 hz ) , 110.0 ( d , jc f = 8 hz ) , 91.9 , 82.4 , 74.4 , 71.8 , 70.5 , 70.0 , 59.6 , 57.6 , 31.0 ; hrms - esi ( m / z ) [ m + h ] calcd for c23h25fno3 382.1818 , found 382.1213 . prepared according to general continuous flow procedure using indole 18 ( 0.015 g , 0.031 mmol ) in ch3cn ( 3.1 ml ) with a 290340 nm band - pass filter and a 10 min residence time at room temperature . purification by flash chromatography ( 5:1 hexanes / etoac ) afforded indole 19 ( 0.0059 g , 40% ) as a yellow oil : rf = 0.71 ( 1:1 hexanes / etoac ) ; ir ( thin film ) ( cm ) 3429 , 2971 , 2930 , 2881 , 1707 , 1479 , 1452 , 1369 , 1260 , 1140 , 1089 ; h nmr ( cdcl3 , 500 mhz ) 7.70 ( bs , 1h ) , 7.387.43 ( m , 4h ) , 7.337.37 ( m , 1h ) , 6.71 ( t , j = 7.8 hz , 1h ) , 5.29 ( dd , j = 8.1 , 2.6 hz , 1h ) , 5.00 ( d , j = 4.0 hz , 1h ) , 4.42 ( t , j = 5.0 hz , 1h ) , 4.064.15 ( m , 2h ) , 3.703.75 ( m , 3h ) , 3.60 ( d , j = 7.0 hz , 1h ) , 3.343.39 ( m , 4h ) , 2.58 ( td , j = 7.7 , 4.1 hz , 1h ) , 1.58 ( s , 9h ) ; c nmr ( cdcl3 , 125 mhz ) 161.0 , 159.9 , 158.1 , 151.9 , 140.3 , 128.6 , 127.6 , 125.6 , 116.1 ( d , jc f = 8 hz ) , 113.7 ( d , jc f = 22 hz ) , 111.2 ( d , jc f = 30 hz ) , 81.5 , 74.2 , 73.6 , 73.2 , 72.4 , 69.9 , 59.4 , 43.7 , 39.9 , 38.6 , 28.8 ; hrms - esi ( m / z ) [ m + na ] calcd for c27h30fnnao5 490.2006 , found 490.2013 . prepared according to the general continuous flow procedure using indole 20 ( 0.016 g , 0.038 mmol ) in ch3cn ( 0.01 m , 3.8 ml ) with a 290340 nm band - pass filter and a 10 min residence time at room temperature . prepared according to the general continuous flow procedure using indole 22(34 ) ( 0.014 g , 0.03 mmol ) in ch3cn ( 3 ml ) with a 305 nm long - pass filter and a 10 min residence time at room temperature .

colonial and post - colonial anthropological literature has paid inadequate attention to the involvement of the aristocracy in large scale irrigation in pre - colonial bali . it has one - sidedly emphasized the cooperative and even autonomous nature of local irrigation networks ( grader 1960 ; geertz 1980 ) . he also emphasizes the autonomous and bottom - up nature of local irrigation management , but adds an important qualification by arguing that regional irrigation management centered upon extended temple networks which culminated in the batur temple complex in central bali . in doing so , he seems to downplay , or even ignore , the role played by regional dynasties and noble lords in the pre - colonial period , while he also seems to underestimate the structural changes which took place at the beginning of the colonial period during the first decades of the twentieth century . although he is aware that i wrote about irrigation in bali during the pre - colonial era and the colonial period , for reasons which are not entirely clear to me , he only refers to this in a very restricted manner.1 it is true that my work is focused on another area in bali i.e . , the southern rice plains of mengwi and badung where conditions were perhaps different from the findings of his research i.e . , in uphill bangli and gianyar ( central bali)but my conclusions contradict both lansing s model and the implicit claim in his work that it is applicable for the whole of bali . in the following pages i will therefore summarize my findings because they offer an alternative picture of irrigation management in pre - colonial bali . control of manpower was one of the main objectives of the pre - colonial contest states in southeast asia and the south balinese kingdom of mengwi ( 17101891 ad ) was no exception in this respect . parallel to the numerous vertical relations between lords and followers unstable connections developed between a number of large dams and the irrigated rice fields of the ordinary people . there was a correspondence between the expanding control of the dynasty over manpower and an increased grip on the irrigation systems in the region . contrary to lansing s model there is ample evidence that the mengwi dynasty was closely involved in the construction , upkeep , and the mostly ritual regulation of the irrigations systems . the irrigation order can only be understood in connection with the royal hierarchy . three rivers , the sungi , penet and ayung , descend the sloping mengwi region which fans out from the mountains to the sea . because the rivers cut deep gorges it was no easy to lead the water to the rice fields . sometimes the water level of the river was dozens of feet below the rice fields , so that in order to irrigate them a dam had to be constructed many miles upstream . the water that was collected at those dams had to be diverted via lengthy conduits and tunnels before it reached the downstream rice fields . the construction of these central dams required the mobilization of a large amount of manpower which was controlled and coordinated by either the dynasty or other local princes . in many oral histories , which i collected during my fieldwork in the early 1980s throughout the mengwi region , mention is made that the king or a local lord ordered the clearing of land and organized the construction of upstream dams . the rise of the dynasty went hand in hand with the expansion of irrigated rice fields in the region . dynastic involvement was required because most villages had a limited range of action and cooperation between villages was often lacking . dynastic involvement was therefore necessary in order to protect and maintain the vulnerable irrigation systems . upkeep demanded manpower and so did reconstruction work since the large earthen dams quickly eroded in the fast - flowing rivers and were often wiped out suddenly at the start of the rainy season by a flash flood . as an example of the mechanisms by which this happened , one of the large dams in the river ayung , dam oongan which was some 55 ft in height , tended to undermine itself because overflow water was diverted at the downstream side of the dam . moreover , major conduits and tunnels required regular inspection , while the entire system had to be guarded against sabotage and external attacks . it is true that small - scale cooperation at the local level was an important feature of irrigation in bali and this is certainly true for the twentieth century . however , without water such cooperation could not be materialized . none but regional dynasties and local lords were able to guarantee the continuity of this fragile system.3 the geographical location of the dynastic centre of mengwi and a number of its satellites it is said that the first king of mengwi together with a local lord who resided north of the dynastic centre built a dam in the river sungi and proceeded to expand rice fields in his domain . as a result the dynastic centre was located in a wide fertile plain some 200 m above sea level where surplus for the dynasty was produced . while the newly constructed dam was guarded by the northern lord , an important side effect was that it also allowed the dynastic centre to influence downstream irrigation . in case of emergency the king was able to cut off the water supply to the domains of southern lords . this happened in the nineteenth century during prolonged conflicts between the royal centres of mengwi and badung , and bangli and gianyar . because the political system in bali was fragmented there was no monopoly of power at the centre . oriental despotism. satellites of the dynasty and other lords were in control of their own irrigations systems . the lord of sibang , for instance , had his own dam twenty miles upstream in the river ayung which was located in the domain of another lord . the lord of sibang had moved a group of trusted followers with their families to the north in order to maintain and protect the dam . the lord of sibang not only looked after his own dam , but he was also responsible for the upkeep of another big dam in the ayung river . little is known about the construction of this dam but in the course of the eighteenth century it became the biggest irrigation project in south bali providing water for thousands of hectares of rice fields in the southern part of the mengwi kingdom.4 the entire system was spread over the domains of several lords . much of the upkeep of this complex system , which included a long tunnel , was delegated to lesser lords and lower units , but , the dynastic centre was ultimately responsible for the overall supervision . this supervision was vital because the system went beyond the restricted interests of its constituent parts . in this context neither bottom up coordination based on autonomous local irrigation units was able to manage this huge system , nor is there any indication that it was solely managed by temples without any dynastic involvement . on the contrary , without the mengwi dynasty the irrigation system of the gumasih dam was simply unthinkable . lansing ( 2007 : 34 ) seems to downplay the importance of dynastic involvement in south bali but offers no evidence that contradicts my findings . the king and local lords had a group of functionaries ( or sedahan ) at their disposal who were responsible for timely repair of the dams and conduits , water allocation to irrigation units , and the collection of taxes . most of the tax was levied from rice fields which were distributed among followers who were given access to rice fields in exchange for labor and loyalty . on this point , clifford geertz ( 1980 : 67 ) presented a contrasting view regarding pre - colonial bali that there was no systematic congruence [ ] between the structure of political authority , the structure of land tenure , and the distribution of land tenancy. however , all the data i was able to collect indicate the opposite . in pre - colonial mengwi there was a strong coherence between political power , the distribution of land , and between the owner and the worker of the land , which is illustrated by the following example from the area of sibang : when the dam at sibang was built , many rice fields around sibang were opened up . each of them received a larger or smaller ( side ) canal , and they could allocate the land on either side of it to their personal followers . trusted favorites received a larger piece of land , while ordinary followers were given about two acres.the lord of sibang selected the best field for his own use and his slaves grew the crop for his household . they were obliged to perform services for their lord , but were allowed to retain the complete harvest . only a small portion of it had to be paid to the lord as tax . a follower whose loyalty and service towards the lord were found wanting would lose access to his land and was to be ousted from the circle of followers.5 when the dam at sibang was built , many rice fields around sibang were opened up . each of them received a larger or smaller ( side ) canal , and they could allocate the land on either side of it to their personal followers . trusted favorites received a larger piece of land , while ordinary followers were given about two acres . the lord of sibang selected the best field for his own use and his slaves grew the crop for his household . the other rice fields were called pecatu and were distributed among his personal followers . they were obliged to perform services for their lord , but were allowed to retain the complete harvest . only a small portion of it had to be paid to the lord as tax . a follower whose loyalty and service towards the lord were found wanting would lose access to his land and was to be ousted from the circle of followers.5 in the course of the eighteenth century irrigation expanded rapidly under the encouragement of the mengwi dynasty . his control over manpower in his kingdom was limited to his own domain and so was his direct control over irrigation matters . local lords , like the one in sibang , managed their own decentralized systems and were in control of their own followers . as a result the fragmented control of manpower paralleled the distribution of irrigation water in fact the top of the irrigation systems was as vulnerable as the apex of the dynastic hierarchy . a weak king or a weak dam conversely , the irrigation order and the dynastic hierarchy could only be maintained if the royal centre was strong ( see fig . 1mengwi in the eighteenth century ( from schulte nordholt 1996 : 54 ) mengwi in the eighteenth century ( from schulte nordholt 1996 : 54 ) the correlation between these two hierarchies was expressed in a telling way . until recently it was custom that in the villages of kapal and sibang , prior to starting to work on the rice fields , a procession would carry a sacred dynastic dagger ( or keris ) to the central dam . there the blade was pushed into the dam accompanied by various rituals , a result of which the powers that resided in the dagger would be infused into the dam . there is indirect evidence that similar rituals used to be held in connection with the dam north of the dynastic centre of mengwi . this suggests that the relation between a strong ruler and a sturdy dam was crucial , because dynastic hierarchy and a smooth irrigation order supported and reinforced each other . the relationship between dynasty and irrigation management also confirms the important role kingship played in guaranteeing the continuity of fertility in the realm , which found its expression in the parallel circulation of water and the human soul between mountains , downstream valleys and the sea.6 how vulnerable both the dynastic and the irrigation orders were is nicely illustrated by a balinese historical song , the kidung nderet , in which a major crisis of the mengwi dynasty during the early 1820s is told an analyzed.7 due to poor leadership in the centre of the dynasty both the hierarchy of lords and followers and the irrigation order fell apart . an important part of the story concerns the mismanagement of the dam gumasih near mambal . during the reign of king cokorda munggu ( 174?1780 ) the responsibility for the dam , the tunnel and conduits , and part of the rice fields that received water from the dam was given to one of his sons . this man was also entitled to levy tax with which the upkeep of the dam and tunnel was paid . under the new king , however , the dynastic centre withdrew this privilege and resumed direct control over the irrigation system of dam gumasih while it also centralized the taxation . but , when the dam and the tunnel needed repair , the dynastic centre did not act appropriately , as a result of which large parts of the southern rice fields did not receive any water , whereas peasants still had to pay tax . unrest evolved eventually into protest and warfare with neighboring kingdoms , which led to a humiliating defeat . the interesting point here is that this balinese text written in the nineteenth century also emphasizes the close connection between dynasty and irrigation.8 around the middle of the nineteenth century the mengwi dynasty managed to recover . part of the recovery was the extension of rice fields in the northeastern region of the mengwi kingdom which involved the building of dams , tunnels and temples.9 this was to a large extent a top - down operation coordinated by the leading lineage of the dynastic centre . most likely , the actual organization of the various irrigation systems in pre - colonial bali differed widely per locality depending on the political and ecological context . in most instances , however , lord - follower relationships informed the lines along which these systems were organized . as far as can be determined , extension of irrigated fields did not automatically result in the establishment of ( semi ) autonomous local irrigation units , or subak , which operated independently from villages . oral traditions concerning this period suggest a different picture . without exception , informants emphasized the leading role of the nobility in the project while they did not mention subak as separate organizations . the term subak did exist though , but referred to a person rather than to an autonomous irrigation association . this official was the lowest link in the regional irrigation hierarchy who was also responsible for the collection of a royal tax on the use of irrigation water , the suwinih . the dynastic centre used this tax to finance large rituals in the newly built irrigation temples . these temples fostered in the first place the fertility of the land and averted plagues . the temples and rituals were supervised by irrigation officials while the dynastic involvement was restricted to the granting of permission to stage certain rituals . however , as soon as ritual and maintenance were neglected and disorder could erupt , it was the duty of the power holders to restore order and hierarchy . it is for this reason that pura arantaja , the main irrigation temple of the northeastern region of mengwi , was commonly called accordingly , this temple represented not only the link between gods , fertility , and men , but was also a reminder of royal authority in the region . so far , i have found no evidence that supports lansing s bottom up model of irrigation management exclusively handled by temple systems . instead , we see a complex but integrated system in which dynasties , temples , and peasants participated and hierarchical relationships and horizontal collaboration were combined . in this system upstream irrigation was primarily coordinated and protected by the dynasty , while downstream irrigation was to a large extent a local affair . labor was mobilized both for local purposes as well as for large scale upstream projects on dams and conduits . in terms of daily routine the system was as much as possible decentralized but the system as a whole depended in the end on dynastic coordination . dynasties , and not temples , were the key institutions that guaranteed the continuity of the system . soon after the dutch had conquered south bali at the beginning of the twentieth century , colonial administrators started to reorganize local society according to orientalist models of a supposed authentic bali in which the village played a central role . since the arbitrary royal rule.10 apart from the reorganization of village administration , which also served to mobilize a reservoir of labor , irrigation became another object of colonial interference , which ultimately aimed to levy an increasing amount of tax.11 based on research conducted by the colonial administrator f.a . his description of the northern balinese irrigation association ( subak ) was taken as the normative standard for the reorganization of irrigation management in south bali . the north featured small - scale irrigation systems without direct aristocratic interference , whereas the south had large scale systems which depended on big dams and dynastic involvement.13 contrary to north bali , the organization of irrigation in the south varied by locality while boundaries between village and subak were often fluid . liefrinck had presented a uniform model in which no consideration was given to local variation or direct aristocratic involvement . as he saw it , dynastic interference was essentially unrelated to the subak , but it had penetrated into the local networks in order to appropriate taxes . this point of view reflected a very common orientalist view of that time which favored the autonomy of local institutions which had to be protected under benign colonial rule . it seems that lansing s ideas were at least partially influenced by this colonial perception . lansing argues that the dutch deliberately sought to restore the centralized irrigation hierarchy of the former kingdoms and that they even employed archaeologists to legitimize this project ( lansing 2007 : 3536 ) . i wonder on what evidence this statement is based . actually , the dutch did the opposite , at least on paper . using liefrinck s model , the colonial administrators in the south observed that little was left of the supposed restored in conformity with the north balinese model.14 what in fact happened , however , was that this so - called restoration fitted the needs of the colonial state . a memorandum on the former kingdom of klunkung by the dutch administrator of bali , resident g.f . de bruyn kops in 1908 illustrates this nicely : the irrigation system , too , ought to be overhauled drastically . in the days of the kings [ ] all the nobles and prominent people had their own tax collector who made sure that his own land was watered aplenty . urgently needed is a separation between irrigation workers and administrative personnel ; a division of irrigation areas , headed by a supervisor must be designed ; the subak have to be assigned in groups to avoid too large a number of subak heads which would impede administrative control.15 the irrigation system , too , ought to be overhauled drastically . in the days of the kings [ ] all the nobles and prominent people had their own tax collector who made sure that his own land was watered aplenty . urgently needed is a separation between irrigation workers and administrative personnel ; a division of irrigation areas , headed by a supervisor must be designed ; the subak have to be assigned in groups to avoid too large a number of subak heads which would impede administrative control.15 within a few years the dutch succeeded in replacing the plurality of relationships from the pre - colonial period with an overarching colonial bureaucracy . apart from administrative amalgamations , they were confronted with new officials and new or redefined terms and titles . in each former kingdom , which had become an administrative district , a balinese official ( sedahan agung ) was appointed who supervised a number of lower officials ( sedahan ) who on their turn both supervised the distribution of irrigation water and the levying of new colonial taxes . while the term subak was known as a local irrigation leader in the mengwi area , it now became the name of the local irrigation association all over bali . the dutch introduced a new term for the subak leader , i.e. , pekaseh , which was taken from the neighboring kingdom of gianyar . these terminological shifts were not insurmountable , but great confusion ensued when the dutch started to reorganize the actual irrigation networks . bureaucratic and administrative criteria prevailed to the extent that irrigation networks had to coincide with administrative borders . this implied that quite a number of irrigation districts no longer formed coherent ecological systems as a result of which the proper flow of water was seriously hampered . moreover , small irrigation units were amalgamated to form fewer and bigger irrigation associations which were organized according to a uniform model . in this way the twelve irrigation districts that had been formed in the former kingdoms of badung and mengwi in 1907 were reduced to six . these amalgamations led to irrigation areas of such a size that proper coordination was no longer possible . whereas in former days a large number of sedahan had coordinated these tasks , these fell now to a single official who was often not familiar with local conditions . the distance between peasants and officials at the regional level increased due to a process of ongoing bureaucratization . since the dutch preferred candidates with bureaucratic skills in order to collect taxes countless small irrigation associations were amalgamated into new and bigger subak . to some extent however , the old cooperative arrangements continued to function as sub - units , or munduk or tempek , within larger subak . the difference was , however , that these sub - units depended for water supply on the larger subak and its head . perhaps the most important structural change took place from 1914 onwards when the old vulnerable earthen dams and conduits were gradually replaced by new strong concrete constructions . since the dutch had conquered the south balinese kingdoms , dynastic control of dams and main conduits was discontinued . as a result some large dams collapsed between 1909 and 1914 and hundreds of hectares of rice fields fell dry . from now on the dutch were responsible for irrigation at the regional level . between 1914 and 1931 four concrete dams were build in the ayung river which together irrigated 12,763 hectares of rice fields.16 lansing does not seem to be fully aware of the fundamental changes this brought to irrigation management . in contrast to the pre - colonial period these new permanent constructions guaranteed a steady supply of water flowing to the rice fields . the responsibility for the building and maintenance of the new concrete structures fell under a separate institution of the colonial state , the public utilities service , which replaced the mobilization of labor under dynastic supervision . consequently , the manifold connections between local irrigation associations and central water supply , which involved in the pre - colonial period the regular mobilization of hundreds of people by dynastic officials to repair the central dam and the main conduits , fell into disuse . moreover , colonial pacification made an end to regional warfare which had often threatened the continuity of the irrigation process . it was only because of these new conditions that for the peasants in south bali irrigation became primarily a local affair . ironically , it was this colonial intervention which was instrumental in achieving the colonial image of the original balinese subak as a strictly local and autonomous institution . as the dutch did not interfere in the ritual activities that accompanied the flow of the seasons , this consequently irrigation management at the local level and ritual affairs remained outside the domain of the colonial state . according to lansing ( 2006 , 2007 ) irrigation management in bali has been a bottom - up process which was and still is supported by networks of autonomous associations while cooperation and coordination is structured by temple networks . since i have not conducted research in central bali , i will refrain from criticizing lansing s findings in that area.17 what i want to emphasize is that south bali offered a different picture . here , to say this , of course , is not to deny the important role that peasant associations play in day - to - day irrigation management at the local level.18 structural changes which occurred during the colonial period created more local autonomy in irrigation management while temples still played a crucial role . hence , it could very well be the case that what lansing tends to perceive as authentic self organizing processes , were actually the result of a colonial transformation which took place at the beginning of the twentieth century .

this article takes issue with stephen lansing s bottom - up model of balinese irrigation management . based on archival research and extensive fieldwork in the former south balinese kingdom of mengwi , it is argued that in pre - colonial days large scale irrigation depended largely on dynastic involvement . during the colonial period ( 19061942 ) the dutch took over the role of regional irrigation management while they strengthened the autonomy of local irrigation associations .

Introduction Dynasty and Irrigation in Pre-colonial South Bali Colonial Transformations

colonial and post - colonial anthropological literature has paid inadequate attention to the involvement of the aristocracy in large scale irrigation in pre - colonial bali . it has one - sidedly emphasized the cooperative and even autonomous nature of local irrigation networks ( grader 1960 ; geertz 1980 ) . he also emphasizes the autonomous and bottom - up nature of local irrigation management , but adds an important qualification by arguing that regional irrigation management centered upon extended temple networks which culminated in the batur temple complex in central bali . in doing so , he seems to downplay , or even ignore , the role played by regional dynasties and noble lords in the pre - colonial period , while he also seems to underestimate the structural changes which took place at the beginning of the colonial period during the first decades of the twentieth century . although he is aware that i wrote about irrigation in bali during the pre - colonial era and the colonial period , for reasons which are not entirely clear to me , he only refers to this in a very restricted manner.1 it is true that my work is focused on another area in bali i.e . , the southern rice plains of mengwi and badung where conditions were perhaps different from the findings of his research i.e . , in uphill bangli and gianyar ( central bali)but my conclusions contradict both lansing s model and the implicit claim in his work that it is applicable for the whole of bali . in the following pages i will therefore summarize my findings because they offer an alternative picture of irrigation management in pre - colonial bali . control of manpower was one of the main objectives of the pre - colonial contest states in southeast asia and the south balinese kingdom of mengwi ( 17101891 ad ) was no exception in this respect . there was a correspondence between the expanding control of the dynasty over manpower and an increased grip on the irrigation systems in the region . contrary to lansing s model there is ample evidence that the mengwi dynasty was closely involved in the construction , upkeep , and the mostly ritual regulation of the irrigations systems . sometimes the water level of the river was dozens of feet below the rice fields , so that in order to irrigate them a dam had to be constructed many miles upstream . the water that was collected at those dams had to be diverted via lengthy conduits and tunnels before it reached the downstream rice fields . the construction of these central dams required the mobilization of a large amount of manpower which was controlled and coordinated by either the dynasty or other local princes . in many oral histories , which i collected during my fieldwork in the early 1980s throughout the mengwi region , mention is made that the king or a local lord ordered the clearing of land and organized the construction of upstream dams . the rise of the dynasty went hand in hand with the expansion of irrigated rice fields in the region . dynastic involvement was required because most villages had a limited range of action and cooperation between villages was often lacking . dynastic involvement was therefore necessary in order to protect and maintain the vulnerable irrigation systems . upkeep demanded manpower and so did reconstruction work since the large earthen dams quickly eroded in the fast - flowing rivers and were often wiped out suddenly at the start of the rainy season by a flash flood . as an example of the mechanisms by which this happened , one of the large dams in the river ayung , dam oongan which was some 55 ft in height , tended to undermine itself because overflow water was diverted at the downstream side of the dam . it is true that small - scale cooperation at the local level was an important feature of irrigation in bali and this is certainly true for the twentieth century . however , without water such cooperation could not be materialized . none but regional dynasties and local lords were able to guarantee the continuity of this fragile system.3 the geographical location of the dynastic centre of mengwi and a number of its satellites it is said that the first king of mengwi together with a local lord who resided north of the dynastic centre built a dam in the river sungi and proceeded to expand rice fields in his domain . as a result the dynastic centre was located in a wide fertile plain some 200 m above sea level where surplus for the dynasty was produced . while the newly constructed dam was guarded by the northern lord , an important side effect was that it also allowed the dynastic centre to influence downstream irrigation . this happened in the nineteenth century during prolonged conflicts between the royal centres of mengwi and badung , and bangli and gianyar . because the political system in bali was fragmented there was no monopoly of power at the centre . satellites of the dynasty and other lords were in control of their own irrigations systems . the lord of sibang , for instance , had his own dam twenty miles upstream in the river ayung which was located in the domain of another lord . the lord of sibang not only looked after his own dam , but he was also responsible for the upkeep of another big dam in the ayung river . little is known about the construction of this dam but in the course of the eighteenth century it became the biggest irrigation project in south bali providing water for thousands of hectares of rice fields in the southern part of the mengwi kingdom.4 the entire system was spread over the domains of several lords . much of the upkeep of this complex system , which included a long tunnel , was delegated to lesser lords and lower units , but , the dynastic centre was ultimately responsible for the overall supervision . in this context neither bottom up coordination based on autonomous local irrigation units was able to manage this huge system , nor is there any indication that it was solely managed by temples without any dynastic involvement . on the contrary , without the mengwi dynasty the irrigation system of the gumasih dam was simply unthinkable . lansing ( 2007 : 34 ) seems to downplay the importance of dynastic involvement in south bali but offers no evidence that contradicts my findings . the king and local lords had a group of functionaries ( or sedahan ) at their disposal who were responsible for timely repair of the dams and conduits , water allocation to irrigation units , and the collection of taxes . on this point , clifford geertz ( 1980 : 67 ) presented a contrasting view regarding pre - colonial bali that there was no systematic congruence [ ] between the structure of political authority , the structure of land tenure , and the distribution of land tenancy. however , all the data i was able to collect indicate the opposite . in pre - colonial mengwi there was a strong coherence between political power , the distribution of land , and between the owner and the worker of the land , which is illustrated by the following example from the area of sibang : when the dam at sibang was built , many rice fields around sibang were opened up . each of them received a larger or smaller ( side ) canal , and they could allocate the land on either side of it to their personal followers . trusted favorites received a larger piece of land , while ordinary followers were given about two acres.the lord of sibang selected the best field for his own use and his slaves grew the crop for his household . only a small portion of it had to be paid to the lord as tax . trusted favorites received a larger piece of land , while ordinary followers were given about two acres . the lord of sibang selected the best field for his own use and his slaves grew the crop for his household . only a small portion of it had to be paid to the lord as tax . a follower whose loyalty and service towards the lord were found wanting would lose access to his land and was to be ousted from the circle of followers.5 in the course of the eighteenth century irrigation expanded rapidly under the encouragement of the mengwi dynasty . local lords , like the one in sibang , managed their own decentralized systems and were in control of their own followers . a weak king or a weak dam conversely , the irrigation order and the dynastic hierarchy could only be maintained if the royal centre was strong ( see fig . 1mengwi in the eighteenth century ( from schulte nordholt 1996 : 54 ) mengwi in the eighteenth century ( from schulte nordholt 1996 : 54 ) the correlation between these two hierarchies was expressed in a telling way . until recently it was custom that in the villages of kapal and sibang , prior to starting to work on the rice fields , a procession would carry a sacred dynastic dagger ( or keris ) to the central dam . there the blade was pushed into the dam accompanied by various rituals , a result of which the powers that resided in the dagger would be infused into the dam . there is indirect evidence that similar rituals used to be held in connection with the dam north of the dynastic centre of mengwi . this suggests that the relation between a strong ruler and a sturdy dam was crucial , because dynastic hierarchy and a smooth irrigation order supported and reinforced each other . the relationship between dynasty and irrigation management also confirms the important role kingship played in guaranteeing the continuity of fertility in the realm , which found its expression in the parallel circulation of water and the human soul between mountains , downstream valleys and the sea.6 how vulnerable both the dynastic and the irrigation orders were is nicely illustrated by a balinese historical song , the kidung nderet , in which a major crisis of the mengwi dynasty during the early 1820s is told an analyzed.7 due to poor leadership in the centre of the dynasty both the hierarchy of lords and followers and the irrigation order fell apart . during the reign of king cokorda munggu ( 174?1780 ) the responsibility for the dam , the tunnel and conduits , and part of the rice fields that received water from the dam was given to one of his sons . under the new king , however , the dynastic centre withdrew this privilege and resumed direct control over the irrigation system of dam gumasih while it also centralized the taxation . the interesting point here is that this balinese text written in the nineteenth century also emphasizes the close connection between dynasty and irrigation.8 around the middle of the nineteenth century the mengwi dynasty managed to recover . part of the recovery was the extension of rice fields in the northeastern region of the mengwi kingdom which involved the building of dams , tunnels and temples.9 this was to a large extent a top - down operation coordinated by the leading lineage of the dynastic centre . most likely , the actual organization of the various irrigation systems in pre - colonial bali differed widely per locality depending on the political and ecological context . in most instances , however , lord - follower relationships informed the lines along which these systems were organized . as far as can be determined , extension of irrigated fields did not automatically result in the establishment of ( semi ) autonomous local irrigation units , or subak , which operated independently from villages . oral traditions concerning this period suggest a different picture . without exception , informants emphasized the leading role of the nobility in the project while they did not mention subak as separate organizations . this official was the lowest link in the regional irrigation hierarchy who was also responsible for the collection of a royal tax on the use of irrigation water , the suwinih . the dynastic centre used this tax to finance large rituals in the newly built irrigation temples . these temples fostered in the first place the fertility of the land and averted plagues . the temples and rituals were supervised by irrigation officials while the dynastic involvement was restricted to the granting of permission to stage certain rituals . however , as soon as ritual and maintenance were neglected and disorder could erupt , it was the duty of the power holders to restore order and hierarchy . it is for this reason that pura arantaja , the main irrigation temple of the northeastern region of mengwi , was commonly called accordingly , this temple represented not only the link between gods , fertility , and men , but was also a reminder of royal authority in the region . so far , i have found no evidence that supports lansing s bottom up model of irrigation management exclusively handled by temple systems . instead , we see a complex but integrated system in which dynasties , temples , and peasants participated and hierarchical relationships and horizontal collaboration were combined . in this system upstream irrigation was primarily coordinated and protected by the dynasty , while downstream irrigation was to a large extent a local affair . labor was mobilized both for local purposes as well as for large scale upstream projects on dams and conduits . in terms of daily routine the system was as much as possible decentralized but the system as a whole depended in the end on dynastic coordination . dynasties , and not temples , were the key institutions that guaranteed the continuity of the system . soon after the dutch had conquered south bali at the beginning of the twentieth century , colonial administrators started to reorganize local society according to orientalist models of a supposed authentic bali in which the village played a central role . since the arbitrary royal rule.10 apart from the reorganization of village administration , which also served to mobilize a reservoir of labor , irrigation became another object of colonial interference , which ultimately aimed to levy an increasing amount of tax.11 based on research conducted by the colonial administrator f.a . his description of the northern balinese irrigation association ( subak ) was taken as the normative standard for the reorganization of irrigation management in south bali . the north featured small - scale irrigation systems without direct aristocratic interference , whereas the south had large scale systems which depended on big dams and dynastic involvement.13 contrary to north bali , the organization of irrigation in the south varied by locality while boundaries between village and subak were often fluid . liefrinck had presented a uniform model in which no consideration was given to local variation or direct aristocratic involvement . as he saw it , dynastic interference was essentially unrelated to the subak , but it had penetrated into the local networks in order to appropriate taxes . this point of view reflected a very common orientalist view of that time which favored the autonomy of local institutions which had to be protected under benign colonial rule . it seems that lansing s ideas were at least partially influenced by this colonial perception . lansing argues that the dutch deliberately sought to restore the centralized irrigation hierarchy of the former kingdoms and that they even employed archaeologists to legitimize this project ( lansing 2007 : 3536 ) . actually , the dutch did the opposite , at least on paper . using liefrinck s model , the colonial administrators in the south observed that little was left of the supposed restored in conformity with the north balinese model.14 what in fact happened , however , was that this so - called restoration fitted the needs of the colonial state . a memorandum on the former kingdom of klunkung by the dutch administrator of bali , resident g.f . in the days of the kings [ ] all the nobles and prominent people had their own tax collector who made sure that his own land was watered aplenty . in the days of the kings [ ] all the nobles and prominent people had their own tax collector who made sure that his own land was watered aplenty . urgently needed is a separation between irrigation workers and administrative personnel ; a division of irrigation areas , headed by a supervisor must be designed ; the subak have to be assigned in groups to avoid too large a number of subak heads which would impede administrative control.15 within a few years the dutch succeeded in replacing the plurality of relationships from the pre - colonial period with an overarching colonial bureaucracy . in each former kingdom , which had become an administrative district , a balinese official ( sedahan agung ) was appointed who supervised a number of lower officials ( sedahan ) who on their turn both supervised the distribution of irrigation water and the levying of new colonial taxes . while the term subak was known as a local irrigation leader in the mengwi area , it now became the name of the local irrigation association all over bali . the dutch introduced a new term for the subak leader , i.e. , pekaseh , which was taken from the neighboring kingdom of gianyar . these terminological shifts were not insurmountable , but great confusion ensued when the dutch started to reorganize the actual irrigation networks . bureaucratic and administrative criteria prevailed to the extent that irrigation networks had to coincide with administrative borders . this implied that quite a number of irrigation districts no longer formed coherent ecological systems as a result of which the proper flow of water was seriously hampered . moreover , small irrigation units were amalgamated to form fewer and bigger irrigation associations which were organized according to a uniform model . in this way the twelve irrigation districts that had been formed in the former kingdoms of badung and mengwi in 1907 were reduced to six . these amalgamations led to irrigation areas of such a size that proper coordination was no longer possible . the distance between peasants and officials at the regional level increased due to a process of ongoing bureaucratization . since the dutch preferred candidates with bureaucratic skills in order to collect taxes countless small irrigation associations were amalgamated into new and bigger subak . to some extent however , the old cooperative arrangements continued to function as sub - units , or munduk or tempek , within larger subak . the difference was , however , that these sub - units depended for water supply on the larger subak and its head . since the dutch had conquered the south balinese kingdoms , dynastic control of dams and main conduits was discontinued . as a result some large dams collapsed between 1909 and 1914 and hundreds of hectares of rice fields fell dry . from now on the dutch were responsible for irrigation at the regional level . between 1914 and 1931 four concrete dams were build in the ayung river which together irrigated 12,763 hectares of rice fields.16 lansing does not seem to be fully aware of the fundamental changes this brought to irrigation management . in contrast to the pre - colonial period these new permanent constructions guaranteed a steady supply of water flowing to the rice fields . the responsibility for the building and maintenance of the new concrete structures fell under a separate institution of the colonial state , the public utilities service , which replaced the mobilization of labor under dynastic supervision . consequently , the manifold connections between local irrigation associations and central water supply , which involved in the pre - colonial period the regular mobilization of hundreds of people by dynastic officials to repair the central dam and the main conduits , fell into disuse . moreover , colonial pacification made an end to regional warfare which had often threatened the continuity of the irrigation process . ironically , it was this colonial intervention which was instrumental in achieving the colonial image of the original balinese subak as a strictly local and autonomous institution . as the dutch did not interfere in the ritual activities that accompanied the flow of the seasons , this consequently irrigation management at the local level and ritual affairs remained outside the domain of the colonial state . according to lansing ( 2006 , 2007 ) irrigation management in bali has been a bottom - up process which was and still is supported by networks of autonomous associations while cooperation and coordination is structured by temple networks . since i have not conducted research in central bali , i will refrain from criticizing lansing s findings in that area.17 what i want to emphasize is that south bali offered a different picture . here , to say this , of course , is not to deny the important role that peasant associations play in day - to - day irrigation management at the local level.18 structural changes which occurred during the colonial period created more local autonomy in irrigation management while temples still played a crucial role . hence , it could very well be the case that what lansing tends to perceive as authentic self organizing processes , were actually the result of a colonial transformation which took place at the beginning of the twentieth century .

observed maximum in ocean heat content trend in early 2000s is likely spuriousnet incoming radiation ( n ) reduced by 0.31 0.21 w m during the warming pausepresent - day estimates of n may contain opposing errors in radiative components observed maximum in ocean heat content trend in early 2000s is likely spurious net incoming radiation ( n ) reduced by 0.31 0.21 w m during the warming pause present - day estimates of n may contain opposing errors in radiative components the recent slowdown in the rate of global surface temperature rise [ easterling and wehner , 2009 , knight et al . , 2009 ] has highlighted a need for a better quantification of earth 's energy imbalance [ hansen et al . , 2011 ; katsman and van oldenborgh , 2011 ; balmaseda et al . , 2013 ; trenberth et al . , 2014 ] and improved understanding of the flow of energy in the climate system [ trenberth , 2009 ; meehl et al . , 2011 ; england et al . , earth 's energy imbalance is defined here as the net downward radiative energy flux ( n , expressed in w m averaged over the entire surface of the earth ) through the top of the atmosphere ( toa ) , representing the residual between incoming solar radiation and outgoing reflected ( mainly short - wavelength visible ) and emitted ( mainly long - wavelength infrared ) radiation . n varies naturally on all timescales due to internal variability of the climate system , volcanic eruptions , and variations in incoming solar radiation . however , increased levels of greenhouse gases initially increase n by reducing outgoing longwave radiation , driving climate change until radiative equilibrium is restored and n returns to zero on average . humans also influence n by changing outgoing shortwave radiation through emissions of aerosol particles and changes to land usage which affect surface albedo . furthermore , subsequent changes in n in response to initial perturbations may be amplified or reduced by feedbacks within the climate system [ boucher et al . , 2013 ; myhre et al . , quantifying the time mean and variability of n is therefore fundamental for understanding climate variability and change [ trenberth , 2009 ; hansen et al . allan et al . produced a homogenized satellite data set of n covering the period since 1985 . satellites provide reasonably accurate estimates of interannual variations in n [ loeb et al . , 2012 ] , but absolute values can not be determined accurately [ stephens et al . , however , on multiyear timescales , n is expected to be largely balanced by changes in global ocean heat content [ palmer et al . , 2011 ; palmer and mcneall , 2014 ] , since changes in energy of the atmosphere , land , and cryosphere are much smaller [ levitus et al . , 2001 observations of trends in ocean heat content ( hereafter ht ) therefore potentially enable the satellite observations to be anchored to obtain absolute values [ loeb et al . , 2012 ; allan et al . , 2014 ] , as well as providing independent estimates of variations in n. however , the subsurface ocean was poorly observed historically , with less than 20% of 1 latitude by 1 longitude regions sampled yearly in the upper 700 m before 1990 [ abraham et al . , 2013 ] , and estimates of ht from different ocean analyses appear to show little consistency , at least on interannual timescales [ trenberth et al . , observational coverage increased to 3040% of 1 regions after 1990 , but observation - based estimates of ht for the period 1998 to 2008 vary by a factor of 2 , from 0.31 ( full depth ocean [ church et al . , 2011 ] ) to 0.64 w m ( upper 700 m only , [ lyman et al . , 2010 ] ) . even in the period since 2005 in which more than 70% of 1 regions are sampled by argo floats [ roemmich et al . , 2009 ] , estimates of ht in the upper 700 m range from 0.16 to 0.39 w m over the period 20052012 [ abraham et al . , 2013 ] . furthermore , annual values of ht appear to be inconsistent with satellite observations of n in some years [ trenberth et al . , 2014 ] , suggesting that earth 's energy budget can not be closed with observations even over the relatively well - observed period since 2005 [ trenberth and fasullo , 2010 ] . here we investigate earth 's energy imbalance over the period since 1960 in observations , atmospheric models forced by observed sea surface temperatures and radiative changes ( atmosphere model intercomparison project , amip ) , and fully coupled model simulations ( coupled model intercomparison project , cmip5 ) . we build on allan et al . by including estimates of ht and developing an observation - based record of n extending back to 1960 . we show that two reanalyses of ht in the early 2000s are inconsistent with satellite observations and amip simulations of n and are therefore likely unreliable , helping to explain our inability to close the energy budget . however , prior to 2000 variations in ht in ocean reanalyses are in reasonable agreement with observation - based estimates of n and with cmip5 models , as earth 's energy imbalance was dominated by volcanic eruptions over this period . we also highlight an observation - based reduction in n that likely contributed to the recent slowdown in surface warming . observational estimates of n since 1985 are based on the clouds and the earth 's radiant energy system ( ceres ) instruments since 2000 and the earth radiation budget satellite ( erbs ) wide field of view nonscanning instrument from 1985 to 1999 [ allan et al . , 2014 ] . these two data sets were homogenized , and gaps filled , using the era interim reanalysis [ dee et al . , 2011 ] and high - resolution atmosphere model simulations driven by observed sea surface temperatures and sea ice concentrations ( see figure s1 in the supporting information and allan et al . for full details ) . this satellite - based n is adjusted to be 0.58 w m over the period july 2005 to june 2010 [ loeb et al . , 2012 ] based on ocean heat content changes ( ht ) of 0.47 w m in the upper 1800 m obtained from a linear fit through argo data [ following lyman and johnson , 2008 ] and 0.07 w m below 1800 m [ purkey and johnson , 2010 ] , plus 0.04 w m estimated for heating and melting of ice and heating of the land and atmosphere . we also use n from amip model simulations form ( i ) the upscale project [ mizielinski et al . , 2014 ] over the period 1986 to 2011 and ( ii ) multimodel simulations of the climate of the twentieth century project ( c20c ) [ scaife et al . , 2009 ] for the period 1950 to 2002 . three of the 16 c20c models were eliminated from our study due to unrealistic anomalies of n. amip simulations can not provide absolute values of n because the sea surface temperature ( sst ) forcing potentially provides unrealistic sources or sinks of energy . amip simulations are in good agreement with satellite observations ( r > 0.6 ) for monthly deseasonalized variability in n over the period 1986 to 2011 ( allan et al . and figure s1 ) , showing that sst observations provide useful additional constraints on n. we therefore create an extended observation - based estimate of n ( hereafter no ) by averaging the satellite observations with the ensemble means of the c20c and upscale simulations ( with each data set given an equal weight where available ) . we use no even when satellite data are available since errors are likely to be reduced by including independent information from the sst observations ( via the amip simulations ) . we compute uncertainties from differences between upscale , c20c , and satellite data ( 0.12 w m ) as well as the average spread of the c20c simulations ( 0.09 w m ) added in quadrature to the 1 sigma anchoring uncertainty ( 0.24 w m ) estimated in allan et al . to obtain a total uncertainty on no of 0.28 w m. ocean heat content trends ( ht ) are computed from the slope of a least squares regression using annual time series of heat content and expressed as a flux relative to the total surface area of the earth [ following lyman and johnson , 2008 ] . we compute ht over 5 year periods and compare with rolling 5 year mean n , thereby smoothing out interannual variability related to el nio . absolute values of n can not be determined accurately from satellite observations [ stephens et al . , we therefore also assess rates of change of ht and n ( htt = dht / dt and nt = dn / dt , again computed with least squares regression over 5 year periods ) , since nt does not depend on absolute values . ocean data are taken primarily from the met office statistical ocean reanalysis ( hereafter mosora , updated from smith and murphy ; see supporting information ) . several published analyses of ht for the upper 700 m appear to capture signals associated with major volcanic eruptions , but the timing is not always consistent , and there is little agreement at other times [ trenberth et al . , however , the ora - s4 analysis , in which temperature and salinity observations are assimilated into a dynamical ocean model [ balmaseda et al . , 2013 ] , was found to be in reasonable agreement with model simulations of volcanic impacts and also simulated plausible changes in ht associated with el nio [ trenberth et al . , because full - depth values are available , we also include ora - s4 in our study . time - varying biases in expendable bathythermographs ( xbts ) and mechanical bathythermographs were corrected in mosora based on an average of adjustments proposed by wijffels et al . this approach was tested , and uncertainties estimated , by performing data withholding experiments for the data - rich period 20072013 , which was not used for the covariances . in these experiments , analyses created using all available observations were taken as the reference and compared with analyses created using subsampled observations at locations typical of historical periods . a total of 12 tests were performed using data locations from 1950 to 1956 , 1955 to 1961 , 1960 to 1966 , , 2005 to 2011 . patterns of ht reconstructed with historical observations agree well with the reference ( figure1 ) . for example , the spatial correlation is 0.88 and 0.76 using the 1990s and 1960s observations , respectively ( compare figure1b with figures1d and 1f ) . uncertainties ( which we quote as 1 sigma throughout ) in global heat content range from around 28 zj ( zj = 10 j ) using 19501956 observations to 8 zj using 20052011 observations , corresponding to uncertainties in ht of 0.7 to 0.2 w m ( table s1 ) . we stress that these estimates only assess uncertainties arising from the observational coverage relative to the recent argo era and do not include unknown instrument errors or the potentially large contributions from regions not sampled adequately by argo floats , such as the shelf seas , ice - covered regions , and ocean below 2000 m [ e.g. , von schuckmann et al . , furthermore , our subsampling experiments suggest that the magnitude of ht is not fully captured using the historical data , especially in the southern ocean ( figure1f ) so that ht might be underestimated in the historical period prior to argo , as suggested by durack et al . . ( left ) total number of temperature observations at 1000 m depth during the period 2008 to 2012 using ( a ) all data and subsampled data typical of ( c ) the 1990s and ( e ) the 1960s . ( right ) patterns of ocean heat content trends ( ht , w m ) for the period 2008 to 2012 computed using ( b ) all observations and subsampled observations typical of ( d ) the 1990s and ( f ) the 1960s . we compare observational estimates of ht and n with 21 cmip5 coupled model simulations ( taylor et al . and table s2 ) driven by observed variations in greenhouse gases , aerosols , solar radiation , and land use until 2005 and following representative concentration pathway ( rcp4.5 ) thereafter . there are long - term drifts in the cmip5 models because the preindustrial control simulations were not necessarily in radiative equilibrium [ sen gupta et al . we therefore compute anomalies in the historical and rcp4.5 simulations relative to preindustrial control simulations ( using at least 100 years of the control simulations ) . global ocean heat content , ht and htt from mosora and ora - s4 are in good agreement ( r = 0.94 , 0.80 , and 0.82 respectively for running 5 year averages ) over their overlapping period ( compare red and dashed magenta curves in figure2 ) , despite the lack of historical ocean observations and additional uncertainties involved in computing derivatives for ht and htt . furthermore , over the period 19601999 , ht and htt are in broad agreement with n ( r = 0.58 and 0.56 between mosora and ora - s4 and no ) and nt , ( r = 0.63 and 0.69 ) . these correlations are significantly greater than zero with 99% confidence based on bootstrapping in 5 year blocks to take serial correlation into account [ e.g. , goddard et al . , ( first panel ) annual mean total ocean heat content anomalies ( relative to 19612010 , zj = 10 j ) from mosora ( red ) [ smith and murphy , 2007 ] and ora - s4 ( dashed magenta ) [ balmaseda et al . , 2013 ] . ( second panel ) five year running mean ocean heat content trends ( ht , red and magenta ) and net incoming radiation ( n ) at the top of the atmosphere from satellites [ allan et al . , 2014 ] ( blue curves ) and atmosphere models driven by observed surface temperatures for the climate of the twentieth century ( c20c ) [ scaife et al . , 2009 ] ( cyan ) and upscale [ mizielinski et al . , 2014 ] ( blue shading ) projects . ( third panel ) trends in ht ( htt , red and magenta ) and n ( blue and cyan ) . red shading shows uncertainties ( 1 sigma ) in mosora arising from observational coverage ( see text and table s1 ) . grey shading highlights potentially spurious ocean reanalyses ( see text ) , with significant differences ( 90% level ) between mosora and n shown by black squares . in figure2 ( second to third panels ) the year represents the center of the time period used to compute trends . after 2000 , ht and htt show pronounced variability that is not present in n and nt ( figure2 , second and third panels ) . this is largely caused by the rapid increase in ocean heat content between 2001 and 2004 resulting in a peak ht in 2002 followed by a trough in 20062007 in both mosora and ora - s4 ( figure2 , second and third panels ) . a peak in ht around 2002 is also evident in other ocean analyses [ trenberth et al . , 2014 ] . however , such an increase in heat content is not supported by thermosteric sea level observations , because total sea level increased at a fairly constant rate [ church et al . , 2013 ] while freshwater input from glaciers and ice sheets likely increased [ marzeion et al . , 2012 ; shepherd et al . , 2012 ; church et al . , 2013 ] , implying a reduction in the rate of thermosteric sea level rise ( and hence ocean heat content trend ) over the period 2001 to 2004 . although interpretation of sea level changes is complicated by the nonuniformity of thermal expansion coefficients , warming is not consistent with a reduction in thermosteric sea level trends . given this , and the agreement between independent estimates of n ( and nt ) from satellite observations and amip simulations ( figure2 ) , and from forced ocean model experiments ( figure s2 ) , we suggest that there are likely to be errors in the observational reanalysis of ocean heat content between 2001 and 2007 ( grey - shaded region in figure2 ) . cheng and zhu drew similar conclusions from a different approach , suggesting that these errors are caused by the transition from xbt to argo data . however , this does not appear to be the sole cause in our study since mosora shows a similar peak in ht in 2002 even when argo observations are omitted ( figure2 , black asterisks ) , although the subsequent trough is reduced . further work is therefore needed to understand the cause of these errors in ocean reanalyses . possible explanations include inadequate vertical or horizontal sampling and/or unresolved biases in the instruments themselves . cmip5 coupled models simulate variations in n and ht that are in good agreement with observations ( r = 0.82 between 5 year running mean cmip5 ensemble mean and no , and 0.68 with mosora excluding the period after 2000 , figure3a ) . since internal variability is largely removed in the ensemble mean of the cmip5 models , this agreement suggests that much of the multiyear variability in earth 's energy imbalance from 1960 to 2000 was externally forced by the volcanic eruptions of agung ( 1963 ) , el chichon ( 1982 ) , and pinatubo ( 1991 ) , as noted by trenberth et al . . furthermore , the cmip5 models do not simulate a peak in ht in 2002 , and the observed peak is significantly different from the cmip5 models , consistent with our earlier conclusion that estimates of ht based on ocean analyses between 2001 and 2007 ( grey shading in figure2 ) are likely unreliable . earth 's energy imbalance . ( a ) time series of 5 year running mean n and ht ( as figure2 , second panel ) for 21 cmip5 coupled model simulations ( n in green , ht in orange , ensemble mean in thick lines ) compared with ht from mosora ( red ) and no ( blue , see text ) . black squares ( diamonds ) show where differences between mosora and no ( cmip5 ) are significant with 90% confidence . ( b ) n averaged over different periods in no ( blue , with 1 sigma uncertainties ) compared to the cmip5 models ( green , box showing the mean 1 sigma and whiskers showing the range ) and estimates from the ipcc fifth assessment ( red ) [ rhein et al . , 2013 , box 3.1 ] . averaged over different periods , no is in broad agreement with the intergovernmental panel on climate change ( ipcc ) estimates [ rhein et al . , 2013 , box 3.1 ] and the cmip5 models ( figure3b ) , but it is difficult to draw firm conclusions because uncertainties in the absolute values of no are large . these uncertainties are dominated by the anchoring estimate of ht and could potentially be reduced by averaging ht over longer periods . we do not attempt this because ocean observations in the early 2000s are potentially unreliable ( as discussed above ) , historical analyses do not capture the full magnitude of ht , especially in the southern ocean ( figure1 ) [ durack et al . , 2014 ] , and uncertainties are evident even in the most recent 5 year means which show a decrease in no but an increase in ht ( figure3a ) . we therefore investigate changes in n and anomalies relative to the period 19602011 since these are less affected by anchoring uncertainties . in general , there is good agreement between the cmip5 ensemble mean and observation - based anomalies of n ( r = 0.82 for 5 year running means ) and its components , absorbed shortwave ( asr , r = 0.87 ) and outgoing longwave ( olr , r = 0.80 ) radiation ( figure4 ) . the cmip5 ensemble mean therefore appears to simulate a realistic magnitude of variability associated with volcanic eruptions . , 1997 ; power et al . , 1999 ] may have contributed to no . however , an increase in n is also apparent in the cmip5 ensemble mean in the late 1970s ( especially in asr ; red curves in figure4 ) , suggesting that external forcing may have played a role . time series of 5 year running mean anomalies ( relative to 1960 to 2011 ) of toa - absorbed shortwave radiation ( asr , red ) , outgoing longwave radiation ( olr , green ) , and net radiation ( n = asr - olr , blue ) in no ( dashed ) and the cmip5 models ( solid , with thick line showing the ensemble mean ) . models and observations both show a rapid increase in n during the recovery from the eruption of mount pinatubo , reaching a peak in the late 1990s . however , there is a subsequent decline of 0.43 0.21 w m in no between 1999 and 2005 in the early part of the global warming slowdown which is larger in magnitude than the model ensemble mean ( 0.12 0.06 w m ) and at the lower end of individual models ( + 0.38 to 0.32 w m ) . both the satellite observations and the amip simulations show a decline over this period ( figures2 , middle , 2 bottom , and s3 ) , showing that the observed signal is not an artifact of our averaging procedure . it is also not caused by the transition from erbs to ceres satellites since all six upscale amip simulations show reductions , ranging from 0.30 to 0.52 w m. furthermore , a similar decline in surface flux between 1999 and 2005 is simulated in forced ocean model experiments ( figure s2 ) . we therefore compute the difference averaged over 20042006 minus 19982000 , giving a reduction of 0.31 0.21 w m between the late 1990s and mid-2000s . we find significant correlation ( r 0.6 , p < 0.01 ) between rolling 5 year mean ocean heat content trends ( ht ) in two ocean analyses and net incoming radiation at the top of the atmosphere ( n ) from satellite observations and amip model simulations over the period 1960 to 1999 . ocean reanalyses of this historical period are therefore potentially useful for further studies of the physical processes of ocean heat uptake . however , ocean reanalyses of ht in the early 2000s are inconsistent with observation- and model - based constraints on n. this helps to explain our inability to close earth 's energy budget [ trenberth and fasullo , 2010 ; trenberth et al . , 2014 ] and suggests that observed estimates of ht covering this period [ e.g. , lyman et al . , 2010 , levitus et al . , 2012 , abraham et al . , 2013 ; rhein et al . , 2013 ] should be treated with caution . it also calls into question whether ocean reanalyses can be used to robustly attribute increased ht below the ocean mixed layer in the early 2000s as an explanation for the onset of the recent slowdown in surface warming [ guemas et al . , we create an observation - based estimate of n ( no ) extending back to 1960 , using satellite data and amip model simulations , and compare with 21 cmip5 coupled climate model simulations . variations in n over this period are dominated by the volcanic eruptions of agung in 1963 , el chichon in 1982 , and pinatubo in 1991 [ trenberth et al . , 2014 ] , with good agreement ( r = 0.82 ) between no and the cmip5 ensemble mean . absolute values of no are in broad agreement with the models , but uncertainties are large and improved estimates of ht are needed to provide a more accurate anchor for satellite observations [ loeb et al . , we find a reduction of 0.31 0.21 w m in no between the late 1990s and the mid-2000s which may have contributed to the recent slowdown in global surface warming . , 2011 ; fyfe et al . , 2013 ; haywood et al . , 2014 ; santer et al . , 2014 ] , an extended and deeper solar minimum [ lean , 2009 ; kaufmann et al . , 2011 ] , and possible nitrate and indirect aerosol effects [ shindell et al . , 2013 ; bellouin et al . , 2011 ] that were not included in the cmip5 models [ flato et al . , 2013 , however , the reduction in no is caused by an increase in olr rather than a reduction in asr which would be expected from these factors ( compare green and red dashed curves in figures4 and s3 ) , though asr may have been reduced relative to the models . internal variability may also reduce n during cooling decades [ katsman and van oldenborgh , 2011 ; palmer and mcneall , 2014 ; brown et al . , 2014 ] , but the cmip5 ensemble mean does simulate a weak reduction ( 0.12 0.06 w m , mainly in asr ) , suggesting a potential role for external forcing . further work is therefore needed to unravel the relative roles of internal variability and external factors on earth 's energy imbalance during the recent warming pause . while present - day anomalies in n in the cmip5 ensemble mean are in good agreement with observations , this is potentially achieved through a cancelation of errors ( in models and/or observations ) since both asr and olr are larger in almost all of the models than the satellite observations ( figures4 and s3 ) . both asr and olr are projected to increase further over the coming decades , with anomalies in asr about twice as large as those in olr on average ( in agreement with cmip3 models [ trenberth and fasullo , 2009 ] ) . further analysis of the roles of forcing and feedback on these signals is needed , along with continued satellite and in situ observations of earth 's energy imbalance to monitor future climate variability and change .

observational analyses of running 5 year ocean heat content trends ( ht ) and net downward top of atmosphere radiation ( n ) are significantly correlated ( r 0.6 ) from 1960 to 1999 , but a spike in ht in the early 2000s is likely spurious since it is inconsistent with estimates of n from both satellite observations and climate model simulations . variations in n between 1960 and 2000 were dominated by volcanic eruptions and are well simulated by the ensemble mean of coupled models from the fifth coupled model intercomparison project ( cmip5 ) . we find an observation - based reduction in n of 0.31 0.21 w m2 between 1999 and 2005 that potentially contributed to the recent warming slowdown , but the relative roles of external forcing and internal variability remain unclear . while present - day anomalies of n in the cmip5 ensemble mean and observations agree , this may be due to a cancelation of errors in outgoing longwave and absorbed solar radiation.key pointsobserved maximum in ocean heat content trend in early 2000s is likely spuriousnet incoming radiation ( n ) reduced by 0.31 0.21 w m2 during the warming pausepresent - day estimates of n may contain opposing errors in radiative components

Key Points 1. Introduction 2. Data and Methods 3. Observation-Based Estimates 4. Comparison With CMIP5 Model Simulations 5. Summary and Conclusions Supporting Information

observed maximum in ocean heat content trend in early 2000s is likely spuriousnet incoming radiation ( n ) reduced by 0.31 0.21 w m during the warming pausepresent - day estimates of n may contain opposing errors in radiative components observed maximum in ocean heat content trend in early 2000s is likely spurious net incoming radiation ( n ) reduced by 0.31 0.21 w m during the warming pause present - day estimates of n may contain opposing errors in radiative components the recent slowdown in the rate of global surface temperature rise [ easterling and wehner , 2009 , knight et al . , earth 's energy imbalance is defined here as the net downward radiative energy flux ( n , expressed in w m averaged over the entire surface of the earth ) through the top of the atmosphere ( toa ) , representing the residual between incoming solar radiation and outgoing reflected ( mainly short - wavelength visible ) and emitted ( mainly long - wavelength infrared ) radiation . n varies naturally on all timescales due to internal variability of the climate system , volcanic eruptions , and variations in incoming solar radiation . furthermore , subsequent changes in n in response to initial perturbations may be amplified or reduced by feedbacks within the climate system [ boucher et al . , quantifying the time mean and variability of n is therefore fundamental for understanding climate variability and change [ trenberth , 2009 ; hansen et al . produced a homogenized satellite data set of n covering the period since 1985 . satellites provide reasonably accurate estimates of interannual variations in n [ loeb et al . , however , on multiyear timescales , n is expected to be largely balanced by changes in global ocean heat content [ palmer et al . , 2001 observations of trends in ocean heat content ( hereafter ht ) therefore potentially enable the satellite observations to be anchored to obtain absolute values [ loeb et al . , 2014 ] , as well as providing independent estimates of variations in n. however , the subsurface ocean was poorly observed historically , with less than 20% of 1 latitude by 1 longitude regions sampled yearly in the upper 700 m before 1990 [ abraham et al . , observational coverage increased to 3040% of 1 regions after 1990 , but observation - based estimates of ht for the period 1998 to 2008 vary by a factor of 2 , from 0.31 ( full depth ocean [ church et al . , 2009 ] , estimates of ht in the upper 700 m range from 0.16 to 0.39 w m over the period 20052012 [ abraham et al . furthermore , annual values of ht appear to be inconsistent with satellite observations of n in some years [ trenberth et al . here we investigate earth 's energy imbalance over the period since 1960 in observations , atmospheric models forced by observed sea surface temperatures and radiative changes ( atmosphere model intercomparison project , amip ) , and fully coupled model simulations ( coupled model intercomparison project , cmip5 ) . by including estimates of ht and developing an observation - based record of n extending back to 1960 . we show that two reanalyses of ht in the early 2000s are inconsistent with satellite observations and amip simulations of n and are therefore likely unreliable , helping to explain our inability to close the energy budget . however , prior to 2000 variations in ht in ocean reanalyses are in reasonable agreement with observation - based estimates of n and with cmip5 models , as earth 's energy imbalance was dominated by volcanic eruptions over this period . we also highlight an observation - based reduction in n that likely contributed to the recent slowdown in surface warming . observational estimates of n since 1985 are based on the clouds and the earth 's radiant energy system ( ceres ) instruments since 2000 and the earth radiation budget satellite ( erbs ) wide field of view nonscanning instrument from 1985 to 1999 [ allan et al . , 2011 ] and high - resolution atmosphere model simulations driven by observed sea surface temperatures and sea ice concentrations ( see figure s1 in the supporting information and allan et al . this satellite - based n is adjusted to be 0.58 w m over the period july 2005 to june 2010 [ loeb et al . , 2012 ] based on ocean heat content changes ( ht ) of 0.47 w m in the upper 1800 m obtained from a linear fit through argo data [ following lyman and johnson , 2008 ] and 0.07 w m below 1800 m [ purkey and johnson , 2010 ] , plus 0.04 w m estimated for heating and melting of ice and heating of the land and atmosphere . we also use n from amip model simulations form ( i ) the upscale project [ mizielinski et al . three of the 16 c20c models were eliminated from our study due to unrealistic anomalies of n. amip simulations can not provide absolute values of n because the sea surface temperature ( sst ) forcing potentially provides unrealistic sources or sinks of energy . amip simulations are in good agreement with satellite observations ( r > 0.6 ) for monthly deseasonalized variability in n over the period 1986 to 2011 ( allan et al . and figure s1 ) , showing that sst observations provide useful additional constraints on n. we therefore create an extended observation - based estimate of n ( hereafter no ) by averaging the satellite observations with the ensemble means of the c20c and upscale simulations ( with each data set given an equal weight where available ) . we use no even when satellite data are available since errors are likely to be reduced by including independent information from the sst observations ( via the amip simulations ) . to obtain a total uncertainty on no of 0.28 w m. ocean heat content trends ( ht ) are computed from the slope of a least squares regression using annual time series of heat content and expressed as a flux relative to the total surface area of the earth [ following lyman and johnson , 2008 ] . we compute ht over 5 year periods and compare with rolling 5 year mean n , thereby smoothing out interannual variability related to el nio . absolute values of n can not be determined accurately from satellite observations [ stephens et al . , we therefore also assess rates of change of ht and n ( htt = dht / dt and nt = dn / dt , again computed with least squares regression over 5 year periods ) , since nt does not depend on absolute values . several published analyses of ht for the upper 700 m appear to capture signals associated with major volcanic eruptions , but the timing is not always consistent , and there is little agreement at other times [ trenberth et al . , 2013 ] , was found to be in reasonable agreement with model simulations of volcanic impacts and also simulated plausible changes in ht associated with el nio [ trenberth et al . patterns of ht reconstructed with historical observations agree well with the reference ( figure1 ) . uncertainties ( which we quote as 1 sigma throughout ) in global heat content range from around 28 zj ( zj = 10 j ) using 19501956 observations to 8 zj using 20052011 observations , corresponding to uncertainties in ht of 0.7 to 0.2 w m ( table s1 ) . we stress that these estimates only assess uncertainties arising from the observational coverage relative to the recent argo era and do not include unknown instrument errors or the potentially large contributions from regions not sampled adequately by argo floats , such as the shelf seas , ice - covered regions , and ocean below 2000 m [ e.g. ( right ) patterns of ocean heat content trends ( ht , w m ) for the period 2008 to 2012 computed using ( b ) all observations and subsampled observations typical of ( d ) the 1990s and ( f ) the 1960s . we compare observational estimates of ht and n with 21 cmip5 coupled model simulations ( taylor et al . and table s2 ) driven by observed variations in greenhouse gases , aerosols , solar radiation , and land use until 2005 and following representative concentration pathway ( rcp4.5 ) thereafter . there are long - term drifts in the cmip5 models because the preindustrial control simulations were not necessarily in radiative equilibrium [ sen gupta et al . we therefore compute anomalies in the historical and rcp4.5 simulations relative to preindustrial control simulations ( using at least 100 years of the control simulations ) . global ocean heat content , ht and htt from mosora and ora - s4 are in good agreement ( r = 0.94 , 0.80 , and 0.82 respectively for running 5 year averages ) over their overlapping period ( compare red and dashed magenta curves in figure2 ) , despite the lack of historical ocean observations and additional uncertainties involved in computing derivatives for ht and htt . furthermore , over the period 19601999 , ht and htt are in broad agreement with n ( r = 0.58 and 0.56 between mosora and ora - s4 and no ) and nt , ( r = 0.63 and 0.69 ) . these correlations are significantly greater than zero with 99% confidence based on bootstrapping in 5 year blocks to take serial correlation into account [ e.g. , ( first panel ) annual mean total ocean heat content anomalies ( relative to 19612010 , zj = 10 j ) from mosora ( red ) [ smith and murphy , 2007 ] and ora - s4 ( dashed magenta ) [ balmaseda et al . ( second panel ) five year running mean ocean heat content trends ( ht , red and magenta ) and net incoming radiation ( n ) at the top of the atmosphere from satellites [ allan et al . ( third panel ) trends in ht ( htt , red and magenta ) and n ( blue and cyan ) . after 2000 , ht and htt show pronounced variability that is not present in n and nt ( figure2 , second and third panels ) . this is largely caused by the rapid increase in ocean heat content between 2001 and 2004 resulting in a peak ht in 2002 followed by a trough in 20062007 in both mosora and ora - s4 ( figure2 , second and third panels ) . a peak in ht around 2002 is also evident in other ocean analyses [ trenberth et al . however , such an increase in heat content is not supported by thermosteric sea level observations , because total sea level increased at a fairly constant rate [ church et al . , 2013 ] , implying a reduction in the rate of thermosteric sea level rise ( and hence ocean heat content trend ) over the period 2001 to 2004 . although interpretation of sea level changes is complicated by the nonuniformity of thermal expansion coefficients , warming is not consistent with a reduction in thermosteric sea level trends . given this , and the agreement between independent estimates of n ( and nt ) from satellite observations and amip simulations ( figure2 ) , and from forced ocean model experiments ( figure s2 ) , we suggest that there are likely to be errors in the observational reanalysis of ocean heat content between 2001 and 2007 ( grey - shaded region in figure2 ) . cheng and zhu drew similar conclusions from a different approach , suggesting that these errors are caused by the transition from xbt to argo data . however , this does not appear to be the sole cause in our study since mosora shows a similar peak in ht in 2002 even when argo observations are omitted ( figure2 , black asterisks ) , although the subsequent trough is reduced . further work is therefore needed to understand the cause of these errors in ocean reanalyses . possible explanations include inadequate vertical or horizontal sampling and/or unresolved biases in the instruments themselves . cmip5 coupled models simulate variations in n and ht that are in good agreement with observations ( r = 0.82 between 5 year running mean cmip5 ensemble mean and no , and 0.68 with mosora excluding the period after 2000 , figure3a ) . since internal variability is largely removed in the ensemble mean of the cmip5 models , this agreement suggests that much of the multiyear variability in earth 's energy imbalance from 1960 to 2000 was externally forced by the volcanic eruptions of agung ( 1963 ) , el chichon ( 1982 ) , and pinatubo ( 1991 ) , as noted by trenberth et al . furthermore , the cmip5 models do not simulate a peak in ht in 2002 , and the observed peak is significantly different from the cmip5 models , consistent with our earlier conclusion that estimates of ht based on ocean analyses between 2001 and 2007 ( grey shading in figure2 ) are likely unreliable . ( a ) time series of 5 year running mean n and ht ( as figure2 , second panel ) for 21 cmip5 coupled model simulations ( n in green , ht in orange , ensemble mean in thick lines ) compared with ht from mosora ( red ) and no ( blue , see text ) . black squares ( diamonds ) show where differences between mosora and no ( cmip5 ) are significant with 90% confidence . ( b ) n averaged over different periods in no ( blue , with 1 sigma uncertainties ) compared to the cmip5 models ( green , box showing the mean 1 sigma and whiskers showing the range ) and estimates from the ipcc fifth assessment ( red ) [ rhein et al . , 2013 , box 3.1 ] and the cmip5 models ( figure3b ) , but it is difficult to draw firm conclusions because uncertainties in the absolute values of no are large . these uncertainties are dominated by the anchoring estimate of ht and could potentially be reduced by averaging ht over longer periods . we do not attempt this because ocean observations in the early 2000s are potentially unreliable ( as discussed above ) , historical analyses do not capture the full magnitude of ht , especially in the southern ocean ( figure1 ) [ durack et al . , 2014 ] , and uncertainties are evident even in the most recent 5 year means which show a decrease in no but an increase in ht ( figure3a ) . we therefore investigate changes in n and anomalies relative to the period 19602011 since these are less affected by anchoring uncertainties . in general , there is good agreement between the cmip5 ensemble mean and observation - based anomalies of n ( r = 0.82 for 5 year running means ) and its components , absorbed shortwave ( asr , r = 0.87 ) and outgoing longwave ( olr , r = 0.80 ) radiation ( figure4 ) . the cmip5 ensemble mean therefore appears to simulate a realistic magnitude of variability associated with volcanic eruptions . , 1999 ] may have contributed to no . however , an increase in n is also apparent in the cmip5 ensemble mean in the late 1970s ( especially in asr ; red curves in figure4 ) , suggesting that external forcing may have played a role . time series of 5 year running mean anomalies ( relative to 1960 to 2011 ) of toa - absorbed shortwave radiation ( asr , red ) , outgoing longwave radiation ( olr , green ) , and net radiation ( n = asr - olr , blue ) in no ( dashed ) and the cmip5 models ( solid , with thick line showing the ensemble mean ) . models and observations both show a rapid increase in n during the recovery from the eruption of mount pinatubo , reaching a peak in the late 1990s . however , there is a subsequent decline of 0.43 0.21 w m in no between 1999 and 2005 in the early part of the global warming slowdown which is larger in magnitude than the model ensemble mean ( 0.12 0.06 w m ) and at the lower end of individual models ( + 0.38 to 0.32 w m ) . both the satellite observations and the amip simulations show a decline over this period ( figures2 , middle , 2 bottom , and s3 ) , showing that the observed signal is not an artifact of our averaging procedure . it is also not caused by the transition from erbs to ceres satellites since all six upscale amip simulations show reductions , ranging from 0.30 to 0.52 w m. furthermore , a similar decline in surface flux between 1999 and 2005 is simulated in forced ocean model experiments ( figure s2 ) . we therefore compute the difference averaged over 20042006 minus 19982000 , giving a reduction of 0.31 0.21 w m between the late 1990s and mid-2000s . we find significant correlation ( r 0.6 , p < 0.01 ) between rolling 5 year mean ocean heat content trends ( ht ) in two ocean analyses and net incoming radiation at the top of the atmosphere ( n ) from satellite observations and amip model simulations over the period 1960 to 1999 . however , ocean reanalyses of ht in the early 2000s are inconsistent with observation- and model - based constraints on n. this helps to explain our inability to close earth 's energy budget [ trenberth and fasullo , 2010 ; trenberth et al . , 2014 ] and suggests that observed estimates of ht covering this period [ e.g. it also calls into question whether ocean reanalyses can be used to robustly attribute increased ht below the ocean mixed layer in the early 2000s as an explanation for the onset of the recent slowdown in surface warming [ guemas et al . , we create an observation - based estimate of n ( no ) extending back to 1960 , using satellite data and amip model simulations , and compare with 21 cmip5 coupled climate model simulations . variations in n over this period are dominated by the volcanic eruptions of agung in 1963 , el chichon in 1982 , and pinatubo in 1991 [ trenberth et al . , 2014 ] , with good agreement ( r = 0.82 ) between no and the cmip5 ensemble mean . absolute values of no are in broad agreement with the models , but uncertainties are large and improved estimates of ht are needed to provide a more accurate anchor for satellite observations [ loeb et al . , we find a reduction of 0.31 0.21 w m in no between the late 1990s and the mid-2000s which may have contributed to the recent slowdown in global surface warming . , 2011 ] that were not included in the cmip5 models [ flato et al . , 2013 , however , the reduction in no is caused by an increase in olr rather than a reduction in asr which would be expected from these factors ( compare green and red dashed curves in figures4 and s3 ) , though asr may have been reduced relative to the models . internal variability may also reduce n during cooling decades [ katsman and van oldenborgh , 2011 ; palmer and mcneall , 2014 ; brown et al . , 2014 ] , but the cmip5 ensemble mean does simulate a weak reduction ( 0.12 0.06 w m , mainly in asr ) , suggesting a potential role for external forcing . further work is therefore needed to unravel the relative roles of internal variability and external factors on earth 's energy imbalance during the recent warming pause . while present - day anomalies in n in the cmip5 ensemble mean are in good agreement with observations , this is potentially achieved through a cancelation of errors ( in models and/or observations ) since both asr and olr are larger in almost all of the models than the satellite observations ( figures4 and s3 ) . further analysis of the roles of forcing and feedback on these signals is needed , along with continued satellite and in situ observations of earth 's energy imbalance to monitor future climate variability and change .

all of these factors can compromise treatment and quality of life following surgery.1 evidence indicates that patients ' quality of sleep after heart surgery is frequently poor , particularly during the postoperative period , and that patients experience high levels of sleep disruption , irregular sleep cycles , and reductions in slow - wave sleep.2 poor quality of sleep in the postoperative period may be due to several factors , including pain from surgical incision , presence of a thoracic drain , pain caused by prolonged time in bed , and high anxiety levels.1 in addition , muscle pain , particularly in the neck , shoulders and back , may make it difficult for patients to breath , cough , move , and sleep.1 massage therapy ( mt ) is a technique that promotes the manual mobilization of several structures from both muscle and subcutaneous tissue , by applying mechanical force to tissues . this mobilization improves lymph movement and venous return ; reduces swelling ; and mobilizes muscle fibers , tendons and skin . thus , mt may be used to promote muscle relaxation and to reduce pain , stress and anxiety,3 which help patients improve their quality of sleep and speed recover . in addition , mt may enhance patient mobility and recovery from surgery , which allows patients to perform daily activities3 and take part in physiotherapy treatment and rehabilitation . the european respiratory society and the european society of intensive care medicine recognize that mt may improve quality of sleep.4 therefore , in this study , we evaluated whether mt is an effective method for improving quality of sleep in patients following coronary artery bypass graft ( cabg ) surgery . we evaluated patients who were waiting for elective cabg surgery between may 2008 and january 2009 at the heart institute ( incor ) , university of so paulo school of medicine , so paulo , brazil . inclusion criteria included patients of both genders between 40 and 80 years of age . we excluded patients who had a body mass index ( bmi ) 35 kg / m , history of regular alcohol consumption , chronic use of hypnotics , previous diagnosis of sleep disorders , surgery other than cabg performed within the last 24 months , or an illiteracy . we also excluded patients who had combined valve and cabg surgery , a thoracic drain in their return to the ward , or an intensive care unit ( icu ) stay longer than 5 days in the postoperative period . all patients provided written informed consent , and the local ethics committee approved our protocol . the preoperative clinical assessment consisted of obtaining information regarding the patients ' demographics and personal characteristics . we aimed to characterize sleep , pain and fatigue complaints before surgery by applying the following questionnaires : the ess was used to evaluate excessive daytime sleepiness in participants . the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. , 3 to 4 times per week ) for two or more of the questions regarding snoring . in the second category , high risk was defined as having either persistent ( i.e. , 3 to 4 times per week ) wake time sleepiness , persistent drowsiness while driving , or both . in the third category , high risk was defined as a history of high blood pressure or a bmi > 30 kg / m . to be considered at high risk for osa , participants had to be at high risk for at least two symptom categories . those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 the vas was used to evaluate pain and fatigue in participants . participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . the control participants sat in comfortable chairs for three consecutive nights and were not subjected to mt . mt was performed by the same physiotherapist during the protocol and consisted of neck , shoulder and back massages . the massages were initiated with light compression by the inner regions of the fingers and progressed to hard compression . manual kneading , friction ( i.e. , digital compression with the thumb ) on trigger points , cervical traction , and mobilization in all planes ( e.g. , the front , back , and sides ) followed . this intervention was performed around 7 pm , 2 - 3 hours before sleep . trigger points were areas that triggered pain during the massages . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . sleep quality was evaluated by the vas of sleep , which has been previously adapted and validated in portuguese.11 the vas of sleep consisted of 16 items from 3 categories as following : sleep disorder scale , effectiveness scale and supplementation scale . the sleep disorder scale looked at seven items and evaluated the degree of sleep impairment caused by fragmentation and late sleep onset . scores ranged from 0 to 70 , and higher values indicated a worse case of sleep disorder . the supplementation scale looked at four items and measured the degree to which additional periods of sleep ( daily naps ) were necessary to supplement the main period of sleep . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the student t , mann - whitney , or chi - squared tests , when appropriate , were used to compare the control and mt groups at baseline . the two - way repeated measures analysis of variance ( anova ) was used to compare the control and mt groups . we evaluated patients who were waiting for elective cabg surgery between may 2008 and january 2009 at the heart institute ( incor ) , university of so paulo school of medicine , so paulo , brazil . inclusion criteria included patients of both genders between 40 and 80 years of age . we excluded patients who had a body mass index ( bmi ) 35 kg / m , history of regular alcohol consumption , chronic use of hypnotics , previous diagnosis of sleep disorders , surgery other than cabg performed within the last 24 months , or an illiteracy . we also excluded patients who had combined valve and cabg surgery , a thoracic drain in their return to the ward , or an intensive care unit ( icu ) stay longer than 5 days in the postoperative period . all patients provided written informed consent , and the local ethics committee approved our protocol . the preoperative clinical assessment consisted of obtaining information regarding the patients ' demographics and personal characteristics . we aimed to characterize sleep , pain and fatigue complaints before surgery by applying the following questionnaires : the ess was used to evaluate excessive daytime sleepiness in participants . the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. , 3 to 4 times per week ) for two or more of the questions regarding snoring . in the second category , high risk was defined as having either persistent ( i.e. , 3 to 4 times per week ) wake time sleepiness , persistent drowsiness while driving , or both . in the third category , high risk was defined as a history of high blood pressure or a bmi > 30 kg / m . to be considered at high risk for osa , participants had to be at high risk for at least two symptom categories . those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 the vas was used to evaluate pain and fatigue in participants . participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . the control participants sat in comfortable chairs for three consecutive nights and were not subjected to mt . mt was performed by the same physiotherapist during the protocol and consisted of neck , shoulder and back massages . the massages were initiated with light compression by the inner regions of the fingers and progressed to hard compression . manual kneading , friction ( i.e. , digital compression with the thumb ) on trigger points , cervical traction , and mobilization in all planes ( e.g. , the front , back , and sides ) followed . this intervention was performed around 7 pm , 2 - 3 hours before sleep . trigger points were areas that triggered pain during the massages . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . sleep quality was evaluated by the vas of sleep , which has been previously adapted and validated in portuguese.11 the vas of sleep consisted of 16 items from 3 categories as following : sleep disorder scale , effectiveness scale and supplementation scale . the sleep disorder scale looked at seven items and evaluated the degree of sleep impairment caused by fragmentation and late sleep onset . scores ranged from 0 to 70 , and higher values indicated a worse case of sleep disorder . the supplementation scale looked at four items and measured the degree to which additional periods of sleep ( daily naps ) were necessary to supplement the main period of sleep . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the preoperative clinical assessment consisted of obtaining information regarding the patients ' demographics and personal characteristics . we aimed to characterize sleep , pain and fatigue complaints before surgery by applying the following questionnaires : the ess was used to evaluate excessive daytime sleepiness in participants . the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. , 3 to 4 times per week ) for two or more of the questions regarding snoring . in the second category , high risk was defined as having either persistent ( i.e. , 3 to 4 times per week ) wake time sleepiness , persistent drowsiness while driving , or both . in the third category , high risk was defined as a history of high blood pressure or a bmi > 30 kg / m . to be considered at high risk for osa , participants had to be at high risk for at least two symptom categories . those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 the vas was used to evaluate pain and fatigue in participants . participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. , 3 to 4 times per week ) for two or more of the questions regarding snoring . in the second category , high risk was defined as having either persistent ( i.e. , 3 to 4 times per week ) wake time sleepiness , persistent drowsiness while driving , or both . in the third category , high risk was defined as a history of high blood pressure or a bmi > 30 kg / m . to be considered at high risk for osa , participants had to be at high risk for at least two symptom categories . those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . the control participants sat in comfortable chairs for three consecutive nights and were not subjected to mt . mt was performed by the same physiotherapist during the protocol and consisted of neck , shoulder and back massages . the massages were initiated with light compression by the inner regions of the fingers and progressed to hard compression . manual kneading , friction ( i.e. , digital compression with the thumb ) on trigger points , cervical traction , and mobilization in all planes ( e.g. , the front , back , and sides ) followed . this intervention was performed around 7 pm , 2 - 3 hours before sleep . trigger points were areas that triggered pain during the massages . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . sleep quality was evaluated by the vas of sleep , which has been previously adapted and validated in portuguese.11 the vas of sleep consisted of 16 items from 3 categories as following : sleep disorder scale , effectiveness scale and supplementation scale . the sleep disorder scale looked at seven items and evaluated the degree of sleep impairment caused by fragmentation and late sleep onset . scores ranged from 0 to 70 , and higher values indicated a worse case of sleep disorder . the supplementation scale looked at four items and measured the degree to which additional periods of sleep ( daily naps ) were necessary to supplement the main period of sleep . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the student t , mann - whitney , or chi - squared tests , when appropriate , were used to compare the control and mt groups at baseline . the two - way repeated measures analysis of variance ( anova ) was used to compare the control and mt groups . a total of 57 cabg surgery patients were invited to participate in the study during the preoperative period . seventeen patients were excluded for the following reasons : icu stay was longer than 5 days ( n = 11 ) , spontaneous withdrawal from the study ( n = 1 ) , thoracic drain put in place upon return to the ward ( n = 1 ) , combined surgical procedures ( n = 2 ) , cancellation of surgery ( n = 1 ) , and death during surgery ( n = 1 ) . the final study sample consisted of 40 patients who were randomized into the control ( n = 20 ) and mt ( n = 20 ) groups ( figure 1 ) . demographic , clinical , and sleep characteristics of the participants are presented in table 1 . no significant differences existed in the anthropometric characteristics between the groups , except for bmi , which was significantly higher in the control group . during the preoperative period , 30% of patients from the control group and 35% from the mt group had complaints of pain in the back and shoulders . the pain intensity was mild in both groups ( p = 0.739 ) . complaints of fatigue were few among both groups ( averaging 1.65 2.55 for control and 1.35 1.45 for mt group , p = 0.152 ) . there were no significant differences between the control and the mt groups regarding the duration of the bypass ( 69.5 min 50.7 min and 58.6 min 45.7 min , respectively , p = 0.29 ) , time on mechanical ventilation in the icu ( 6.8 h 2.4 h and 7.2 h 3.1 h , respectively , p = 0.82 ) , length of icu stay ( 69.2 h 27.6 h and 66.8 h 31.9 h , respectively , p = 0.51 ) , and length of hospital stay ( 7.55 d 1.23 d and 7.75 d 2.38 d , respectively , p = 0.923 ) . the total sleep time of participants during the study period was obtained from their sleep diaries and was similar between groups , with averages per night of 383 min 158 min and 385 min 116 min in the control and the mt groups , respectively ( p = 0.536 ) . participants in both groups received similar quantities of dipyrone and tramadol as analgesics during the study period . vas scores for pain in the chest , back and shoulders during the postoperative period for each group are presented in figures 2 a , b , and c. there was a significant decrease in the complaints of pain in the chest ( p<0.001 ) , back ( p = 0.007 ) and shoulder ( p = 0.036 ) from day 1 to day 3 in both groups . complaints of fatigue among participants were different between the two groups ( figure 3 ) . the control group had high fatigue scores on day 1 , which decreased over the duration of the study ( p = 0.022 ) . in contrast , the mt group had lower fatigue scores on day 1 ( p = 0.006 ) and day 2 ( p = 0.028 ) than the control group . vas scores for sleep characteristics exhibited by participants during the study period are presented in table 2 and figures 4 a , b , and c. the sleep disorder scale scores showed no significant intragroup ( p = 0.936 ) or intergroup ( p = 0.489 ) differences . effectiveness scale scores were higher for the mt group than the control group ( p = 0.019 ) during all the study period . supplementation scale scores decreased ( indicating less need for napping ) on the third day in both groups ( p = 0.031 ) . this study evaluated the effects of mt in patients during the postoperative period after cabg surgery . the results indicate that mt improved the comfort level of the mt group participants in comparison with those patients in the control group . recovery from fatigue was significantly faster in the mt group , reaching statistically significant differences by day 1 and day 2 . sleep effectiveness was also significantly higher in the mt group participants during all the study period . in addition , the results indicate that the beneficial effects of mt were not mediated by reducing pain because the number of complaints about pain was similar in both the control and mt groups . we found that several participants had complaints of daily somnolence and poor quality of sleep during the preoperative period . this observation is consistent with previous findings that showed that poor sleep is common in patients awaiting surgical procedures.2 this poor quality of sleep is likely caused by pain , stress , and anxiety.13 in addition , patients with cardiovascular disease frequently have osa , which is an independent risk factor for cardiovascular disease ( e.g. , hypertension , ischemic heart disease , atrial fibrillation , stroke , heart failure).14 osa may contribute to poor sleep and excessive daytime somnolence during the preoperative period . in our sample , 55% of participants ( n = 21 ) showed symptoms suggestive of osa upon evaluation using the berlin questionnaire ( table 1 ) . thus , osa may play a role in worsening sleep not only in the preoperative but also in the postoperative periods . in addition to the symptoms present in the preoperative period , sleep fragmentation and deprivation are also commonly observed after surgery . sleep deprivation per se is a stress condition , which causes the inappropriate activation of the hypothalamic - pituitary - adrenal axis . this activation results in an increase in cortisol release,15 the worsening of the immune system , and a predisposition to inflammation and infection.16 furthermore , sleep deprivation may increase sympathetic nervous system activity and blood pressure.16 - 18 all of these factors may impact patient recovery . in our study , we observed that all participants reported having a certain degree of sleep disorder following surgery and a necessity for daily naps , both of which significantly decreased from day 2 to day 3 . mt promoted improvement in sleep effectiveness ( figure 4b ) , indicating that it had a positive influence on sleep . similar to our findings , tsay and colleagues ( 2003)19 found that mt improved sleep quality in patients with end - stage renal disease . field and colleagues ( 2002)20 observed that mt decreased anxiety and depressed mood and increased the number of sleep hours in patients with fibromyalgia . thus , previous literature supports our finding that mt improves sleep and recovery in patients following surgery . mt promoted a faster decrease in complaints of fatigue in the mt group participants than in those of the control group , reaching statistical significance by day 1 and day 2 ( figure 2 ) . a previous study has shown that mt promotes a significant decrease in cortisol levels from the baseline ( averaging 31% ) and increase in active neurotransmitters , such as serotonin ( averaging 28% ) and dopamine ( averaging 31%).21 mt may also promote the following : parasympathetic activation,3,22 which causes reductions in heart rate , blood pressure , and breathing ; increases in the release of hormones ( e.g. , endorphins);3 and decreases in stress levels.22 for example , field and colleagues ( 2002)20 found reductions in substance p , which is related to pain complaints , in patients with fibromyalgia who received mt . such positive changes in conjunction with the beneficial effects of mt on sleep quality may play a role in reducing the effects of stress caused by disease or surgery21 and feelings of fatigue . field and colleagues ( 2002)20 evaluated the effects of mt and relaxation therapy in a group of patients with fibromyalgia . in their study , a decrease in complaints of pain among participants assigned to the mt group was associated with a reduction in substance p. in a multi - center randomized clinical trial , kutner and colleagues ( 2008)23 showed that mt had immediate beneficial effects on pain and mood among patients with advanced cancer . in their study , both the massage and the simple touch groups had significant improvements in pain and quality of life over time without any increases in analgesic medication use.23 in the present study , pain in the chest , back and shoulders was similar in both the control and the mt groups at the start of the study and decreased in a similar manner over the 3 days . this decrease in pain during the postoperative period has been previously described in patients following heart surgery.24 reduction in pain during the recovery period following surgery may in part be explained by the generation of humoral mediators ( e.g. , endorphins ) that contribute to an increasing sense of wellbeing.1 in a recent study , albert and colleagues ( 2009)25 found no additional reduction in pain in patients who were subjected to mt both before and after heart surgery . this study suggests that mt is most effective in reducing pain when it is applied post - surgery . therefore , our study allows us to conclude that the beneficial effects of mt on sleep are unlikely to be mediated by pain reduction . the design allowed for the inclusion of two groups that were similar in preoperative , intraoperative , and postoperative characteristics . patients randomized to the control group had a significantly higher bmi than those in the mt group . however , both groups had a mean bmi in the overweight range ( 28.5 kg / m and 25.9 kg / m in the control and mt groups , respectively , table 1 ) . therefore , an imbalance in participant bmi had no influence on the results of the present study . the primary limitation of the study is that we employed self - reported , subjective methods and did not perform polysomnography to evaluate the effects of mt . patients with cardiac disease present a high prevalence of sleep disordered breathing , that was not objectively investigated in this study.26 however , the study evaluated complaints of fatigue , pain , and sleep , which are intrinsically subjective . moreover , our observation that the pain complaints were similar in both the control and the mt groups suggests that our evaluation was consistent and valid . in conclusion , our data suggest that mt is effective at improving the quality of sleep and decreasing fatigue in patients during the recovery period following cabg surgery .

introduction : poor sleep quality is common among patients following cardiopulmonary artery bypass graft surgery . pain , stress , anxiety and poor sleep quality may be improved by massage therapy.objective:this study evaluated whether massage therapy is an effective technique for improving sleep quality in patients following cardiopulmonary artery bypass graft surgery.method:participants included cardiopulmonary artery bypass graft surgery patients who were randomized into a control group and a massage therapy group following discharge from the intensive care unit ( day 0 ) , during the postoperative period . the control group and the massage therapy group comprised participants who were subjected to three nights without massage and three nights with massage therapy , respectively . the patients were evaluated on the following mornings ( i.e. , day 1 to day 3 ) using a visual analogue scale for pain in the chest , back and shoulders , in addition to fatigue and sleep . participants kept a sleep diary during the study period.results:fifty-seven cardiopulmonary artery bypass graft surgery patients were enrolled in the study during the preoperative period , 17 of whom were excluded due to postoperative complications . the remaining 40 participants ( male : 67.5% , age : 61.9 years 8.9 years , body mass index : 27.2 kg / m2 3.7 kg / m2 ) were randomized into control ( n = 20 ) and massage therapy ( n = 20 ) groups . pain in the chest , shoulders , and back decreased significantly in both groups from day 1 to day 3 . the participants in the massage therapy group had fewer complaints of fatigue on day 1 ( p = 0.006 ) and day 2 ( p = 0.028 ) in addition , they reported a more effective sleep during all three days ( p = 0.019 ) when compared with the participants in the control group.conclusion:massage therapy is an effective technique for improving patient recovery from cardiopulmonary artery bypass graft surgery because it reduces fatigue and improves sleep .

INTRODUCTION MATERIALS AND METHODS Patients Data Collection Preoperative evaluation Epworth Sleepiness Scale (ESS) The Pittsburgh Sleep Quality Index Berlin Questionnaire Visual Analogue Scale (VAS) for pain and fatigue Postoperative protocol and evaluation Data Analysis RESULTS DISCUSSION CONCLUSION

all of these factors can compromise treatment and quality of life following surgery.1 evidence indicates that patients ' quality of sleep after heart surgery is frequently poor , particularly during the postoperative period , and that patients experience high levels of sleep disruption , irregular sleep cycles , and reductions in slow - wave sleep.2 poor quality of sleep in the postoperative period may be due to several factors , including pain from surgical incision , presence of a thoracic drain , pain caused by prolonged time in bed , and high anxiety levels.1 in addition , muscle pain , particularly in the neck , shoulders and back , may make it difficult for patients to breath , cough , move , and sleep.1 massage therapy ( mt ) is a technique that promotes the manual mobilization of several structures from both muscle and subcutaneous tissue , by applying mechanical force to tissues . thus , mt may be used to promote muscle relaxation and to reduce pain , stress and anxiety,3 which help patients improve their quality of sleep and speed recover . in addition , mt may enhance patient mobility and recovery from surgery , which allows patients to perform daily activities3 and take part in physiotherapy treatment and rehabilitation . the european respiratory society and the european society of intensive care medicine recognize that mt may improve quality of sleep.4 therefore , in this study , we evaluated whether mt is an effective method for improving quality of sleep in patients following coronary artery bypass graft ( cabg ) surgery . we evaluated patients who were waiting for elective cabg surgery between may 2008 and january 2009 at the heart institute ( incor ) , university of so paulo school of medicine , so paulo , brazil . we excluded patients who had a body mass index ( bmi ) 35 kg / m , history of regular alcohol consumption , chronic use of hypnotics , previous diagnosis of sleep disorders , surgery other than cabg performed within the last 24 months , or an illiteracy . we also excluded patients who had combined valve and cabg surgery , a thoracic drain in their return to the ward , or an intensive care unit ( icu ) stay longer than 5 days in the postoperative period . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. in the second category , high risk was defined as having either persistent ( i.e. those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 the vas was used to evaluate pain and fatigue in participants . participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the student t , mann - whitney , or chi - squared tests , when appropriate , were used to compare the control and mt groups at baseline . we evaluated patients who were waiting for elective cabg surgery between may 2008 and january 2009 at the heart institute ( incor ) , university of so paulo school of medicine , so paulo , brazil . we excluded patients who had a body mass index ( bmi ) 35 kg / m , history of regular alcohol consumption , chronic use of hypnotics , previous diagnosis of sleep disorders , surgery other than cabg performed within the last 24 months , or an illiteracy . we also excluded patients who had combined valve and cabg surgery , a thoracic drain in their return to the ward , or an intensive care unit ( icu ) stay longer than 5 days in the postoperative period . all patients provided written informed consent , and the local ethics committee approved our protocol . the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. in the second category , high risk was defined as having either persistent ( i.e. those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 the vas was used to evaluate pain and fatigue in participants . participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . , the front , back , and sides ) followed . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the preoperative clinical assessment consisted of obtaining information regarding the patients ' demographics and personal characteristics . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. in the second category , high risk was defined as having either persistent ( i.e. participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 the participants rated the probability of dozing , on a scale of 0 to 3 , in 8 different situations . a score over 10 indicated the presence of excessive daytime sleepiness.5 a portuguese version of this scale has been previously validated for use in brazil.6 this index was used to evaluate the quality of sleep of the participants , according to seven components , during the thirty days leading up to the study . the participants ' final scores were interpreted as follows : 0 - 4 indicated good sleep quality , 5 - 10 indicated poor sleep quality , over 10 indicated the presence of a sleep disorder.7 a portuguese version of this questionnaire has been previously validated for use in brazil.8 this questionnaire was used to classify participants at low- and high - risk for obstructive sleep apnea ( osa ) , based on the following three symptom categories : ( 1 ) snoring , ( 2 ) tiredness , and ( 3 ) clinical characteristics . in the first category , high risk was defined as having persistent symptoms ( i.e. in the second category , high risk was defined as having either persistent ( i.e. in the third category , high risk was defined as a history of high blood pressure or a bmi > 30 kg / m . those patients who were at high risk for only one symptom category or denied having persistent symptoms were considered at low - risk for osa.9 participants indicated the intensity of pain and fatigue on a scale of 0 to 10 , where 0 and 10 corresponded to minimum and maximum , respectively.10 following discharge from the icu to the ward during the postoperative period ( day 0 ) , the participants were randomized into control and mt groups . over the duration of the study , participants completed a sleep diary in which they recorded the times at which they went to sleep , woke up , and took daytime naps . both , the mt and control participants were evaluated for their pain and fatigue levels and their sleep quality each morning following the previous night pain and fatigue were evaluated by the vas , as described above , during the preoperative period . scores ranged from 0 to 40 , and higher values indicated that additional naps were necessary during the day.12 the student t , mann - whitney , or chi - squared tests , when appropriate , were used to compare the control and mt groups at baseline . a total of 57 cabg surgery patients were invited to participate in the study during the preoperative period . seventeen patients were excluded for the following reasons : icu stay was longer than 5 days ( n = 11 ) , spontaneous withdrawal from the study ( n = 1 ) , thoracic drain put in place upon return to the ward ( n = 1 ) , combined surgical procedures ( n = 2 ) , cancellation of surgery ( n = 1 ) , and death during surgery ( n = 1 ) . the final study sample consisted of 40 patients who were randomized into the control ( n = 20 ) and mt ( n = 20 ) groups ( figure 1 ) . demographic , clinical , and sleep characteristics of the participants are presented in table 1 . no significant differences existed in the anthropometric characteristics between the groups , except for bmi , which was significantly higher in the control group . during the preoperative period , 30% of patients from the control group and 35% from the mt group had complaints of pain in the back and shoulders . the pain intensity was mild in both groups ( p = 0.739 ) . complaints of fatigue were few among both groups ( averaging 1.65 2.55 for control and 1.35 1.45 for mt group , p = 0.152 ) . there were no significant differences between the control and the mt groups regarding the duration of the bypass ( 69.5 min 50.7 min and 58.6 min 45.7 min , respectively , p = 0.29 ) , time on mechanical ventilation in the icu ( 6.8 h 2.4 h and 7.2 h 3.1 h , respectively , p = 0.82 ) , length of icu stay ( 69.2 h 27.6 h and 66.8 h 31.9 h , respectively , p = 0.51 ) , and length of hospital stay ( 7.55 d 1.23 d and 7.75 d 2.38 d , respectively , p = 0.923 ) . the total sleep time of participants during the study period was obtained from their sleep diaries and was similar between groups , with averages per night of 383 min 158 min and 385 min 116 min in the control and the mt groups , respectively ( p = 0.536 ) . participants in both groups received similar quantities of dipyrone and tramadol as analgesics during the study period . vas scores for pain in the chest , back and shoulders during the postoperative period for each group are presented in figures 2 a , b , and c. there was a significant decrease in the complaints of pain in the chest ( p<0.001 ) , back ( p = 0.007 ) and shoulder ( p = 0.036 ) from day 1 to day 3 in both groups . complaints of fatigue among participants were different between the two groups ( figure 3 ) . the control group had high fatigue scores on day 1 , which decreased over the duration of the study ( p = 0.022 ) . in contrast , the mt group had lower fatigue scores on day 1 ( p = 0.006 ) and day 2 ( p = 0.028 ) than the control group . vas scores for sleep characteristics exhibited by participants during the study period are presented in table 2 and figures 4 a , b , and c. the sleep disorder scale scores showed no significant intragroup ( p = 0.936 ) or intergroup ( p = 0.489 ) differences . effectiveness scale scores were higher for the mt group than the control group ( p = 0.019 ) during all the study period . supplementation scale scores decreased ( indicating less need for napping ) on the third day in both groups ( p = 0.031 ) . this study evaluated the effects of mt in patients during the postoperative period after cabg surgery . the results indicate that mt improved the comfort level of the mt group participants in comparison with those patients in the control group . recovery from fatigue was significantly faster in the mt group , reaching statistically significant differences by day 1 and day 2 . sleep effectiveness was also significantly higher in the mt group participants during all the study period . in addition , the results indicate that the beneficial effects of mt were not mediated by reducing pain because the number of complaints about pain was similar in both the control and mt groups . we found that several participants had complaints of daily somnolence and poor quality of sleep during the preoperative period . this observation is consistent with previous findings that showed that poor sleep is common in patients awaiting surgical procedures.2 this poor quality of sleep is likely caused by pain , stress , and anxiety.13 in addition , patients with cardiovascular disease frequently have osa , which is an independent risk factor for cardiovascular disease ( e.g. , hypertension , ischemic heart disease , atrial fibrillation , stroke , heart failure).14 osa may contribute to poor sleep and excessive daytime somnolence during the preoperative period . in our sample , 55% of participants ( n = 21 ) showed symptoms suggestive of osa upon evaluation using the berlin questionnaire ( table 1 ) . thus , osa may play a role in worsening sleep not only in the preoperative but also in the postoperative periods . in addition to the symptoms present in the preoperative period , sleep fragmentation and deprivation are also commonly observed after surgery . this activation results in an increase in cortisol release,15 the worsening of the immune system , and a predisposition to inflammation and infection.16 furthermore , sleep deprivation may increase sympathetic nervous system activity and blood pressure.16 - 18 all of these factors may impact patient recovery . in our study , we observed that all participants reported having a certain degree of sleep disorder following surgery and a necessity for daily naps , both of which significantly decreased from day 2 to day 3 . similar to our findings , tsay and colleagues ( 2003)19 found that mt improved sleep quality in patients with end - stage renal disease . thus , previous literature supports our finding that mt improves sleep and recovery in patients following surgery . mt promoted a faster decrease in complaints of fatigue in the mt group participants than in those of the control group , reaching statistical significance by day 1 and day 2 ( figure 2 ) . a previous study has shown that mt promotes a significant decrease in cortisol levels from the baseline ( averaging 31% ) and increase in active neurotransmitters , such as serotonin ( averaging 28% ) and dopamine ( averaging 31%).21 mt may also promote the following : parasympathetic activation,3,22 which causes reductions in heart rate , blood pressure , and breathing ; increases in the release of hormones ( e.g. , endorphins);3 and decreases in stress levels.22 for example , field and colleagues ( 2002)20 found reductions in substance p , which is related to pain complaints , in patients with fibromyalgia who received mt . such positive changes in conjunction with the beneficial effects of mt on sleep quality may play a role in reducing the effects of stress caused by disease or surgery21 and feelings of fatigue . in their study , both the massage and the simple touch groups had significant improvements in pain and quality of life over time without any increases in analgesic medication use.23 in the present study , pain in the chest , back and shoulders was similar in both the control and the mt groups at the start of the study and decreased in a similar manner over the 3 days . this decrease in pain during the postoperative period has been previously described in patients following heart surgery.24 reduction in pain during the recovery period following surgery may in part be explained by the generation of humoral mediators ( e.g. , endorphins ) that contribute to an increasing sense of wellbeing.1 in a recent study , albert and colleagues ( 2009)25 found no additional reduction in pain in patients who were subjected to mt both before and after heart surgery . patients randomized to the control group had a significantly higher bmi than those in the mt group . however , both groups had a mean bmi in the overweight range ( 28.5 kg / m and 25.9 kg / m in the control and mt groups , respectively , table 1 ) . patients with cardiac disease present a high prevalence of sleep disordered breathing , that was not objectively investigated in this study.26 however , the study evaluated complaints of fatigue , pain , and sleep , which are intrinsically subjective . moreover , our observation that the pain complaints were similar in both the control and the mt groups suggests that our evaluation was consistent and valid . in conclusion , our data suggest that mt is effective at improving the quality of sleep and decreasing fatigue in patients during the recovery period following cabg surgery .

even after successful weight loss , weight regain is very common [ 1 , 2 ] . therefore it is important to identify factors that are associated with and could enhance the self - regulation of food intake and other behaviours related to weight management . because obesity is always a result of an imbalance between the energy intake and energy expenditure , decreased energy intake regulation of energy balance is very complex , however , with multiple mechanisms maintaining homeostasis and resisting changes in the energy balance . therefore , during negative energy balance , that is , when energy intake is lower than energy requirements , orexigenic pathways in the organism are activated , a usual consequence of which is a regain of reduced body weight . successful long - term management of weight thus requires safe and effective means to counteract these compensatory regulatory mechanisms to reduce appetite and enhance satiety . various foods , even regardless of their energy content , may differ in their capacity to regulate satiety . this can be accounted for multiple characteristics of food , such as energy density , macro- and micronutrient composition , palatability , food form , and structure [ 610 ] . among different food characteristics , especially dietary fibre and protein have recently raised much interest as potential factors capable of increasing the satiating value of food [ 11 , 12 ] . indeed , higher protein intake has been shown to sustain or increase satiety both during the periods of energy restriction or during the periods of isocaloric or ad libitum energy intake . greater satiating effect of protein has been ascribed to greater meal - induced thermogenesis after protein - rich meals than after meals rich in carbohydrates or fat [ 16 , 17 ] . protein also efficiently stimulates the expression of various gastrointestinal satiety hormones , and amino acids serve as precursors for specific neurotransmitters involved in appetite or are directly involved in pathways regulating food intake . high dietary fibre intake has also been shown to contribute to increased satiety and reduced energy intake [ 12 , 18 ] . among key mechanisms , dietary fibre increases satiety and reduces energy intake by decreasing the energy density of the diet as well as by retarding nutrient absorption , gastric emptying , and intestinal passage times in the gastrointestinal tract . several studies have shown that higher intake of dietary protein and fibre can contribute to greater weight loss or better maintenance of reduced weight [ 11 , 12 , 19 , 20 ] . greater weight loss with high - protein or high - fibre diets has been ascribed in part to their higher satiating effects and thereby to better dietary compliance and ability to reduce spontaneous energy intake . however , in spite of the large number of short - term studies linking various food properties to increased satiety , it is still poorly known whether the satiety value of food has an impact on the self - regulation of food intake and thereby on the regulation of body weight also at the longer term . different features of eating behaviour , such as restraint and disinhibition of eating , are also strong determinants of weight management [ 2124 ] . dietary restraint and disinhibition are psychological constructs that assess behavioural control and attitudes toward food and eating [ 25 , 26 ] . dietary restraint refers to a tendency to consciously restrict or control food intake , whereas dietary disinhibition is a tendency to overeat in the presence of , for example , palatable foods , emotional stress , or other disinhibiting stimuli . dietary disinhibition has been linked to higher body weight and increased risk of weight regain [ 22 , 24 ] , whereas increased dietary restraint with greater weight loss or better maintenance of reduced body weight [ 23 , 27 , 28 ] . other features of eating behaviour , such as emotional eating and greater susceptibility to sensations of hunger or external food - related cues , have also been associated with greater risk of obesity and difficulties in weight management [ 2932 ] . therefore , the aim of the present study was to investigate factors that are associated with successful weight management in middle - aged obese men and women . we were especially interested in investigating whether foods with higher predetermined satiety values , when ingested as a part of a weight - maintenance diet , contribute to better self - regulation of food intake and reduced body weight . the study consisted thus of two distinct , consecutive phases : determination of satiating values of foods in a controlled laboratory setting to select foods with lower and higher satiating values for the intervention and an intervention including weight - loss and weight - maintenance periods whereby the satiety - tested foods were used as a part of a weight - maintenance diet . originally 99 ( 28 males , 71 females ) obese ( inclusion criteria being body mass index ( bmi ) 3040 kg / m , age 3065 years ) subjects were recruited into the intervention study . they were recruited by an announcement in a local newspaper and among the eligible subjects who had participated previously in the studies performed at the university of kuopio , kuopio , finland ( currently university of eastern finland ) . the exclusion criteria of the subjects were bmi > 40 or < 30 kg / m , pregnancy , type 1 or 2 diabetes , abnormal liver , thyroid or kidney function , polycystic ovary syndrome , less than 6 months since coronary event or operation , myocardial infarction , susceptibility to arrhythmia , diagnosed eating disorder , neuroleptic or oral cortisone medication , and excess alcohol consumption ( women > 16 , men > 24 portions / week ) . the subjects could not have any other diseases , medications , or life situations that would have potentially prevented them to successfully complete the study . altogether the ethics committee of the district hospital region of northern savo and the kuopio university hospital approved the study plan , and all participants gave written informed consent for their participation . because the satiating value of food can not be directly estimated based on the nutritional composition or other characteristics of foods [ 20 , 33 ] , satiety tests were performed to identify foods with different satiety values for the intervention . satiety tests were performed in a laboratory setting to control for potential confounders known to affect satiety measurements [ 32 , 33 ] . satiety values were determined for food products from seven categories ( yoghurt - type dairy products , crisp bread , soft bread , cheese , cold cuts , beverages , vegetable meal components ) . these food categories were selected so that the test foods would represent diversely different parts of the mixed diet . however , due to practical reasons a limited number of foods ( altogether 22 foods , 25 foods per food category ) could be included into the satiety tests . the foods were provided by finnish food manufacturers , and they represented both commercial products as well as products at the stage of product development . in the selection of foods for satiety testing , the following factors were considered : the difference between foods regarding the amount of dietary fibre or protein per 700 kj portion should be as large as possible , since based on literature dietary fibre and protein play an important role in the satiating effect of foods [ 11 , 20 ] . furthermore , we chose an isocaloric portion size of 700 kj , which was in our previous study shown to be an energy level at which different satiety effects of test foods can be distinguished . at this energy level we tried to keep portion sizes ( weight or volume ) as similar as possible however , if the sizes of tested isocaloric portions differed , the volume or weight of food with potentially greater satiety value should be greater than that with potentially lower satiety value , due to possible independent effect of volume or weight on postprandial satiety . also , within each food category , the foods with potentially different satiety values should otherwise be as similar as possible . the satiety values of the test foods were assessed at vtt , espoo , finland , before the intervention study . altogether 35 subjects ( 20 males , 15 females , aged 23.3 2.2 ( range 1928 ) years ) recruited mainly from the helsinki university of technology participated in the tests . the subjects were healthy , normal - weight ( n = 27 ) , or at most moderately overweight ( n = 8 , mean bmi 23.3 2.3 ( range 19.129.7 ) kg / m ) due to potentially attenuated satiety responses in obese as compared with lean individuals . the subjects followed normal dietary and exercise patterns and did not have any dietary restrictions or abnormal eating behaviour based on individual interviews and the three - factor eating questionnaire : cognitive restraint 7.5 4.2 ( mean sd ) , disinhibition of eating 5.1 2.8 , hunger 4.9 2.5 . subjects participated in the satiety tests in the morning , after a minimum of 8 hours overnight fast . before the actual satiety tests , the subjects were familiarized with the procedure and trained to use the rating scales using typical finnish rye bread as the practice product . the satiety tests lasted for 3 hours , during which the subjects rated their satiety - related perceptions before consuming the test product and after 20 , 40 , 60 , 90 , 120 , 150 , and 180 min from the beginning of the consumption . at each session , subjects ate the whole portion of the test food with 2 dl of water . with crisp breads subjects consumed 50 g of slices of cucumber and 1.5 dl of water . the satiety - related sensations were evaluated using a 10-unit scale ( 0 = not at all , 10 = extremely ) before the consumption of the test product . immediately after the consumption and during the following 180 min , sensations were rated as a change from the starting value with a 100+10 scale ( 10 = a lot less than that in the beginning , 0 = as much as that in the beginning , + 10 = a lot more than that in the beginning ) where 0 represents the baseline value . a total of six satiety / hunger and thirst - related attributes were rated ( hunger , thirst , desire to eat something , satiety , fullness , desire to eat the test food ) . the data were collected using a computerized data - collecting system ( csa , computerized sensory analysis system , compusense , guelph , canada , compusense 5 , version 4.6.702 sp3 ) . results were analyzed as the changes in the satiety - related sensations from the baseline levels as a function of time , and the area under curve ( auc , cm min ) was calculated . all curves followed the same pattern with the lowest or highest ratings 20 min after eating the sample , and then scores increased or decreased , respectively , during the 3-hour follow - up time . finally , 8 food pairs with significant difference in the overall 3 h response of the feelings of hunger or satiety were selected to be the test foods in the intervention ( table 2 ) . subjects were informed that the aim of the study was to examine the effect of various food characteristics on the management of body weight and food intake and related mechanisms . since satiety is a highly subjective sensation , the subjects were not told about the different satiety values of the test foods to avoid any bias due to that information . the weight - loss period was performed by using very - low - calorie - diet ( vlcd ) products ( nutrifast , leiras finland ltd ) providing 600 kcal / day . in addition , the subjects were allowed to consume ad libitum low - energy vegetables and noncaloric beverages . the vlcd products were given free of charge , and they were consumed for 7 weeks , followed by a 2-week transition phase during which subjects gradually finished using the vlcd products and switched back to the mixed food diet . during the weight - loss period , the subjects were given dietary counselling in group sessions , 7 times during the whole period . the group sessions , lasting for 1.5 hours , were held in the evenings . altogether 810 subjects participated in each group . after the weight - loss period , the subjects were randomized , stratified by age and sex , into two diet groups : higher - satiety food group ( hsf ) and lower - satiety food group ( lsf ) ( table 4 ) . the baseline characteristics of the subjects in the hsf and lsf groups are presented in tables 4 , 6 , and 7 ( baseline ) . there were no significant differences at the baseline between the groups except for external eating , which was greater in the hsf than in the lsf group ( p = 0.02 ) , and the proportion of daily energy from carbohydrates , which was greater in the lsf than in the hsf group ( p = 0.03 ) . neither of the diets was considered a control diet . during the 24-week weight - maintenance period , subjects were instructed to maintain their weight loss but not to continue actively losing weight . the subjects in the hsf consumed the test foods with higher satiety value the subjects in the lsf consumed the test foods with lower satiety value as a part of their weight - management diet . the way of using the test foods and all dietary counselling was otherwise the same in both groups . the test foods aimed to cover about 30% of the individually estimated daily energy requirements . the proportion of test foods was tested in the pilot study performed prior to the intervention study to find out what was the highest realistic amount of the test foods that could be consumed as a part of the diet . 19 subjects ( 5 men , 14 women ) of same age ( 51.2 10.0 years ) and bmi ( 31.3 4.4 kg / m ) as the subjects in the intervention study participated in the pilot and used the test foods for two weeks as a part of their habitual diet . at the end of this period , they were interviewed about their experiences , and the intervention protocol was further modified using this information . the individual energy requirements were estimated by calculating the basal metabolic rate by the formula of mifflin st - jeor . to take into account the likely reduction of the energy expenditure due to the recent marked weight loss , the resultant values were deducted by 5% . the results were then multiplied by factor 1.3 to take into account the energy requirements due to any physical activity . based on the experiences from the pilot study , the resultant values were then deducted by the value 333.45 kcal to take into account the difference between the calculated energy requirements and the reported energy intakes . thus the final equations for the calculation of the energy requirements were [ ( 9.99 weight ( kg ) + 6.25 height ( cm ) 4.92 age ( yrs ) + 5 ) 0.95 1.3 ] 335.45 kcal for men and [ ( 89.99 weight ( kg ) + 6.25 height ( cm ) 4.92 age ( yrs)161 ) 0.95 1.3 ] 335.45 kcal for women . the resultant individual energy levels were rounded up to the nearest 100 kcal and varied from 1200 kcal to 2300 kcal / day . the amounts of portions of different test foods used per day were calculated and advised based on these energy levels . the subjects received the test foods free of charge from the university of kuopio ( current the university of eastern finland ) every two weeks . the test foods were given as the blank packages so that the subjects could not identify their exact content on the basis of , for example , comparable commercial products . during every visit , the subjects ' body weight was measured , and they were given written instructions about the use of the test foods as well as the weight - management diet in general . the written instructions included the numbers of the portions of each test food and the recommendations of the number of servings within each food group ( vegetables / berries / fruit , dairy , potatoes and cereals , meat / fish / poultry , fat ) to be consumed each day so that the total energy intake would cover the individually estimated daily energy requirement . in addition , to allow the possible satiety effect of test foods to function , subjects were told that they could increase or decrease the number of food servings ( other than test foods ) if still hungry or if sated , respectively . table 5 shows the portion sizes and typical numbers of daily portions of each test food as instructed to be consumed during the intervention . the portion sizes could be different from those used in the standard satiety tests because the foods were used as a part of a diet and were therefore targeted to represent typical portion sizes of each food . the number of the portions / day varied among different energy levels and from day to day . in addition , in order to reduce the risk of monotony and boredom due to the continued and frequent use of a quite limited number of test products , test foods within each list varied so that each two weeks list included 6 out of 8 test foods . on each week , the subjects were allowed to have one day free of the use of the test foods , if they wished . the subjects recorded the use of the test foods for the whole duration of the weight - maintenance period . these recordings were used when calculating the compliance ( i.e. , the use of the test foods as compared with the instructed use ) . the subjects were not given any specific instructions about physical activity but were advised to keep it at their habitual level . body weight was measured in the morning after 12 hours fast after voiding in the normal indoor clothing using a digital scale ( vogel & halke , hamburg , germany ) with the weighing accuracy of 0.1 kg . the measurements were done at the separate study visits in the beginning of the weight - loss period before any weight loss had occurred , at the end of the weight - loss period ( i.e. , the beginning of the weight - maintenance period ) , and at 12 and 24 weeks after the beginning of the weight - maintenance period ( figure 1 ) . height was measured using a wall - mounted stadiometer to the nearest 0.1 cm in the beginning of the weight - loss period . body mass index ( bmi ) was calculated ( weight ( kg)/height ( m ) ) . subjects completed 4-day food records altogether five times during the study : before the weight - loss period and at 6 , 12 , 18 , and 24 weeks from the beginning of the weight - maintenance period . nutrient intake was calculated with diet32 analysis program ( aivo finland oy ) using the finnish food composition database , fineli ( national institute for health and welfare ) . various features of eating behaviour were evaluated by standardized and validated self - report questionnaires . three - factor eating questionnaire ( tfeq ) was used to measure cognitive restraint of eating and divided also to the flexible and rigid parts of the restraint , disinhibition of eating , and susceptibility to hunger . the dutch eating behaviour questionnaire ( debq ) was used to measure restraint of eating , emotional eating , and external eating . the subjects filled in the questionnaires in the beginning of the weight - loss period and after 12 and 24 weeks of weight maintenance . psychological distress was evaluated by the general health questionnaire in the beginning and at the end of the weight - loss period as well as after 12 and 24 weeks of weight maintenance . during the weight - maintenance period , the subjects evaluated at the end of each week their overall well - being , bowel function , hunger , satiety , and easiness of control of food intake by the 9-point scale ( 1 = not at all / extremely bad , , 9 = a lot / extremely good ) . in addition , at the end of each dietary data collection day , the subjects rated how difficult / easy they had experienced control of food intake that day by using 7-point scale ( 1 = very difficult , , 7= very easy ) . at the end of the study , the subjects filled in a final questionnaire to assess how they had experienced the weight - maintenance period in general ( 1 = very difficult , , 10 = very easy ) and what they had liked about the test foods ( 1 = did not like at all , , 9 = liked very much ) . the statistical analyses were performed with spss for windows software ( spss for windows , version 14.0 , usa ) . the results are expressed as mean and standard deviation ( sd ) with a value p < 0.05 as a criterion for the statistical significance , unless not otherwise specified . in the satiety tests , paired samples t - test was used to evaluate the differences between the food pairs within each food category . in the intervention study , independent samples t - test ( student 's t - test ) and chi - square test were used to compare the baseline values between the study groups . linear mixed - effect modelling was used to compare the effect of the study group on the changes in the examined variables . the associations between the variables in the whole study population were analyzed by spearman 's correlation test and by partial correlation adjusted by the study group . altogether 82 out of 99 subjects completed the intervention . there were 17 subjects who dropped out , three subjects ( 2 men , 1 woman ) during the weight - loss period and 14 subjects ( 6 men , 9 women ) during the weight - maintenance period . there were no differences in the dropouts during the weight - maintenance period between the hsf ( 8 ( 3 men , 5 women ) ) and the lsf groups ( 6 ( 2 men , 4 women ) ) . the use of test foods , as an indicator of compliance , was equal in both groups ( % of the instructed use , calculated from the individual recordings during the weight - maintenance period : 100.8 9.0% versus 99.1 9.1% , hsf versus lsf ) , as well as the proportion of energy ingested from the test foods ( mean during the weight - maintenance period : 30.7 6.1% versus 29.0 5.0% , hsf versus lsf ) . the subjects in the hsf group generally liked the test foods they used more than in the subjects in the lsf group ( 7.1 1.0 versus 6.4 1.0 , p = 0.003 ) . weight changes were comparable between the hsf and the lsf groups ( figure 1 ) . the weight reduced by about 12 kg during the weight - loss period in both groups ( hsf 12.5 2.4% , lsf 12.4 2.6% of the body weight at the baseline ) . during the weight - maintenance period , there was only a very slight regain in the mean body weight with no significant difference between the groups ( hsf 1.3 3.5% , lsf 0.9 3.5% ) . the interindividual variation in the success to maintain the reduced body weight during the weight - maintenance period was large , with weight changes varying from 8.8% further weight reduction to 9.8% weight regain . the overall energy intake was similar in both groups with no significant differences in overall energy intake during the intervention ( table 6 ) . only at the end of the study , the energy intake changed differently in the hsf and lsf groups , with continuing gradual increase in the energy intake in the hsf group and a small decrease in the lsf group ( p = 0.02 ) . instead , the macronutrient composition of the diet changed differently in the hsf and the lsf groups . the intakes of dietary protein and fibre increased at the beginning of the weight - maintenance period in the hsf group whereas they remained about the same in the lsf group ( p < 0.001 for all ) . the intakes of dietary fiber and protein were thus higher in the hsf than those in the lsf group for the whole duration of the weight - maintenance period ( p < 0.001 for all ) . the intake of dietary fat decreased from the beginning of the study to the weight - maintenance period in both groups , the decrease being greater and thus overall fat intake lower in the hsf than in the lsf group during the weight - maintenance period ( p < 0.001 ) . the total carbohydrate intake decreased from the beginning of the study to the weight maintenance in both groups . the reduction was greater in the hsf group than that in the lsf group ( p = 0.04 ) . as the percentage of energy , carbohydrate intake remained about the same during the whole study being greater in the lsf group ( p < 0.001 ) . the alcohol intake decreased from the beginning of the study in both groups , being , as the percentage of energy , somewhat greater in the hsf group than that in the lsf group ( p = 0.04 ) . the observed differences in dietary protein , fibre , fat , and carbohydrate intakes during the weight - maintenance period were due to the different macronutrient contents of the test foods ( table 5 ) . the composition and energy intake of the freely selected part of the diet was the same in both groups ( data not shown ) . the subjects in the hsf and the lsf groups experienced the study very similarly based on their responses in the final questionnaire at the end of the study ( estimated easiness of the weight maintenance period : hsf 7.1 1.8 , lsf 7.1 1.9 ) . neither did they report any differences in the easiness of the control of food intake or other conditions that were evaluated along the study ( overall wellbeing , bowel function , hunger , satiety ) . cognitive restraint of eating , both flexible and rigid , increased in both the hsf and the lsf groups after the weight - loss period , as measured by the tfeq and the debq questionnaires ( table 7 ) . at the same time , disinhibition of eating , susceptibility to hunger , and binge eating as well as emotional and external eating decreased in both groups . there were no significant differences in the changes between the groups , except in the external eating which decreased more in the hsf group than those in the lsf group ( p = 0.007 ) most likely due to higher scores in the hsf at the beginning of the study . neither were there any significant differences between the groups in the changes in the psychological distress , as assessed by ghq , which alleviated in both groups after the weight loss then gradually returning towards the baseline level . due to great interindividual variation in the success to maintain reduced body weight , irrespective of the study group , the predictors of weight maintenance were analysed in the whole study population . the changes in the eating behaviour during the study were significantly associated with the weight maintenance ( table 8) . the success in weight maintenance was associated with the greater increase in the cognitive restraint of eating , especially in the flexible control of eating as well as the greater decrease in the disinhibition of eating , susceptibility to hunger , binge eating behaviour , external eating , and psychological distress . most of these changes , that is , increase in the flexible control of eating and the decrease in susceptibility to hunger , binge eating behaviour , external eating , and psychological distress , were associated with better weight maintenance also after adjusting for group membership during the weight - maintenance period ( table 8) . the self - reported easiness of the weight - maintenance period resulted to be a single factor which was most strongly associated with the successful maintenance of reduced body weight during the weight - maintenance period ( r = 0.47 , p < 0.001 , n = 82 ) . the association was significant , even slightly stronger , also after making adjustment for study group ( partial correlation r = 0.52 , p < 0.001 ) . those subjects who experienced the weight - maintenance period the easiest also managed better than those experiencing it more difficult . similarly , the self - reported easiness of the control of food intake , as assessed at the different phases of the weight - maintenance period , correlated significantly with the weight maintenance ( mean of weekly assessments , r = 0.39 , p < 0.001 , n = 82 ) , also after adjusting for group membership ( partial correlation r = 0.39 , p < 0.001 ) . the easier the subjective control of food intake , the more successful the weight maintenance . among the dietary determinants of successful weight maintenance , the compliance ( i.e. , the use of the test foods ) correlated with the final outcome ; the higher the compliance , the better the reduced body weight maintained during the weight - maintenance period ( r = 0.27 , p = 0.02 , n = 82 ) . this association was seen also after adjusting for the study group ( partial correlation r = 0.23 , p = 0.05 ) . lower energy intake according to the food diaries during the weight - maintenance period was also associated with the better maintenance of reduced body weight ( r = 0.24 , p = 0.03 , n = 82 ) also when adjusted by group membership ( partial correlation r = 0.22 , p = 0.047 ) . more specifically , we were interested in whether foods with predetermined satiety values consumed in free - living conditions as a part of a weight - maintenance diet would affect self - regulation of food intake and thereby weight management . to our knowledge , no previous studies have been published on the issue . in contrast to expectation , the diet containing foods with higher predetermined satiety value did not contribute to better weight management . this was even despite the obvious differences in the macronutrient contents of the diets , especially the differences in the amounts of dietary protein and fibre , which by themselves could also have been expected to influence satiety or weight management [ 11 , 12 ] . nevertheless , the present results were interestingly in line with the findings of a recent large intervention comparing the effects of weight - loss diets differing in the proportions of dietary fat , protein , or carbohydrate . in that study , there were also no significant differences in satiety , hunger , or diet satisfaction among the diets . instead , the strongest determinant of successful weight loss was an attendance at the group sessions , suggesting that behavioural factors rather than the composition of the diet are more important for the success of weight loss . also in the present study the successful maintenance of reduced weight was not primarily associated with the diet - related factor , but rather with the psychobehavioural variables related especially to eating behaviour . also participants ' subjective experiences about the easiness of the intervention and control of food intake were associated with better weight maintenance . the only diet - related factors that were significantly associated with the weight management were lower energy intake and better compliance of the use of the test products , which , on the other hand , could be seen also as behavioural factors similarly as the attendance at group sessions in the sacks et al . thus , as sacks et al . also pointed out , behavioural and psychological factors rather than macronutrient metabolism seem to have the main influence on the success of longer - term weight management . changes in various features of eating behaviour were associated with the better success in weight management . all the observed changes , that is , increase in the cognitive restraint of eating and decrease in binge eating , external eating , disinhibition of eating , and susceptibility to hunger , were into the direction that could be suggested to represent better self - control of eating . these changes were also well in line with several previous findings , thus further confirming the important role of eating behaviour in the successful weight management [ 27 , 28 , 30 , 31 , 43 ] . when distinguishing cognitive restraint of eating into flexible and rigid forms of restraint behaviour , it was specifically the flexible restraint that was associated with the better maintenance of reduced weight . this is in accordance with the earlier findings [ 22 , 27 , 31 , 38 , 44 ] and also with the concept of flexible restraint . by definition , flexible restraint of eating is a form of eating behaviour characterized by a more graduated approach to eating , dieting , and weight in contrast to rigid control which is characterized by a dichotomous , all - or - nothing approach to eating , dieting , and weight . a form of restrained eating in which flexible approach is adopted seems thus to be beneficial to effective weight control [ 31 , 45 ] . the beneficial effect of flexible control of eating on weight management has been documented earlier both in cross - sectional and in prospective analysis . in cross - sectional settings , flexible restraint has been shown to predict lower fatness and bmi [ 22 , 46 ] . in prospective studies , successful weight change has been more strongly associated with flexible than with rigid form of cognitive restraint of eating [ 31 , 38 , 46 , 47 ] . concluded , sustained weight loss seems to require that subjects adopt a flexible eating self - regulation pattern that allows them to modify their eating behaviour and quite likely also their physical activity by appropriate ways in this complex food environment . successful restraint of eating can also be linked with higher self - control capacity in general . therefore attempts to enhance self - regulation could be of value to those attempting to control body weight and food intake . also recent brain imaging studies have confirmed links between body weight , restraint , and self - regulation . individuals with high dietary restraint showed increased neural activity in the cortical and subcortical control and reward areas , especially at the dorsal prefrontal cortex and dorsal striatum , in response to meal ingestion or pictures of palatable foods . this suggests that the cognitive control of food intake is achieved by modulating neural circuits controlling inappropriate behavioural responses and food reward [ 48 , 49 ] . this may also be the way to counterbalance the physiological compensatory changes , like reductions in energy expenditure and circulating mediators of appetite , which do promote weight regain after weight loss . as a concept , self - control is close to the concept of self - efficacy which refers to an individual 's belief that she or he can successfully execute a sequence of actions in a specified context . self - efficacy has been shown to be an important predictor of successful weight management [ 31 , 51 , 52 ] . however , interestingly , the experience of greater easiness of weight maintenance as well as easiness of control of food intake was associated with the more successful maintenance of reduced body weight . this suggests that those experiencing intervention easier might also have felt themselves more competent and efficient in managing their attempts to maintain reduced body weight . as further support for this , however , it should be noticed that the ratings of easiness of weight management or control of food intake are highly subjective measures and strongly susceptible to reverse causality or confounding . they could thus also be simply consequences of changes in other psychobehavioural variables or influenced by the body weight change itself . nevertheless , tools and approaches which could enhance subjective experience of self - control and thereby likely also the experienced easiness of weight management should thus be taken into account to increase the long - term success of weight management . in current behavioural interventions , still fairly little attention has been devoted to , for example , psychological resources needed for the long - term management of body weight or other health - related lifestyle habits . in the present study , except for the lower energy intake and better compliance of the use of the test foods , other dietary factors were not associated with better weight management . this was despite the fact that the differences in the protein and fibre contents of the study diets were comparable with those reported in previous studies yet demonstrating better weight management on the diets with higher protein [ 14 , 5456 ] or fibre content [ 12 , 18 ] . indeed , in a recent diogenes study even smaller difference ( 4.56.5% versus 7.5% , diogenes versus present study ) in the dietary protein intake between the study groups was followed by significant , although relatively small ( 0.9 kg ) , difference in body weight regain after a successful weight loss . the authors ascribed this to be due to too subtle satiety effects of the diets to be subjectively measured . on the other hand , the positive association between protein intake and satiety has been suggested to be evident only when protein intake is greater than protein requirements [ 16 , 57 ] . indeed , lejeune et al . reported that the greater satiating effect of dietary protein was seen only when protein intake was as much as 2.6 g / kg . this is much more than what was the protein intake ( 1.3 g / kg ) in the hsf group in the present study . it could therefore be argued that the protein intake was not enough to contribute to possible significant effect on long - term satiety . however , much higher protein intake would have been impractical to achieve and would not have represented a normal balanced diet . estimated that about 14 g increase in daily dietary fibre intake in ad libitum conditions was associated with about 10% decrease in dietary energy intake and consequent decrease in body weight . in the present study the hsf group increased their daily dietary fibre intake approximately by 10 g. it could therefore be argued that the dietary fibre content of the study diet was still not enough to contribute to more successful weight management , although already smaller amounts of dietary fibre have been demonstrated to be effective . when interpreting the results , it should be noticed that the weight - maintenance diet was not totally ad libitum since the participants were given instructions about the recommended number of portions to be consumed within each food group . this could naturally have diminished the possible influence of dietary fibre and protein and their potential satiety effect on the self - regulation of food intake and thereby on the maintenance of reduced body weight . in line , most studies reporting greater weight loss after high - protein than low - protein diet have been those allowing ad libitum food intake , whereas only few of the studies that have provided isocaloric high- versus low - protein diets have shown significant weight loss results [ 14 , 19 , 32 ] . we can not rule out that if the proportion of foods with different satiety values would have been greater than 30% of energy intake , a possible satiety effect could have been seen . this was , however , the highest amount of test foods that could be realistically included into the diet without an excessive risk of dietary noncompliance due to the continuous use of only limited number of test foods . on the other hand , the proportion of test foods should not be too high to limit freedom in the remaining diet to show potential differences in energy intake . finally , all the other dietary counselling given to the subjects during the study , especially in the group sessions before the weight - maintenance period , might also have diminished the possible differences between the groups . another central methodological issue is that , to our knowledge , this was the first time to examine whether foods with predetermined satiety values could contribute to weight management . thus , at the same time , the study examined the predictive value of a single satiety measurement of food on satiety during continued use in a free - living context . as the results suggest , the different predetermined satiety values of the test foods did not influence weight management in a free - living situation . no differences were seen either in energy intake or in the weekly ratings of hunger or satiety . this confirms how much more difficult it is to get reliable data about satiety outside a controlled laboratory setting . it also raises issues as to how far laboratory data can really be extrapolated to free - living conditions . yet controlled laboratory testing is a general and recommended practice when assessing the satiety values of foods . , the satiety values of the test foods were determined in a separate group representing mostly relatively young , normal - weight subjects . the demographic characteristics of the subjects in the satiety tests and in the intervention were thus quite different . normal - weight and overweight subjects were , however , selected for satiety testing instead of obese individuals due to potentially attenuated satiety responses in obese as compared with lean individuals , to be able to demonstrate actual differences in satiety values between the test products . furthermore , in the satiety tests , the foods were tested as isocaloric portions , and , during the intervention , the recommended portion sizes were adjusted to represent typical portion sizes of each food relative to everyday food consumption . the test foods also varied in some other dimensions , such as macronutrient content and palatability . all this could have affected the behaviours observed over the weight - maintenance period and perhaps obscured the potential effect of satiety value of foods . nevertheless , although satiety value of foods should ideally be tested in subjects and conditions representing the target situation as close as possible , controlling all the prevailing conditions comparable to free - living conditions may be unrealistic . furthermore , most of the foods are rarely eaten as such but usually as a part of a mixed meal or a snack . thus , the satiety values do vary also according to other prevailing , mostly uncontrollable circumstance - related factors . it could be argued that factors that predict subsequent weight maintenance are not necessarily the same if weight loss is initially achieved rapidly with a vlcd compared with methods inducing more gradual weight loss . a recent study comparing factors associated with successful weight maintenance in relation to initial method of weight loss ( i.e. , vlcd versus commercial program or self - guided approach ) did not , however , observe any differences across the methods on the changes in eating behaviour or dietary intake , which further supports the relevance of the present findings on the success of weight management . foods with higher predetermined satiety value , when ingested as a part of a controlled weight - maintenance diet , did not contribute to better weight maintenance . instead , success in weight maintenance was associated with a greater increase in flexible control of eating and greater decrease in uncontrollable eating and psychological distress . thus , means to support changes in these psychobehavioural factors are of essential importance in successful long - term weight management .

this study aimed to investigate factors associated with weight management , especially whether satiety value of food as a part of a weight - maintenance diet would affect self - regulation of food intake and weight management . altogether 82 obese subjects completed the study consisting of weight - loss and weight - maintenance ( wm ) periods . during the wm , subjects were randomized into higher- and lower - satiety food groups . no differences were observed in the changes in body weight , energy intake , or eating behaviour between the groups , even despite the different macronutrient compositions of the diets . however , when regarding all study subjects , success in wm was most strongly associated with a greater increase in the flexible control of eating and experience of greater easiness of wm and control of food intake and a greater decrease in uncontrollable eating and psychological distress . psychobehavioural factors seem to be more strongly associated with successful weight management than the predetermined satiety effect or other characteristics of the diet .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion 5. Conclusions

therefore it is important to identify factors that are associated with and could enhance the self - regulation of food intake and other behaviours related to weight management . because obesity is always a result of an imbalance between the energy intake and energy expenditure , decreased energy intake regulation of energy balance is very complex , however , with multiple mechanisms maintaining homeostasis and resisting changes in the energy balance . therefore , during negative energy balance , that is , when energy intake is lower than energy requirements , orexigenic pathways in the organism are activated , a usual consequence of which is a regain of reduced body weight . among key mechanisms , dietary fibre increases satiety and reduces energy intake by decreasing the energy density of the diet as well as by retarding nutrient absorption , gastric emptying , and intestinal passage times in the gastrointestinal tract . however , in spite of the large number of short - term studies linking various food properties to increased satiety , it is still poorly known whether the satiety value of food has an impact on the self - regulation of food intake and thereby on the regulation of body weight also at the longer term . different features of eating behaviour , such as restraint and disinhibition of eating , are also strong determinants of weight management [ 2124 ] . dietary restraint refers to a tendency to consciously restrict or control food intake , whereas dietary disinhibition is a tendency to overeat in the presence of , for example , palatable foods , emotional stress , or other disinhibiting stimuli . other features of eating behaviour , such as emotional eating and greater susceptibility to sensations of hunger or external food - related cues , have also been associated with greater risk of obesity and difficulties in weight management [ 2932 ] . therefore , the aim of the present study was to investigate factors that are associated with successful weight management in middle - aged obese men and women . we were especially interested in investigating whether foods with higher predetermined satiety values , when ingested as a part of a weight - maintenance diet , contribute to better self - regulation of food intake and reduced body weight . the study consisted thus of two distinct , consecutive phases : determination of satiating values of foods in a controlled laboratory setting to select foods with lower and higher satiating values for the intervention and an intervention including weight - loss and weight - maintenance periods whereby the satiety - tested foods were used as a part of a weight - maintenance diet . because the satiating value of food can not be directly estimated based on the nutritional composition or other characteristics of foods [ 20 , 33 ] , satiety tests were performed to identify foods with different satiety values for the intervention . immediately after the consumption and during the following 180 min , sensations were rated as a change from the starting value with a 100+10 scale ( 10 = a lot less than that in the beginning , 0 = as much as that in the beginning , + 10 = a lot more than that in the beginning ) where 0 represents the baseline value . results were analyzed as the changes in the satiety - related sensations from the baseline levels as a function of time , and the area under curve ( auc , cm min ) was calculated . finally , 8 food pairs with significant difference in the overall 3 h response of the feelings of hunger or satiety were selected to be the test foods in the intervention ( table 2 ) . subjects were informed that the aim of the study was to examine the effect of various food characteristics on the management of body weight and food intake and related mechanisms . during the weight - loss period , the subjects were given dietary counselling in group sessions , 7 times during the whole period . after the weight - loss period , the subjects were randomized , stratified by age and sex , into two diet groups : higher - satiety food group ( hsf ) and lower - satiety food group ( lsf ) ( table 4 ) . during the 24-week weight - maintenance period , subjects were instructed to maintain their weight loss but not to continue actively losing weight . the subjects in the hsf consumed the test foods with higher satiety value the subjects in the lsf consumed the test foods with lower satiety value as a part of their weight - management diet . the proportion of test foods was tested in the pilot study performed prior to the intervention study to find out what was the highest realistic amount of the test foods that could be consumed as a part of the diet . 19 subjects ( 5 men , 14 women ) of same age ( 51.2 10.0 years ) and bmi ( 31.3 4.4 kg / m ) as the subjects in the intervention study participated in the pilot and used the test foods for two weeks as a part of their habitual diet . in addition , to allow the possible satiety effect of test foods to function , subjects were told that they could increase or decrease the number of food servings ( other than test foods ) if still hungry or if sated , respectively . the portion sizes could be different from those used in the standard satiety tests because the foods were used as a part of a diet and were therefore targeted to represent typical portion sizes of each food . the measurements were done at the separate study visits in the beginning of the weight - loss period before any weight loss had occurred , at the end of the weight - loss period ( i.e. height was measured using a wall - mounted stadiometer to the nearest 0.1 cm in the beginning of the weight - loss period . subjects completed 4-day food records altogether five times during the study : before the weight - loss period and at 6 , 12 , 18 , and 24 weeks from the beginning of the weight - maintenance period . three - factor eating questionnaire ( tfeq ) was used to measure cognitive restraint of eating and divided also to the flexible and rigid parts of the restraint , disinhibition of eating , and susceptibility to hunger . the subjects filled in the questionnaires in the beginning of the weight - loss period and after 12 and 24 weeks of weight maintenance . psychological distress was evaluated by the general health questionnaire in the beginning and at the end of the weight - loss period as well as after 12 and 24 weeks of weight maintenance . during the weight - maintenance period , the subjects evaluated at the end of each week their overall well - being , bowel function , hunger , satiety , and easiness of control of food intake by the 9-point scale ( 1 = not at all / extremely bad , , 9 = a lot / extremely good ) . at the end of the study , the subjects filled in a final questionnaire to assess how they had experienced the weight - maintenance period in general ( 1 = very difficult , , 10 = very easy ) and what they had liked about the test foods ( 1 = did not like at all , , 9 = liked very much ) . linear mixed - effect modelling was used to compare the effect of the study group on the changes in the examined variables . there were 17 subjects who dropped out , three subjects ( 2 men , 1 woman ) during the weight - loss period and 14 subjects ( 6 men , 9 women ) during the weight - maintenance period . there were no differences in the dropouts during the weight - maintenance period between the hsf ( 8 ( 3 men , 5 women ) ) and the lsf groups ( 6 ( 2 men , 4 women ) ) . the use of test foods , as an indicator of compliance , was equal in both groups ( % of the instructed use , calculated from the individual recordings during the weight - maintenance period : 100.8 9.0% versus 99.1 9.1% , hsf versus lsf ) , as well as the proportion of energy ingested from the test foods ( mean during the weight - maintenance period : 30.7 6.1% versus 29.0 5.0% , hsf versus lsf ) . the weight reduced by about 12 kg during the weight - loss period in both groups ( hsf 12.5 2.4% , lsf 12.4 2.6% of the body weight at the baseline ) . during the weight - maintenance period , there was only a very slight regain in the mean body weight with no significant difference between the groups ( hsf 1.3 3.5% , lsf 0.9 3.5% ) . the interindividual variation in the success to maintain the reduced body weight during the weight - maintenance period was large , with weight changes varying from 8.8% further weight reduction to 9.8% weight regain . only at the end of the study , the energy intake changed differently in the hsf and lsf groups , with continuing gradual increase in the energy intake in the hsf group and a small decrease in the lsf group ( p = 0.02 ) . the intakes of dietary protein and fibre increased at the beginning of the weight - maintenance period in the hsf group whereas they remained about the same in the lsf group ( p < 0.001 for all ) . the intakes of dietary fiber and protein were thus higher in the hsf than those in the lsf group for the whole duration of the weight - maintenance period ( p < 0.001 for all ) . the intake of dietary fat decreased from the beginning of the study to the weight - maintenance period in both groups , the decrease being greater and thus overall fat intake lower in the hsf than in the lsf group during the weight - maintenance period ( p < 0.001 ) . the alcohol intake decreased from the beginning of the study in both groups , being , as the percentage of energy , somewhat greater in the hsf group than that in the lsf group ( p = 0.04 ) . the observed differences in dietary protein , fibre , fat , and carbohydrate intakes during the weight - maintenance period were due to the different macronutrient contents of the test foods ( table 5 ) . the composition and energy intake of the freely selected part of the diet was the same in both groups ( data not shown ) . the subjects in the hsf and the lsf groups experienced the study very similarly based on their responses in the final questionnaire at the end of the study ( estimated easiness of the weight maintenance period : hsf 7.1 1.8 , lsf 7.1 1.9 ) . neither did they report any differences in the easiness of the control of food intake or other conditions that were evaluated along the study ( overall wellbeing , bowel function , hunger , satiety ) . cognitive restraint of eating , both flexible and rigid , increased in both the hsf and the lsf groups after the weight - loss period , as measured by the tfeq and the debq questionnaires ( table 7 ) . there were no significant differences in the changes between the groups , except in the external eating which decreased more in the hsf group than those in the lsf group ( p = 0.007 ) most likely due to higher scores in the hsf at the beginning of the study . neither were there any significant differences between the groups in the changes in the psychological distress , as assessed by ghq , which alleviated in both groups after the weight loss then gradually returning towards the baseline level . due to great interindividual variation in the success to maintain reduced body weight , irrespective of the study group , the predictors of weight maintenance were analysed in the whole study population . the changes in the eating behaviour during the study were significantly associated with the weight maintenance ( table 8) . the success in weight maintenance was associated with the greater increase in the cognitive restraint of eating , especially in the flexible control of eating as well as the greater decrease in the disinhibition of eating , susceptibility to hunger , binge eating behaviour , external eating , and psychological distress . most of these changes , that is , increase in the flexible control of eating and the decrease in susceptibility to hunger , binge eating behaviour , external eating , and psychological distress , were associated with better weight maintenance also after adjusting for group membership during the weight - maintenance period ( table 8) . the self - reported easiness of the weight - maintenance period resulted to be a single factor which was most strongly associated with the successful maintenance of reduced body weight during the weight - maintenance period ( r = 0.47 , p < 0.001 , n = 82 ) . similarly , the self - reported easiness of the control of food intake , as assessed at the different phases of the weight - maintenance period , correlated significantly with the weight maintenance ( mean of weekly assessments , r = 0.39 , p < 0.001 , n = 82 ) , also after adjusting for group membership ( partial correlation r = 0.39 , p < 0.001 ) . the easier the subjective control of food intake , the more successful the weight maintenance . , the use of the test foods ) correlated with the final outcome ; the higher the compliance , the better the reduced body weight maintained during the weight - maintenance period ( r = 0.27 , p = 0.02 , n = 82 ) . lower energy intake according to the food diaries during the weight - maintenance period was also associated with the better maintenance of reduced body weight ( r = 0.24 , p = 0.03 , n = 82 ) also when adjusted by group membership ( partial correlation r = 0.22 , p = 0.047 ) . more specifically , we were interested in whether foods with predetermined satiety values consumed in free - living conditions as a part of a weight - maintenance diet would affect self - regulation of food intake and thereby weight management . in contrast to expectation , the diet containing foods with higher predetermined satiety value did not contribute to better weight management . this was even despite the obvious differences in the macronutrient contents of the diets , especially the differences in the amounts of dietary protein and fibre , which by themselves could also have been expected to influence satiety or weight management [ 11 , 12 ] . nevertheless , the present results were interestingly in line with the findings of a recent large intervention comparing the effects of weight - loss diets differing in the proportions of dietary fat , protein , or carbohydrate . instead , the strongest determinant of successful weight loss was an attendance at the group sessions , suggesting that behavioural factors rather than the composition of the diet are more important for the success of weight loss . also in the present study the successful maintenance of reduced weight was not primarily associated with the diet - related factor , but rather with the psychobehavioural variables related especially to eating behaviour . also participants ' subjective experiences about the easiness of the intervention and control of food intake were associated with better weight maintenance . the only diet - related factors that were significantly associated with the weight management were lower energy intake and better compliance of the use of the test products , which , on the other hand , could be seen also as behavioural factors similarly as the attendance at group sessions in the sacks et al . changes in various features of eating behaviour were associated with the better success in weight management . all the observed changes , that is , increase in the cognitive restraint of eating and decrease in binge eating , external eating , disinhibition of eating , and susceptibility to hunger , were into the direction that could be suggested to represent better self - control of eating . these changes were also well in line with several previous findings , thus further confirming the important role of eating behaviour in the successful weight management [ 27 , 28 , 30 , 31 , 43 ] . the beneficial effect of flexible control of eating on weight management has been documented earlier both in cross - sectional and in prospective analysis . in prospective studies , successful weight change has been more strongly associated with flexible than with rigid form of cognitive restraint of eating [ 31 , 38 , 46 , 47 ] . therefore attempts to enhance self - regulation could be of value to those attempting to control body weight and food intake . also recent brain imaging studies have confirmed links between body weight , restraint , and self - regulation . this suggests that the cognitive control of food intake is achieved by modulating neural circuits controlling inappropriate behavioural responses and food reward [ 48 , 49 ] . self - efficacy has been shown to be an important predictor of successful weight management [ 31 , 51 , 52 ] . however , interestingly , the experience of greater easiness of weight maintenance as well as easiness of control of food intake was associated with the more successful maintenance of reduced body weight . as further support for this , however , it should be noticed that the ratings of easiness of weight management or control of food intake are highly subjective measures and strongly susceptible to reverse causality or confounding . nevertheless , tools and approaches which could enhance subjective experience of self - control and thereby likely also the experienced easiness of weight management should thus be taken into account to increase the long - term success of weight management . in the present study , except for the lower energy intake and better compliance of the use of the test foods , other dietary factors were not associated with better weight management . this was despite the fact that the differences in the protein and fibre contents of the study diets were comparable with those reported in previous studies yet demonstrating better weight management on the diets with higher protein [ 14 , 5456 ] or fibre content [ 12 , 18 ] . indeed , in a recent diogenes study even smaller difference ( 4.56.5% versus 7.5% , diogenes versus present study ) in the dietary protein intake between the study groups was followed by significant , although relatively small ( 0.9 kg ) , difference in body weight regain after a successful weight loss . estimated that about 14 g increase in daily dietary fibre intake in ad libitum conditions was associated with about 10% decrease in dietary energy intake and consequent decrease in body weight . in the present study the hsf group increased their daily dietary fibre intake approximately by 10 g. it could therefore be argued that the dietary fibre content of the study diet was still not enough to contribute to more successful weight management , although already smaller amounts of dietary fibre have been demonstrated to be effective . when interpreting the results , it should be noticed that the weight - maintenance diet was not totally ad libitum since the participants were given instructions about the recommended number of portions to be consumed within each food group . this could naturally have diminished the possible influence of dietary fibre and protein and their potential satiety effect on the self - regulation of food intake and thereby on the maintenance of reduced body weight . finally , all the other dietary counselling given to the subjects during the study , especially in the group sessions before the weight - maintenance period , might also have diminished the possible differences between the groups . thus , at the same time , the study examined the predictive value of a single satiety measurement of food on satiety during continued use in a free - living context . as the results suggest , the different predetermined satiety values of the test foods did not influence weight management in a free - living situation . no differences were seen either in energy intake or in the weekly ratings of hunger or satiety . normal - weight and overweight subjects were , however , selected for satiety testing instead of obese individuals due to potentially attenuated satiety responses in obese as compared with lean individuals , to be able to demonstrate actual differences in satiety values between the test products . all this could have affected the behaviours observed over the weight - maintenance period and perhaps obscured the potential effect of satiety value of foods . furthermore , most of the foods are rarely eaten as such but usually as a part of a mixed meal or a snack . a recent study comparing factors associated with successful weight maintenance in relation to initial method of weight loss ( i.e. , vlcd versus commercial program or self - guided approach ) did not , however , observe any differences across the methods on the changes in eating behaviour or dietary intake , which further supports the relevance of the present findings on the success of weight management . foods with higher predetermined satiety value , when ingested as a part of a controlled weight - maintenance diet , did not contribute to better weight maintenance . instead , success in weight maintenance was associated with a greater increase in flexible control of eating and greater decrease in uncontrollable eating and psychological distress .

the prevalence of hyperphagia in patients with dementia is 23%51% and tends to occur in patients in the middle stage of dementia.1,2 the main features of hyperphagia include increased appetite ( eg , frequent food searches or requests for food ) , faster eating speed , and increased amount of food consumed.1,3,4 hyperphagia is usually complicated with altered eating habits , inadequate eating behaviors , and pica ; this may easily lead to risks of upper airway obstruction , nutritional imbalance , or poisoning caused by erroneous eating and also increase the conflicts between caregivers and patients.48 the literature shows that the hyperphagic behaviors of patients with dementia may originate from damage to the frontotemporal lobe or an impaired satiety center in the ventromedial nucleus of the hypothalamus , thereby stimulating the neurological effects of starvation , or increasingly agitated behavior of patients with dementia , thereby requiring constant feeding to supplement calories.6,7,9,10 however , reports have also suggested that hyperphagia in patients with dementia is caused by memory degradation , which causes the patients to forget what food they have eaten or how to take the food accurately.11 non - declarative memory ( sometimes termed as implicit memory ) has always been the focus of dementia memory training . it is involved with the unconscious recognition of an object ( eg , priming ) and the correct completion of the steps in a task ( eg , procedural memory ) . some studies found that individuals in the mild or moderate stages of alzheimer s disease ( ad ) show improvement in non - declarative tasks.1214 in this sense , interventions in patients with ad have the potential to maintain function through changed speed or accuracy when completing a task . two approaches that can be used for training non - declarative memory are spaced retrieval ( sr ) and montessori - based activities.15,16 sr is a learning approach developed to facilitate recall of a variety of information in individuals with dementia . when learning using sr , the patient repeatedly rehearses and recalls the information to be learnt at set intervals , with the interval between rehearsals constantly increasing.1719 if the patient can still accurately recall information after more than 16 minutes , the information is considered to be successfully stored in their long - term memory system.20 creighton et al concluded camp previous reported that the technique integrates four different methods of learning.21 first , sr is a form of ecologically valid priming , spacing effect information that is learned and retrieved more effectively when trials are distributed over time rather than mass practiced . the utilization of both these methods is believed to be particularly beneficial to individuals with dementia , as they capitalize on relatively spared and automatic implicit memory system . third , sr is regarded as a form of conditioning ; strong associations are formed between the target question and response through repetition , and successful recall trials may serve as intrinsic reinforcers . finally , sr can also be considered a form of errorless learning , as it minimizes the possibility of errors during the acquisition phase of learning by ensuring that mistakes are repeatedly corrected.22,23 de ajuriaguerra and tissot observed that decline of certain capacities in dementia appeared to reverse piaget s developmental stages.24 cognition and behavior development in children and loss in people with dementia were found to be remarkably similar . the cognitive capability of most elders with dementia has deteriorated to the preoperational stage ( which is similar to mild and moderate dementia ) or the sensorimotor stage ( which is similar to severe dementia ) of piaget s theory of cognitive development.25 piaget s theory also provides direction for the selection of appropriate activities to foster the development of cognitive skills at various ages by programmed montessori - based activities . all montessori - based activities , which were originally used with children aged 06 years , can break down each step of activities of daily living in order to enhance the procedural memory . moreover , these activities can also provide sensory stimulation and movement coordination training to help the person with dementia to have a successful experience and create more control of behaviors within an orderly environment.25,26 both sr and montessori activities have been verified to alleviate the cognitive degeneration of patients with dementia , improve problem behaviors , and increase positive emotions and activities of daily living.23,2729 lin et al successfully used sr and montessori - based activities to improve eating difficulties in patients with dementia , and wu et al found that applying standardized and individualized sr in combination with montessori activities training could significantly improve eating difficulties in patients with dementia.30,31 the causes of eating difficulties in patients with dementia are similar to those of hyperphagia , including impaired functioning of the satiety center of hypothalamus , which causes patients to forget how to take , chew , and swallow food , as well as worsening the ability to use tableware . however , the memory training processes in these studies involved using food as the teaching material and provided the participants with food they enjoyed as a reward after the end of each training period . during the process of behavior shaping , the provision of food items that patients like reinforces the learning process . repeated provision of highly reinforcing food ( especially food with a high sugar and fat content ) can reinforce the social pleasure of the patients receiving training and constantly reinforce their behavior.3234 the aim of this study was to investigate and compare the long - term effectiveness of sr and sr + m on improving hyperphagic behaviors of special care unit residents with dementia . this was to establish whether memory training can still improve abnormal behaviors of patients with dementia without the reinforcement of food and to investigate the difference in improvement of eating - related behaviors between sr alone and sr training combined with montessori - based activities ( sr + m ) . further , this study was to understand whether montessori activities in combination with sr can improve the effectiveness of message memorization . the participants were recruited from eight dementia special care units in taiwan , republic of china ( four long - term care facilities and four veterans nursing homes ) . the inclusion criteria were as follows : 1 ) a diagnosis of dementia by a physician ; 2 ) hyperphagia - related behavior scoring of at least 3 points on the scale used ; 3 ) successfully clearing the sr screening test and the ability to maintain memory for 20 seconds ; 4 ) able to see , listen , and read ; and 5 ) possessing sufficiently fine hand movement and muscle power , scoring 45 points on the scale used . the exclusion criteria were as follows : 1 ) placed with a feeding tube or needing to be fed , 2 ) a body mass index of < 18.5 kg / m , 3 ) vascular dementia , or 4 ) a recent brain injury or acute gastrointestinal infection . the study was reviewed and approved by taipei city hospital institutional review board ( tchirb no : 991226-e ) . a researcher personally explained the research and its purpose to institutional agents and family members and obtained their written informed consent ( trial registration : chictr - ior-15007531 ) . this study had a longitudinal two - step cluster - randomized single - blind design and was conducted from june 2012 to october 2014 . after the institutions identified research subjects who met the inclusion criteria , trained research assistants collected pretest data of all the subjects and classified the severity of their hyperphagia as mild , moderate , or severe . the patients in each hyperphagia category were randomized into three groups , and a homogeneity test was applied to confirm that these groups were homogeneous . the patients were then randomly assigned to the sr group ( 48 subjects ) , the sr + m group ( 52 subjects ) , and the control group ( 48 subjects ) . after the grouping of subjects was confirmed , two memory trainers who had received training in sr and montessori activity methods provided the two experimental groups with memory training courses for a total of 30 sessions spread over 6 consecutive weeks ( with each session lasting for at least 40 minutes ) . the control group did not receive any intervention but instead engaged in the routine activities of institutions . following completion of the interventions , the research assistants collected post - training test data and follow - up data at 1 month , 3 months , and 6 months for the subjects in all the three groups . during the research process , a total of eight subjects left the study ( sr group : 2 ; sr + m group : 3 ; control group : 3 ) . the main causes were hospitalization ( five subjects ) , returning home for care ( two subjects ) , and death ( one subject ) . a total of 140 subjects completed the entire research process and follow - up data collection , including 46 subjects in the sr group , 49 in the sr + m group , and 45 in the control group ( figure 1 ) . the objectives for the subjects were as follows : 1 ) to memorize where food is placed ( only food placed in bowls could be taken : four sessions ) ; 2 ) to memorize slowing down their dining speed ( chewing slowly , swallowing slowly , and eating the food bite by bite : eleven sessions ) ; 3 ) to memorize a satiation message ( stopping eating on hearing a 20-second melody and putting down the bowl : eight sessions ) the 20-second melody functions as a warning message to remind the patients with dementia to stop eating when hearing it ; in addition to being applied in classroom training , it was also played 40 minutes after three meals throughout the course of the 6-week intervention to enable all the residents to hear it ; however , it was no longer played after the 6 weeks were up and 4 ) to memorize appropriate dining etiquette behavior ( appropriate behavior while eating and interacting with other people : seven sessions ) . trainers began by introducing themselves to the subjects for reorientation and then gave the memory training , after which they provided a review of the session . the memory training process of each group was as follows : the sr group : during each training session , the subject had to learn ( and review ) a memory message . once they could successfully recall the message immediately ( 0 minute ) , the trainer extended the time before recalling the message to 1 minute ; if the subject still could remember the message , the recall interval was extended sequentially to 2 minutes , 4 minutes , 6 minutes , 8 minutes , and 16 minutes . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . at the end of the session , the training sessions that continued with the same objective started with the longest recall interval of the previous session ; training sessions beginning a new objective started with a recall interval of 0 minute . when the subject failed to immediately recall a message , the trainer provided auxiliary memory clues , such as images or texts , one by one . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1).the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . each activity started with sensory stimulation followed by practicing an action ( eg , gently pressing , scooping , or aligning ) using standard procedure and the cognitive training of matching and identification . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1).the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . the sr group : during each training session , the subject had to learn ( and review ) a memory message . once they could successfully recall the message immediately ( 0 minute ) , the trainer extended the time before recalling the message to 1 minute ; if the subject still could remember the message , the recall interval was extended sequentially to 2 minutes , 4 minutes , 6 minutes , 8 minutes , and 16 minutes . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . at the end of the session , the training sessions that continued with the same objective started with the longest recall interval of the previous session ; training sessions beginning a new objective started with a recall interval of 0 minute . when the subject failed to immediately recall a message , the trainer provided auxiliary memory clues , such as images or texts , one by one . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1 ) . the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . each activity started with sensory stimulation followed by practicing an action ( eg , gently pressing , scooping , or aligning ) using standard procedure and the cognitive training of matching and identification . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1 ) . the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the interview guide included what eating behaviors change the caregiver observed such as increasing eating frequency , preferred food , searching food , etc . ; how the caregiver felt and responded for the abovementioned behaviors as well as managed those behaviors . content analysis was used to analyze the interview data . a 19-item scale of hyperphagia in residents with dementia and a ten - item scale of distress of the caregiver to hyperphagic behaviors content validity index for five experts in first time was 0.870 , because one expert disagreed that pica was included in excessive eating behaviors . the definition of excessive eating may be a variant of hyperorality disturbances which include oral exploration and pica,7 pica was retained . for internal consistency , to determine interrater reliability , intraclass coefficient was 0.7930.796.38 to evaluate hyperphagic behaviors , each subject was observed for 7 consecutive days , including observations of at least ten lunch or dinner dining situations . a 19-item scale of hyperphagia in residents with dementia was used to facilitate this.32 the scale covered four categories behaviors : intention to eat , rapid eating , increased eating , and inappropriate dining etiquette . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . if the score for a single behavior was 3 points or more or the total score exceeded 3 points , the subject was considered suffering from hyperphagia . the severity of hyperphagia was determined from the total score : 3 points , mild hyperphagia ; 46 points , moderate hyperphagia ; > 7 points , severe hyperphagia . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . pica was scored as yes ( 1 point ) or no ( 0 point ) according to whether the subjects ate food that was inedible ( eg , soap or stone ) or inappropriate ( eg , raw meat , food waste , or spoiled food ) . the changes in eating habits assessed using record forms covered eight types of changes ( eg , prefer sweet foods more than before and tend to eat food in the same order ) . the short meal frequency form was used to record the frequency of meals that lasted for < 10 minutes . all the observation records were collected by six research assistants who had cleared the 24-hour observer training and completed an interrater reliability test . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . i m bothered that residents keep asking for food when i did nt provide them with it ; i m worried that residents eat what they should not eat ) were scored on a likert scale ( 03 ) according to the level of distress . the internal consistency was shown to be 0.706.38 spss for windows version 20.0 was used for the statistical analysis , and p - values < 0.05 were considered significant . generalized estimating equation was used to analyze the changes from the pretraining test data in the post - training test and 1-month , 3-month , and 6-month follow - up data for all three groups of subjects . differences between the two experimental groups in the performance of memory training were evaluated with independent sample t - tests , the friedman test , and the wilcoxon signed - rank test . the participants were recruited from eight dementia special care units in taiwan , republic of china ( four long - term care facilities and four veterans nursing homes ) . the inclusion criteria were as follows : 1 ) a diagnosis of dementia by a physician ; 2 ) hyperphagia - related behavior scoring of at least 3 points on the scale used ; 3 ) successfully clearing the sr screening test and the ability to maintain memory for 20 seconds ; 4 ) able to see , listen , and read ; and 5 ) possessing sufficiently fine hand movement and muscle power , scoring 45 points on the scale used . the exclusion criteria were as follows : 1 ) placed with a feeding tube or needing to be fed , 2 ) a body mass index of < 18.5 kg / m , 3 ) vascular dementia , or 4 ) a recent brain injury or acute gastrointestinal infection . the study was reviewed and approved by taipei city hospital institutional review board ( tchirb no : 991226-e ) . a researcher personally explained the research and its purpose to institutional agents and family members and obtained their written informed consent ( trial registration : chictr - ior-15007531 ) . this study had a longitudinal two - step cluster - randomized single - blind design and was conducted from june 2012 to october 2014 . after the institutions identified research subjects who met the inclusion criteria , trained research assistants collected pretest data of all the subjects and classified the severity of their hyperphagia as mild , moderate , or severe . the patients in each hyperphagia category were randomized into three groups , and a homogeneity test was applied to confirm that these groups were homogeneous . the patients were then randomly assigned to the sr group ( 48 subjects ) , the sr + m group ( 52 subjects ) , and the control group ( 48 subjects ) . after the grouping of subjects was confirmed , two memory trainers who had received training in sr and montessori activity methods provided the two experimental groups with memory training courses for a total of 30 sessions spread over 6 consecutive weeks ( with each session lasting for at least 40 minutes ) . the control group did not receive any intervention but instead engaged in the routine activities of institutions . following completion of the interventions , the research assistants collected post - training test data and follow - up data at 1 month , 3 months , and 6 months for the subjects in all the three groups . during the research process , a total of eight subjects left the study ( sr group : 2 ; sr + m group : 3 ; control group : 3 ) . the main causes were hospitalization ( five subjects ) , returning home for care ( two subjects ) , and death ( one subject ) . a total of 140 subjects completed the entire research process and follow - up data collection , including 46 subjects in the sr group , 49 in the sr + m group , and 45 in the control group ( figure 1 ) . the objectives for the subjects were as follows : 1 ) to memorize where food is placed ( only food placed in bowls could be taken : four sessions ) ; 2 ) to memorize slowing down their dining speed ( chewing slowly , swallowing slowly , and eating the food bite by bite : eleven sessions ) ; 3 ) to memorize a satiation message ( stopping eating on hearing a 20-second melody and putting down the bowl : eight sessions ) the 20-second melody functions as a warning message to remind the patients with dementia to stop eating when hearing it ; in addition to being applied in classroom training , it was also played 40 minutes after three meals throughout the course of the 6-week intervention to enable all the residents to hear it ; however , it was no longer played after the 6 weeks were up and 4 ) to memorize appropriate dining etiquette behavior ( appropriate behavior while eating and interacting with other people : seven sessions ) . trainers began by introducing themselves to the subjects for reorientation and then gave the memory training , after which they provided a review of the session . the memory training process of each group was as follows : the sr group : during each training session , the subject had to learn ( and review ) a memory message . once they could successfully recall the message immediately ( 0 minute ) , the trainer extended the time before recalling the message to 1 minute ; if the subject still could remember the message , the recall interval was extended sequentially to 2 minutes , 4 minutes , 6 minutes , 8 minutes , and 16 minutes . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . at the end of the session , the training sessions that continued with the same objective started with the longest recall interval of the previous session ; training sessions beginning a new objective started with a recall interval of 0 minute . when the subject failed to immediately recall a message , the trainer provided auxiliary memory clues , such as images or texts , one by one . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1).the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . each activity started with sensory stimulation followed by practicing an action ( eg , gently pressing , scooping , or aligning ) using standard procedure and the cognitive training of matching and identification . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1).the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . the sr group : during each training session , the subject had to learn ( and review ) a memory message . once they could successfully recall the message immediately ( 0 minute ) , the trainer extended the time before recalling the message to 1 minute ; if the subject still could remember the message , the recall interval was extended sequentially to 2 minutes , 4 minutes , 6 minutes , 8 minutes , and 16 minutes . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . at the end of the session , the training sessions that continued with the same objective started with the longest recall interval of the previous session ; training sessions beginning a new objective started with a recall interval of 0 minute . when the subject failed to immediately recall a message , the trainer provided auxiliary memory clues , such as images or texts , one by one . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1 ) . the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . each activity started with sensory stimulation followed by practicing an action ( eg , gently pressing , scooping , or aligning ) using standard procedure and the cognitive training of matching and identification . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1 ) . the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the interview guide included what eating behaviors change the caregiver observed such as increasing eating frequency , preferred food , searching food , etc . ; how the caregiver felt and responded for the abovementioned behaviors as well as managed those behaviors . content analysis was used to analyze the interview data . a 19-item scale of hyperphagia in residents with dementia and a ten - item scale of distress of the caregiver to hyperphagic behaviors content validity index for five experts in first time was 0.870 , because one expert disagreed that pica was included in excessive eating behaviors . after obtaining the expert s agreement about the definition of excessive eating may be a variant of hyperorality disturbances which include oral exploration and pica,7 pica was retained . for internal consistency , cronbach s alpha was 0.918 . to determine interrater reliability , intraclass coefficient was 0.7930.796.38 to evaluate hyperphagic behaviors , each subject was observed for 7 consecutive days , including observations of at least ten lunch or dinner dining situations . a 19-item scale of hyperphagia in residents with dementia was used to facilitate this.32 the scale covered four categories behaviors : intention to eat , rapid eating , increased eating , and inappropriate dining etiquette . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . if the score for a single behavior was 3 points or more or the total score exceeded 3 points , the subject was considered suffering from hyperphagia . the severity of hyperphagia was determined from the total score : 3 points , mild hyperphagia ; 46 points , moderate hyperphagia ; > 7 points , severe hyperphagia . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . pica was scored as yes ( 1 point ) or no ( 0 point ) according to whether the subjects ate food that was inedible ( eg , soap or stone ) or inappropriate ( eg , raw meat , food waste , or spoiled food ) . the changes in eating habits assessed using record forms covered eight types of changes ( eg , prefer sweet foods more than before and tend to eat food in the same order ) . the short meal frequency form was used to record the frequency of meals that lasted for < 10 minutes . all the observation records were collected by six research assistants who had cleared the 24-hour observer training and completed an interrater reliability test . the research assistants were blinded to the randomization of subjects . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . i m bothered that residents keep asking for food when i did nt provide them with it ; i m worried that residents eat what they should not eat ) were scored on a likert scale ( 03 ) according to the level of distress . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the interview guide included what eating behaviors change the caregiver observed such as increasing eating frequency , preferred food , searching food , etc . ; how the caregiver felt and responded for the abovementioned behaviors as well as managed those behaviors . content analysis was used to analyze the interview data . a 19-item scale of hyperphagia in residents with dementia and a ten - item scale of distress of the caregiver to hyperphagic behaviors content validity index for five experts in first time was 0.870 , because one expert disagreed that pica was included in excessive eating behaviors . after obtaining the expert s agreement about the definition of excessive eating may be a variant of hyperorality disturbances which include oral exploration and pica,7 pica was retained . for internal consistency , cronbach s alpha was 0.918 . to determine interrater reliability , intraclass coefficient was 0.7930.796.38 to evaluate hyperphagic behaviors , each subject was observed for 7 consecutive days , including observations of at least ten lunch or dinner dining situations . a 19-item scale of hyperphagia in residents with dementia was used to facilitate this.32 the scale covered four categories behaviors : intention to eat , rapid eating , increased eating , and inappropriate dining etiquette . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . if the score for a single behavior was 3 points or more or the total score exceeded 3 points , the subject was considered suffering from hyperphagia . the severity of hyperphagia was determined from the total score : 3 points , mild hyperphagia ; 46 points , moderate hyperphagia ; > 7 points , severe hyperphagia . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . pica was scored as yes ( 1 point ) or no ( 0 point ) according to whether the subjects ate food that was inedible ( eg , soap or stone ) or inappropriate ( eg , raw meat , food waste , or spoiled food ) . the changes in eating habits assessed using record forms covered eight types of changes ( eg , prefer sweet foods more than before and tend to eat food in the same order ) . the short meal frequency form was used to record the frequency of meals that lasted for < 10 minutes . all the observation records were collected by six research assistants who had cleared the 24-hour observer training and completed an interrater reliability test . the research assistants were blinded to the randomization of subjects . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . primary caregivers in the institutions were invited to complete a scale of distress of the caregiver to hyperphagic behaviors . reactions to each of ten hyperphagic behaviors ( eg , i m bothered that residents keep asking for food when i did nt provide them with it ; i m worried that residents eat what they should not eat ) were scored on a likert scale ( 03 ) according to the level of distress . spss for windows version 20.0 was used for the statistical analysis , and p - values < 0.05 were considered significant . generalized estimating equation was used to analyze the changes from the pretraining test data in the post - training test and 1-month , 3-month , and 6-month follow - up data for all three groups of subjects . differences between the two experimental groups in the performance of memory training were evaluated with independent sample t - tests , the friedman test , and the wilcoxon signed - rank test . the mean age of the 140 research subjects was 82.55 years ( standard deviation [ sd ] : 5.95 ) , and 70.7% of them were men . the subjects mainly suffered from moderate cognitive impairment , mild dysfunction in daily activities , and mild agitation ( mmse score : 12.09 , sd : 5.15 ; barthel index : 67.25 , sd : 24.90 ; cohen - mansfield agitation inventory score : 39.51 , sd : 7.03 ) . the mean duration of stay in institutions was 3 years , which was also the mean time since the confirmation of diagnosis of dementia . the family members and friends of 54.3% of the subjects visited them at least once every month . the homogeneity test on the three groups showed no significant differences among the groups ( table 1 ) . this study used a generalized estimating equation to compare all the 6-month follow - up data of the three groups . the results showed that the scores of the sr group for hyperphagic behavior and pica continued to decrease for 3 months and the improvement in short meal frequency lasted for 1 month after memory training . for the sr + m group , the improvements in hyperphagic behavior and short meal frequency lasted for 6 months after training . the improvement in distress to the primary caregiver of both the sr and sr + m groups only lasted until the post - training test . there was no significant change in eating habits among the three groups ( table 2 ) . analyses of the effectiveness of improvement in frequency of the four categories behaviors of hyperphagia ( intention to eat , rapid eating , increased eating , and inappropriate dining etiquette ) show that the behaviors of rapid eating and increased eating improved in both the sr and sr + m groups . the improvement lasted for a longer period of time in the sr + m group ( table 3 ) . in memory performance , after 30 session s activity intervention , the recall scores on the mmse in posttest was slightly higher than pretest in both experimental groups , while the recall scores on the mmse in the posttest was lower than pretest in the control group . however , sr and sr + m training programs can improve recall memory performance of patients with dementia that lasted for at least 1 month after training ( table 4 ) . the longest recall intervals in the sessions associated with training objectives 1 , 2 , and 3 were better than those for the sessions for training objective 4 ( to memorize appropriate dining etiquette behavior ) in both groups . the best recall performance in both groups was during sessions for training objective 2 ( to memorize slowing down the dining speed ) ; the performance of the sr + m group was significantly better than that of the sr group ( t=2.052 , p=0.044 ) , particularly in the training for chewing slowly lee et al examined whether sr training could improve cognitive function in people with very mild to mild dementia , but no change in neuropsychological performance was found.39 it was believed that factors leading to this result included a low cognitive effort required for sr training , and the small sample size of their study . in the current study , we combined sr with accumulating cues and montessori - based activities to create an effortful learning condition . we found the same increase in performance on recall memory scores as did wu et al,40 who indicates that applying the sr with accumulating techniques were more likely to at least possibly maintain memory status . although the change in recall memory scores in both experimental groups did not reach statistical significance in the subsequent three follow - ups , there was a trend toward significant improvement . on the other hand , this study found that both the sr and sr + m memory training can improve the hyperphagic behavior of patients with dementia without the need for food incentives . this finding is consistent with that of the study by lin et al30 and wu et al,31 suggesting that sr and montessori activities can improve dysphagia of patients with dementia . the longest recall interval of subjects participating in this study was 31 minutes , which is almost the same as that of patients with dementia in studies by lee et al and wu et al.39,40 thus , non - declarative memory training can be used for patients with moderate dementia , and it can help improve eating behaviors of patients with dementia.12,26,41,42 even though this study revealed positive effects of memory training , every patient with hyperphagia had to receive 40 minutes of memory training , five times per week for a total of 30 times . the caregivers could confirm the suitable time for the daily training of patients with dementia and then referred to the protocols designed in this study to provide one - on - one memory training . the training results showed that the hyperphagic behaviors that were significantly improved were rapid eating , increased eating , and pica . all these behaviors can affect the dietary safety of patients with dementia and lead to conflicts between them and with others . for example , rapid eating may involve swallowing food without chewing , stuffing the mouth with food without swallowing , and eating quickly when seeing food . similarly , increased eating includes stealing food from other s plate , eating on seeing food , and eating continually in the presence of food without a sensation of being full , and pica behavior involves eating inedible or inappropriate food . the results of this study suggest that the reason for the improvement of these specific behaviors was that the relevant training was provided in earlier training sessions ( on knowing where the food is and slowing down the dining speed ) . it could be expected that the subjects would have relatively greater cumulative memories of these . moreover , both rapid eating and pica behavior involved more specific actions and behaviors , and therefore , it was easier for patients with dementia to firmly memorize the associated training . the other two behaviors intention to eat and dining etiquette involved greater psychological and interactive learning , particularly with dining etiquette ; therefore , it was more difficult for patients with moderate dementia to memorize and learn them . this was supported by the results of the longest recall intervals , which were lowest for the sessions about dining etiquette . therefore , if the inadequate behaviors of patients with dementia are deconstructed ( breaking tasks into small components and starting with easier items ) , it will be easier for improvement by providing continuous training for specific actions.43 montessori activities are structured activities that involve everyday practical tasks and engage the learning - by - doing system , thus it can provide patients with dementia with the continuous practice required by procedural memory . beyond that , these activities are designed with rich , tangible sensory stimulation to improve patient s motor coordination , sense of reality , and sense of control over surroundings.16,44,45 therefore , montessori activities are beneficial for learning in patients with dementia , which was reflected in our study results : the slowing down of dining speed was significantly better in the sr + m group than in the sr group , and the improvements in dining speed and short meal frequency also lasted longer in the sr + m group . the baseline scores for caregiver distress for the three groups of subjects were not high . the reason for this may be that the chinese believe that being able to eat is a blessing , and therefore , they usually encourage the elderly to eat food ; they would have greater concerns about elderly people eating too little rather than too much . a previous survey showed that one - third of caregivers worry about swallowing problems.46 however , compared with dysphagia , the chinese are more tolerant toward hyperphagia , and their perceived distress regarding hyperphagia was lower.47 therefore , there was little scope to improve on caregiver distress . once the problem of short meal times was improved , the meal time of a greater number of patients with dementia increased , which would in turn be expected to increase the need for the company of caregivers or the provision of dining assistance.30 this may be the reason why the distress of caregivers was not continuously improved . after memory training , there was no significant change in eating habits in the three groups . changes in eating habits are more common in patients with frontotemporal dementia or severe semantic dementia and may also be associated with hallucinations , change in taste perception , or antipsychotic drugs used to treat severe agitated behaviors.10,11,4850 this study mainly enrolled patients with ad who had fewer behavioral and psychiatric symptoms and moderate cognitive impairment . moreover , it can not be ignored that dementia special care units usually provide regular meals in a closed environment , and therefore , it is less likely that patients independently acquire or choose food . therefore , the obtainment of food source and change in eating habits of the subjects may be affected , leading to no significant difference among the three groups . this retrospective study , unlike investigational studies , used behavioral observation to collect and analyze long - term data . however , some limitations are acknowledged in this study . first , the subjects were recruited from eight dementia special care units in taiwan , republic of china , and therefore , the research result may only be generalized to taiwanese residents with dementia who live in dementia special care units . second , due to the limitations of the institutionalized meal supply culture ( a fixed amount of food is provided at a fixed time or the food source is strictly limited ) , it could be expected that the observed hyperphagic behaviors of residents with dementia were underestimated . however , conversely , these factors also enabled subjects to continue maintaining stable dining during the intervention process . the effect of memory training programs on improving hyperphagic behavior of patients with dementia living in a community should be explored further . finally , eating problems involve extremely complex factors , including physiological and psychological factors , and aspects of interpersonal interaction . future studies should collect other relevant data in multiple ways to confirm the full impact of memory training on hyperphagia of patients with dementia . restricting or prohibiting a certain behavior tends to induce frustration or anger in patients with dementia . therefore , the training in this study did not focus on restricting the food intake of patients with dementia but on improving hyperphagic behaviors instead . the training programs were integrated with real situations to simulate dining procedures . from starting to eat food to slowing down the dining speed , learning to memorize satiation messages , and exhibiting appropriate dining etiquette , daily training enabled residents with dementia to constantly review and gradually adjust their inappropriate behaviors and relearn to accurately and slowly consume the amount of food they were supposed to eat . this study found that sr and sr + m memory training were non - pharmacological interventions that could improve hyperphagic behaviors of patients with dementia and significantly improve rapid eating , increased eating , and pica behavior . when patients with dementia exhibit the hyperphagic problem of rapid eating , sr + m memory training is advised for long - term effectiveness . example of memory training protocols between sr group and sr + m group ( learn item : eating food bite by bite ) the training protocols between sr group and sr + m group only differ in phase ii . abbreviations : sr , spaced retrieval training group ; sr + m , spaced retrieval and montessori - based activities training group .

backgroundhyperphagia increases eating - associated risks for people with dementia and distress for caregivers . the purpose of this study was to compare the long - term effectiveness of spaced retrieval ( sr ) training and sr training combined with montessori activities ( sr + m ) for improving hyperphagic behaviors of special care unit residents with dementia.methodsthe study enrolled patients with dementia suffering from hyperphagia resident in eight institutions and used a cluster - randomized single - blind design , with 46 participants in the sr group , 49 in the sr + m group , and 45 participants in the control group . for these three groups , trained research assistants collected baseline data on hyperphagic behavior , pica , changes in eating habits , short meal frequency , and distress to caregivers . the sr and sr + m groups underwent memory training over a 6-week training period ( 30 sessions ) , and a generalized estimating equation was used to compare data of all the three groups of subjects obtained immediately after the training period and at follow - ups 1 month , 3 months , and 6 months later.resultsresults showed that the hyperphagic and pica behaviors of both the sr and sr + m groups were significantly improved ( p<0.001 ) and that the effect lasted for 3 months after training . the improvement of fast eating was significantly superior in the sr + m group than in the sr group . the improvement in distress to caregivers in both intervention groups lasted only until the posttest . improvement in changes in eating habits of the two groups was not significantly different from that of the control group.conclusionsr and sr + m training programs can improve hyperphagic behavior of patients with dementia . the sr + m training program is particularly beneficial for the improvement of rapid eating . caregivers can choose a suitable memory training program according to the eating problems of their residents .

Introduction Methods Participants Study design Interventions Measures Demographic characteristics Scale of hyperphagia in residents with dementia Scale of distress of the caregiver to hyperphagic behaviors Statistical analysis Results Discussion Limitations Conclusion Supplementary materials

the prevalence of hyperphagia in patients with dementia is 23%51% and tends to occur in patients in the middle stage of dementia.1,2 the main features of hyperphagia include increased appetite ( eg , frequent food searches or requests for food ) , faster eating speed , and increased amount of food consumed.1,3,4 hyperphagia is usually complicated with altered eating habits , inadequate eating behaviors , and pica ; this may easily lead to risks of upper airway obstruction , nutritional imbalance , or poisoning caused by erroneous eating and also increase the conflicts between caregivers and patients.48 the literature shows that the hyperphagic behaviors of patients with dementia may originate from damage to the frontotemporal lobe or an impaired satiety center in the ventromedial nucleus of the hypothalamus , thereby stimulating the neurological effects of starvation , or increasingly agitated behavior of patients with dementia , thereby requiring constant feeding to supplement calories.6,7,9,10 however , reports have also suggested that hyperphagia in patients with dementia is caused by memory degradation , which causes the patients to forget what food they have eaten or how to take the food accurately.11 non - declarative memory ( sometimes termed as implicit memory ) has always been the focus of dementia memory training . two approaches that can be used for training non - declarative memory are spaced retrieval ( sr ) and montessori - based activities.15,16 sr is a learning approach developed to facilitate recall of a variety of information in individuals with dementia . moreover , these activities can also provide sensory stimulation and movement coordination training to help the person with dementia to have a successful experience and create more control of behaviors within an orderly environment.25,26 both sr and montessori activities have been verified to alleviate the cognitive degeneration of patients with dementia , improve problem behaviors , and increase positive emotions and activities of daily living.23,2729 lin et al successfully used sr and montessori - based activities to improve eating difficulties in patients with dementia , and wu et al found that applying standardized and individualized sr in combination with montessori activities training could significantly improve eating difficulties in patients with dementia.30,31 the causes of eating difficulties in patients with dementia are similar to those of hyperphagia , including impaired functioning of the satiety center of hypothalamus , which causes patients to forget how to take , chew , and swallow food , as well as worsening the ability to use tableware . repeated provision of highly reinforcing food ( especially food with a high sugar and fat content ) can reinforce the social pleasure of the patients receiving training and constantly reinforce their behavior.3234 the aim of this study was to investigate and compare the long - term effectiveness of sr and sr + m on improving hyperphagic behaviors of special care unit residents with dementia . this was to establish whether memory training can still improve abnormal behaviors of patients with dementia without the reinforcement of food and to investigate the difference in improvement of eating - related behaviors between sr alone and sr training combined with montessori - based activities ( sr + m ) . this study had a longitudinal two - step cluster - randomized single - blind design and was conducted from june 2012 to october 2014 . after the institutions identified research subjects who met the inclusion criteria , trained research assistants collected pretest data of all the subjects and classified the severity of their hyperphagia as mild , moderate , or severe . the patients were then randomly assigned to the sr group ( 48 subjects ) , the sr + m group ( 52 subjects ) , and the control group ( 48 subjects ) . following completion of the interventions , the research assistants collected post - training test data and follow - up data at 1 month , 3 months , and 6 months for the subjects in all the three groups . a total of 140 subjects completed the entire research process and follow - up data collection , including 46 subjects in the sr group , 49 in the sr + m group , and 45 in the control group ( figure 1 ) . the objectives for the subjects were as follows : 1 ) to memorize where food is placed ( only food placed in bowls could be taken : four sessions ) ; 2 ) to memorize slowing down their dining speed ( chewing slowly , swallowing slowly , and eating the food bite by bite : eleven sessions ) ; 3 ) to memorize a satiation message ( stopping eating on hearing a 20-second melody and putting down the bowl : eight sessions ) the 20-second melody functions as a warning message to remind the patients with dementia to stop eating when hearing it ; in addition to being applied in classroom training , it was also played 40 minutes after three meals throughout the course of the 6-week intervention to enable all the residents to hear it ; however , it was no longer played after the 6 weeks were up and 4 ) to memorize appropriate dining etiquette behavior ( appropriate behavior while eating and interacting with other people : seven sessions ) . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1).the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1).the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . generalized estimating equation was used to analyze the changes from the pretraining test data in the post - training test and 1-month , 3-month , and 6-month follow - up data for all three groups of subjects . this study had a longitudinal two - step cluster - randomized single - blind design and was conducted from june 2012 to october 2014 . after the institutions identified research subjects who met the inclusion criteria , trained research assistants collected pretest data of all the subjects and classified the severity of their hyperphagia as mild , moderate , or severe . the patients were then randomly assigned to the sr group ( 48 subjects ) , the sr + m group ( 52 subjects ) , and the control group ( 48 subjects ) . following completion of the interventions , the research assistants collected post - training test data and follow - up data at 1 month , 3 months , and 6 months for the subjects in all the three groups . a total of 140 subjects completed the entire research process and follow - up data collection , including 46 subjects in the sr group , 49 in the sr + m group , and 45 in the control group ( figure 1 ) . the objectives for the subjects were as follows : 1 ) to memorize where food is placed ( only food placed in bowls could be taken : four sessions ) ; 2 ) to memorize slowing down their dining speed ( chewing slowly , swallowing slowly , and eating the food bite by bite : eleven sessions ) ; 3 ) to memorize a satiation message ( stopping eating on hearing a 20-second melody and putting down the bowl : eight sessions ) the 20-second melody functions as a warning message to remind the patients with dementia to stop eating when hearing it ; in addition to being applied in classroom training , it was also played 40 minutes after three meals throughout the course of the 6-week intervention to enable all the residents to hear it ; however , it was no longer played after the 6 weeks were up and 4 ) to memorize appropriate dining etiquette behavior ( appropriate behavior while eating and interacting with other people : seven sessions ) . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . during the recall interval , the trainers arranged interesting nonstructured activities for the subjects , such as drawing , reading a newspaper , or poker games ( the example is given in table s1 and figure s1).the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . trainers could adjust the complexity of activities according to the subject s learning condition ( the example is given in table s1 and figure s1).the control group : the control group engaged in the routine activities of institutions , with no particular memory training activities . if the subject forgot the message , the trainer retrained them to memorize it and returned to the previous recall interval for the subject to recall the message again and then adjusted the memory training interval backwards or forwards according to the subject s recall performance until the end of the training . the sr + m group : the same procedure described for sr memory training was followed ; however , in this group , the subjects engaged in action and practice - based structured montessori activities during the recall interval . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . in addition to collecting demographic information on age , sex , education , marital status , length of institutionalization , duration of dementia , and family visits , chinese versions of mini - mental state examination ( mmse ) and recall subscale of the mmse , barthel index , and cohen - mansfield agitation inventory were also used to evaluate separately the episodic memory , functional status , and 29 agitated behaviors of subjects because of their good internal consistency , interrater reliability , and concurrent validity.3537 a convenience sample of 21 formal and informal caregivers from day care centers and long - term care facilities were interviewed to determine excessive eating behaviors among elders with dementia . the observed frequencies for each of these hyperphagic behaviors were scored as follows : the subject did not engage in the hyperphagic behavior , 0 points ; one to three occurrences , 1 point ; four to six occurrences , 2 points ; and more than seven occurrences , 3 points . the internal consistency ( cronbach s ) and the interrater reliability ( intraclass correlation coefficient ) of this scale were 0.91 and 0.92 , respectively.38 during the observation period , record forms were also used to collect information on pica , changes in eating habits , and short meal frequency . to avoid the hawthorne effect during the collection of observation data , this study used 68 subjects on the same table as that of the special care unit to perform regional , positional , and whole - day observation , instead of observing the subjects only at meal times in the observation area . generalized estimating equation was used to analyze the changes from the pretraining test data in the post - training test and 1-month , 3-month , and 6-month follow - up data for all three groups of subjects . this study used a generalized estimating equation to compare all the 6-month follow - up data of the three groups . the results showed that the scores of the sr group for hyperphagic behavior and pica continued to decrease for 3 months and the improvement in short meal frequency lasted for 1 month after memory training . for the sr + m group , the improvements in hyperphagic behavior and short meal frequency lasted for 6 months after training . the improvement in distress to the primary caregiver of both the sr and sr + m groups only lasted until the post - training test . analyses of the effectiveness of improvement in frequency of the four categories behaviors of hyperphagia ( intention to eat , rapid eating , increased eating , and inappropriate dining etiquette ) show that the behaviors of rapid eating and increased eating improved in both the sr and sr + m groups . the improvement lasted for a longer period of time in the sr + m group ( table 3 ) . however , sr and sr + m training programs can improve recall memory performance of patients with dementia that lasted for at least 1 month after training ( table 4 ) . the best recall performance in both groups was during sessions for training objective 2 ( to memorize slowing down the dining speed ) ; the performance of the sr + m group was significantly better than that of the sr group ( t=2.052 , p=0.044 ) , particularly in the training for chewing slowly lee et al examined whether sr training could improve cognitive function in people with very mild to mild dementia , but no change in neuropsychological performance was found.39 it was believed that factors leading to this result included a low cognitive effort required for sr training , and the small sample size of their study . on the other hand , this study found that both the sr and sr + m memory training can improve the hyperphagic behavior of patients with dementia without the need for food incentives . this finding is consistent with that of the study by lin et al30 and wu et al,31 suggesting that sr and montessori activities can improve dysphagia of patients with dementia . the longest recall interval of subjects participating in this study was 31 minutes , which is almost the same as that of patients with dementia in studies by lee et al and wu et al.39,40 thus , non - declarative memory training can be used for patients with moderate dementia , and it can help improve eating behaviors of patients with dementia.12,26,41,42 even though this study revealed positive effects of memory training , every patient with hyperphagia had to receive 40 minutes of memory training , five times per week for a total of 30 times . the caregivers could confirm the suitable time for the daily training of patients with dementia and then referred to the protocols designed in this study to provide one - on - one memory training . the training results showed that the hyperphagic behaviors that were significantly improved were rapid eating , increased eating , and pica . therefore , if the inadequate behaviors of patients with dementia are deconstructed ( breaking tasks into small components and starting with easier items ) , it will be easier for improvement by providing continuous training for specific actions.43 montessori activities are structured activities that involve everyday practical tasks and engage the learning - by - doing system , thus it can provide patients with dementia with the continuous practice required by procedural memory . beyond that , these activities are designed with rich , tangible sensory stimulation to improve patient s motor coordination , sense of reality , and sense of control over surroundings.16,44,45 therefore , montessori activities are beneficial for learning in patients with dementia , which was reflected in our study results : the slowing down of dining speed was significantly better in the sr + m group than in the sr group , and the improvements in dining speed and short meal frequency also lasted longer in the sr + m group . once the problem of short meal times was improved , the meal time of a greater number of patients with dementia increased , which would in turn be expected to increase the need for the company of caregivers or the provision of dining assistance.30 this may be the reason why the distress of caregivers was not continuously improved . after memory training , there was no significant change in eating habits in the three groups . changes in eating habits are more common in patients with frontotemporal dementia or severe semantic dementia and may also be associated with hallucinations , change in taste perception , or antipsychotic drugs used to treat severe agitated behaviors.10,11,4850 this study mainly enrolled patients with ad who had fewer behavioral and psychiatric symptoms and moderate cognitive impairment . second , due to the limitations of the institutionalized meal supply culture ( a fixed amount of food is provided at a fixed time or the food source is strictly limited ) , it could be expected that the observed hyperphagic behaviors of residents with dementia were underestimated . the effect of memory training programs on improving hyperphagic behavior of patients with dementia living in a community should be explored further . therefore , the training in this study did not focus on restricting the food intake of patients with dementia but on improving hyperphagic behaviors instead . this study found that sr and sr + m memory training were non - pharmacological interventions that could improve hyperphagic behaviors of patients with dementia and significantly improve rapid eating , increased eating , and pica behavior . when patients with dementia exhibit the hyperphagic problem of rapid eating , sr + m memory training is advised for long - term effectiveness . example of memory training protocols between sr group and sr + m group ( learn item : eating food bite by bite ) the training protocols between sr group and sr + m group only differ in phase ii .

addiction to illicit drug is a serious public health concern with a global prevalence of 3.3% to 6.6% among the adult population ( 1 ) . although , men are more likely to use drugs compared to women , this does not necessarily reflect a lower risk for women ( 2 - 4 ) . however , drug treatment centers are often focused on men and less women are enrolled in and receive services through these centres . while several socio - cultural factors could be at play , a part of this disproportionate enrollment can be explained by the higher prevalence of drug use , involvement in drug - related activities , and therefore higher referral rates of men to these centres ( 4 ) . social role theory highlights that men and women act based on different stereotypes that determine their social roles and expectations . dominant notions of femininity ( e.g. , responsibility of taking care of the family ) and masculinity ( e.g. , macho attitudes ) often influence men s and women s risk taking behaviors ( 5 ) . gender - based differences can not be overlooked when it comes to the pathways to substance use and misuse . dissimilarities in men s and women s drug addiction patterns could be due to a number of socio - cultural , biological , and mental factors ( 4 , 6 ) . for example , risk aversion behaviors vary across the genders due to the effect of nature versus nurture ; men are more likely to take risks while women are often promoted to lead a cautious lifestyle due to their personal preferences or socio - cultural norms and expectations ( 7 ) . the motives and underlying reasons that drive women to illicit drug use and addiction may be different from those in men . while men are mainly introduced to drug abuse through their peer and friendship networks , women may start using drugs due to familial matters and the influence of their intimate relationships ( 3 , 6 ) . men and women are also attracted to different types of drugs ; for example , men are more likely to smoke marijuana , while women are more likely to abuse alcohol and prescription drugs , such as benzodiazepines and sedatives ( 3 , 6 ) . moreover , men and women may abuse drugs to fulfill different desires ( 4 ) . in the middle east and north africa ( mena ) region which faces a large drug use problem , our understanding of gendered experiences of exposure to illicit drug use remains limited ( 8) . among mena countries , iran has one of the largest drug using population , partly due to its long borders with afghanistan and pakistan , two major producers of illicit drugs . although drug use is common in iran , some provinces seem to have a significantly higher rate of drug abuse in comparison with the rest of the country . for example , the kerman province , located in southeast iran , has the highest rate of opium use in the county . drug related problems , in iran s largest province , are usually explained by its proximity to the afghanistan and pakistan borders , as well as the cultural acceptability of opium smoking . based on a recent study , residents of kerman have been ranked as those with the highest acceptance rate of opium use , both subjectively and objectively ( 9 ) . given the paucity of evidence on the gendered - experiences of exposure to drug use in this region , and a possible unique pattern due to the different socio - economic and cultural situation , a qualitative study was carried out on male and female people who use drugs ( pwud ) in a drop - in center ( dic ) and hiv triangulation clinic in kerman . due to the hard - to - reach nature of pwud , particularly women , the stigma attached to addiction , and the criminalization of drug possession in iran , we chose our samples from an hiv triangulation center and a dic for women . the triangular hiv center was established in kerman in 2004 to provide specific services exclusively to people living with hiv . these services include counseling , treatment , and methadone maintenance therapy . as the sample of women at the hiv triangulation center who had a history of drug use this center is the only one to provide services to vulnerable women who are either drug - addicted , sex workers or both ; it provides specific harm reduction services such as methadone therapy , distributing free condoms , serving free meals , and counseling . due to participants ' reluctance to disclose their demographic characteristics , we were not able to collect precise information in this regard . however we know that they were all young or middle aged and had limited years of education . in this qualitative study , a convenience sample of participants ( men and women ) were recruited between september and november 2011 . we prepared an interview guide on the main areas of the study , exploring the high risk behaviours associated with hiv infection , ( e.g. , risky drug use and sexual practices ) . regarding drug use , we asked the participants to share their history of using drugs . we probed the experiences of the participants , their route of drug use and the influential people who encouraged them to use drugs for the first time . interviews were conducted in persian and all participants received a non - monetary incentive of food worth us$ 8 . data were collected through in - depth interviews that took approximately 45 to 60 minutes to complete . interviews were transcribed on the day of the interview and and data were analyzed using thematic analysis ( 10 ) . to analyses the data , initial codes were extracted and categorized to form the main categories and themes of the study . themes and categories were finalized through a group discussion with co - authors . the proposal of this study was reviewed and approved by the ethics committee of kerman university of medical sciences . the participants were given information about the aim of our study and how our results would be disseminated . all interviews were conducted in a private room in the hiv center , where no one else could hear the conversations all participants consented verbally to participate and were reassured that they could skip any question they felt uncomfortable with or stop the interview whenever they desired . data were coded and kept on a password - secured desktop computer , and all audio files were destroyed two weeks after the final analysis . due to the hard - to - reach nature of pwud , particularly women , the stigma attached to addiction , and the criminalization of drug possession in iran , we chose our samples from an hiv triangulation center and a dic for women . the triangular hiv center was established in kerman in 2004 to provide specific services exclusively to people living with hiv . these services include counseling , treatment , and methadone maintenance therapy . as the sample of women at the hiv triangulation center who had a history of drug use this center is the only one to provide services to vulnerable women who are either drug - addicted , sex workers or both ; it provides specific harm reduction services such as methadone therapy , distributing free condoms , serving free meals , and counseling . due to participants ' reluctance to disclose their demographic characteristics , we were not able to collect precise information in this regard . however we know that they were all young or middle aged and had limited years of education . in this qualitative study , a convenience sample of participants ( men and women ) were recruited between september and november 2011 . we prepared an interview guide on the main areas of the study , exploring the high risk behaviours associated with hiv infection , ( e.g. , risky drug use and sexual practices ) . regarding drug use , we asked the participants to share their history of using drugs . we probed the experiences of the participants , their route of drug use and the influential people who encouraged them to use drugs for the first time . interviews were conducted in persian and all participants received a non - monetary incentive of food worth us$ 8 . no personal information or any traceable information were obtained . data were collected through in - depth interviews that took approximately 45 to 60 minutes to complete . interviews were transcribed on the day of the interview and and data were analyzed using thematic analysis ( 10 ) . to analyses the data , initial codes were extracted and categorized to form the main categories and themes of the study . the proposal of this study was reviewed and approved by the ethics committee of kerman university of medical sciences . the participants were given information about the aim of our study and how our results would be disseminated . all interviews were conducted in a private room in the hiv center , where no one else could hear the conversations all participants consented verbally to participate and were reassured that they could skip any question they felt uncomfortable with or stop the interview whenever they desired . data were coded and kept on a password - secured desktop computer , and all audio files were destroyed two weeks after the final analysis . participants preferred to mention their approximate age only ; however , they aged from their mid-20s to their early 50s . most of them were single or divorced and unemployed or in unstable jobs with low incomes . at the time of the interviews , the majority of the participants were living on their own or staying with friends . most had been born into low- or middle - income families and had not completed high school . opium , crystal meth , and heroin were the most frequently reported drugs and most participants had started drugs when they were young . most men ( 13 out of 15 ) in the study were single and had started smoking cigarettes before using drugs . conversely , most women were married ( 13 out of 14 ) when they started using drugs , and had started drug use with opium . only one single woman had begun drug use with heroin . also , except a small proportion of women who had later shifted to crystal meth and heroin , others continued to use opium . on the other hand , men reported a different pattern ; they had started with a light substance ( i.e. , smoking cigarettes ) , and ended up using chemical ( e.g. , crystal meth ) and heavy substances ( e.g. , heroin ) . a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . moreover , male interviewee 3 reported : when my friend offered me to smoke a cigarette , i did . male interviewee 1 who had first experienced smoking with his friend , mentioned starting smoking to look manly and strong . we [ him and his friend ] were in a park and he [ his friend ] lit a cigarette and gave it to me , come on ! i was surprised ; he said " do not be scared " , he sounded okay and comfortable with smoking . his brother and my brother were also in the park , so i did not want to wimp out . he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . i was scared , but i just could not say no to him ! a number of participants used drugs or smoked to cope with their painful emotions . for example , male interviewee 2 whose wife had concealed her genetic disease before marriage and passed it on to their son , stated : when i realized my son has a genetic disease , it was so upsetting . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . my leg was painful , and my husband told me that opium is good for leg pain and back pain . female interviewee 5 who had a drug using mother also stated : i was working hard and my mother [ who was addicted as well ] told me it was good for me to use opium and refresh myself and improve my general health . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . they told me to use opium , because it was good for my sutures and pain . some participants however , reported higher work performance as a motive to start drug use . for instance , female interviewee 3 stated : my aunt told me that opium is good for you , it numbs your pain , it is good for leg pain , it will refresh you and you can work harder . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . female interviewee 7 declared : i was with my boyfriend , whom i married later on . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . they [ relatives ] told me that opium is good for me and would calm me down . for some women , drawing attention from others was the main cause for drug use initiation . female interviewee 6 , a former prisoner outlined : when i was in prison , i asked my husband to forgive me and stay with me ; however , he refused to do so . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . whenever my husband would not look for a job or work , i would give him a hard time about our financial struggles . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . participants preferred to mention their approximate age only ; however , they aged from their mid-20s to their early 50s . most of them were single or divorced and unemployed or in unstable jobs with low incomes . at the time of the interviews , the majority of the participants were living on their own or staying with friends . most had been born into low- or middle - income families and had not completed high school . opium , crystal meth , and heroin were the most frequently reported drugs and most participants had started drugs when they were young . most men ( 13 out of 15 ) in the study were single and had started smoking cigarettes before using drugs . conversely , most women were married ( 13 out of 14 ) when they started using drugs , and had started drug use with opium . only one single woman had begun drug use with heroin . also , except a small proportion of women who had later shifted to crystal meth and heroin , others continued to use opium . on the other hand , men reported a different pattern ; they had started with a light substance ( i.e. , smoking cigarettes ) , and ended up using chemical ( e.g. , crystal meth ) and heavy substances ( e.g. , heroin ) . a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . men s reasons for drug use initiation ranged from curiosity to peer pressure . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . moreover , male interviewee 3 reported : when my friend offered me to smoke a cigarette , i did . male interviewee 1 who had first experienced smoking with his friend , mentioned starting smoking to look manly and strong . we [ him and his friend ] were in a park and he [ his friend ] lit a cigarette and gave it to me , come on ! i was surprised ; he said " do not be scared " , he sounded okay and comfortable with smoking . his brother and my brother were also in the park he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . i was scared , but i just could not say no to him ! a number of participants used drugs or smoked to cope with their painful emotions . for example , male interviewee 2 whose wife had concealed her genetic disease before marriage and passed it on to their son , stated : when i realized my son has a genetic disease , it was so upsetting . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . my leg was painful , and my husband told me that opium is good for leg pain and back pain . female interviewee 5 who had a drug using mother also stated : i was working hard and my mother [ who was addicted as well ] told me it was good for me to use opium and refresh myself and improve my general health . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . they told me to use opium , because it was good for my sutures and pain . some participants however , reported higher work performance as a motive to start drug use . for instance , female interviewee 3 stated : my aunt told me that opium is good for you , it numbs your pain , it is good for leg pain , it will refresh you and you can work harder . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . female interviewee 7 declared : i was with my boyfriend , whom i married later on . he prepared the stuff and asked me to use heroin . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . they [ relatives ] told me that opium is good for me and would calm me down . for some women , drawing attention from others was the main cause for drug use initiation . female interviewee 6 , a former prisoner outlined : when i was in prison , i asked my husband to forgive me and stay with me ; however , he refused to do so . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . whenever my husband would not look for a job or work , i would give him a hard time about our financial struggles . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . moreover , male interviewee 3 reported : when my friend offered me to smoke a cigarette , i did . male interviewee 1 who had first experienced smoking with his friend , mentioned starting smoking to look manly and strong . we [ him and his friend ] were in a park and he [ his friend ] lit a cigarette and gave it to me , come on ! i was surprised ; he said " do not be scared " , he sounded okay and comfortable with smoking . his brother and my brother were also in the park , so i did not want to wimp out . he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . i was scared , but i just could not say no to him ! a number of participants used drugs or smoked to cope with their painful emotions . for example , male interviewee 2 whose wife had concealed her genetic disease before marriage and passed it on to their son , stated : when i realized my son has a genetic disease , it was so upsetting . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . my leg was painful , and my husband told me that opium is good for leg pain and back pain . female interviewee 5 who had a drug using mother also stated : i was working hard and my mother [ who was addicted as well ] told me it was good for me to use opium and refresh myself and improve my general health . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . they told me to use opium , because it was good for my sutures and pain . some participants however , reported higher work performance as a motive to start drug use . for instance , female interviewee 3 stated : my aunt told me that opium is good for you , it numbs your pain , it is good for leg pain , it will refresh you and you can work harder . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . female interviewee 7 declared : i was with my boyfriend , whom i married later on . he prepared the stuff and asked me to use heroin . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . they [ relatives ] told me that opium is good for me and would calm me down . for some women , drawing attention from others was the main cause for drug use initiation . female interviewee 6 , a former prisoner outlined : when i was in prison , i asked my husband to forgive me and stay with me ; however , he refused to do so . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . whenever my husband would not look for a job or work , i would give him a hard time about our financial struggles . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . the results of our study suggest that social roles and stereotypes which define the expected gendered behaviours , could create different drug use patterns and practices across men and women . gender differences are not only observed in drug types amongst men and women , but are also present in their motivations or reasons of drug use initiation . such differences could originate from socio - cultural factors or physiological differences across genders . in the current study , men were mainly single when they started smoking cigarettes or using drugs , however , most women were married . in the conservative and patriarchal context of iran , compared to men , single young women are often subject to more restrictions and supervisions ; reducing their chances of accessing drugs . on the other hand , once they get married , they may be encouraged by their partners or their families , to start using drugs . previous studies support the idea that women s decisions to start drug use are mainly influenced by their relationships with men ( 11 ) . the impact of a man as a husband on a woman s decision regarding drug use could be explained through a complex combination of love , power , and women s status ( 12 ) . nonetheless , family influence can be both negative and positive for women . while some women might start using drugs under pressure from their husbands , others might cease drug use for the sake of their family ( 11 ) . furthermore , different popularity and social acceptance patterns could impact substance abuse practices across genders . the ways in which boys and girls behave to win popularity and social acceptance often vary . for girls , parental socio - economic status and their own physical appearance matter the most , socialization can affect people s behaviors through different mechanisms , including learning from observation and self - concepts , ( i.e , adopting behaviors consistent with a group identity ) . however , whether people start smoking due to peer pressure is the subject of controversy . some studies acknowledge the importance of peer pressure on smoking initiation ( 14 ) , but others argue that it is not a strong predictor for commencing smoking . they suggest identity statements and coolness as important determinants of smoking debut ( 14 ) . in our study , women did not report showing off as a motive for drug use debut ; however , other studies have observed showing off to play a role in smoking initiation among young women ( 15 ) . this could also be the case in iran where about 93.5% of pwud had smoked cigarettes before they started illicit drug use ( 17 ) . however , consistent with our findings , the evidence suggest a different gateway to drug use debut among women where most iranian women who use drugs have often begun with opium ( 17 ) . in iran , several studies have shown boys to be more likely to smoke cigarettes than girls ( 13 , 18 ) . this could stem from different gendered expecatations and socio - cultural norms around smoking cigarettes that disapprove women s cigarette smoking but show a tolerance for men who smoke ( 19 ) . the degree of acceptance of drug use in iran could also be rooted in the belief in the usefulness and benefits of drugs . in our study in particular , women started opium use mainly to alleviate physical pain and refresh themselves ; a finding that could be attributed to the popularity and acceptability of opium in our study setting ( i.e. , kerman ) . based on a study conducted in iran , among iranian people , residents of kerman had a high acceptance of opium use , both subjectively and objectively ( 9 ) . this could point to the difference between men s and women s perceptions of risk . men often evaluate the outcome of a risky behavior less critically and enjoy engaging in risky behaviors more than women ( 20 ) . overall , the pathology of drug abuse in iran is a multifaceted problem and requires a multi - dimensional policy across different public health and law enforcement sectors . different experiences of men and women in entry into illicit drug use calls for gender - specific harm reduction approaches and polices . educating families on the adverse health - related effects of drug use could be a starting point in initiating a socio - cultural shift . youth should also be empowered and educated with a set of skills to say no to drugs . as men have been at the centre of attention in most drug - related policies , scaling up specific interventions catered towards women who use drugs our participants belonged to low- or middle - income families and often had a history of incarceration and drug injection . therefore , the results of this study in two single centres in kerman have limited generalizability to other demographics of pwud across the country . however , given the hard - to - reach nature of this population , female pwud in particular , the current study does contribute to our understanding of different experiences of entry into drug use across men and women in the context of iran .

backgroundaddiction is characterized differently among women and men , and they begin using drugs for different reasons and motives.objectivesthe aim of the study was to explore the gendered experiences and patterns of illicit drug use initiation in an iranian context.patients and methodsa total of 29 participants ( 15 men and 14 women ) took part in in - depth interviews conducted at a hiv triangulation clinic ( for men and women ) and drop - in - center for women in kerman in 2011.resultsthe results of the study suggest that patterns of drug use are different among iranian men and women . men often transit to drug use from cigarette smoking , whereas women s drug use practices often begins with opium . unlike women , men who used drugs were often single at their drug use debut.conclusionsdifferent patterns of first exposure to drug use among men and women highlight the role of gendered expectations and socio - cultural norms in shaping drug use experiences of people who use drugs and call for gender - specific harm reduction interventions .

1. Background 2. Objectives 3. Patients and Methods 3.1. Study Setting 3.2. Data Collection 3.3. Data Analysis 3.4. Ethical Considerations 4. Results 4.1. Study Participants 4.2. Main Themes of the Study 4.2.1. Motivations to Use Drug 5. Discussion

addiction to illicit drug is a serious public health concern with a global prevalence of 3.3% to 6.6% among the adult population ( 1 ) . although , men are more likely to use drugs compared to women , this does not necessarily reflect a lower risk for women ( 2 - 4 ) . however , drug treatment centers are often focused on men and less women are enrolled in and receive services through these centres . while several socio - cultural factors could be at play , a part of this disproportionate enrollment can be explained by the higher prevalence of drug use , involvement in drug - related activities , and therefore higher referral rates of men to these centres ( 4 ) . social role theory highlights that men and women act based on different stereotypes that determine their social roles and expectations . , responsibility of taking care of the family ) and masculinity ( e.g. , macho attitudes ) often influence men s and women s risk taking behaviors ( 5 ) . gender - based differences can not be overlooked when it comes to the pathways to substance use and misuse . dissimilarities in men s and women s drug addiction patterns could be due to a number of socio - cultural , biological , and mental factors ( 4 , 6 ) . for example , risk aversion behaviors vary across the genders due to the effect of nature versus nurture ; men are more likely to take risks while women are often promoted to lead a cautious lifestyle due to their personal preferences or socio - cultural norms and expectations ( 7 ) . the motives and underlying reasons that drive women to illicit drug use and addiction may be different from those in men . while men are mainly introduced to drug abuse through their peer and friendship networks , women may start using drugs due to familial matters and the influence of their intimate relationships ( 3 , 6 ) . men and women are also attracted to different types of drugs ; for example , men are more likely to smoke marijuana , while women are more likely to abuse alcohol and prescription drugs , such as benzodiazepines and sedatives ( 3 , 6 ) . moreover , men and women may abuse drugs to fulfill different desires ( 4 ) . in the middle east and north africa ( mena ) region which faces a large drug use problem , our understanding of gendered experiences of exposure to illicit drug use remains limited ( 8) . among mena countries , iran has one of the largest drug using population , partly due to its long borders with afghanistan and pakistan , two major producers of illicit drugs . although drug use is common in iran , some provinces seem to have a significantly higher rate of drug abuse in comparison with the rest of the country . given the paucity of evidence on the gendered - experiences of exposure to drug use in this region , and a possible unique pattern due to the different socio - economic and cultural situation , a qualitative study was carried out on male and female people who use drugs ( pwud ) in a drop - in center ( dic ) and hiv triangulation clinic in kerman . due to the hard - to - reach nature of pwud , particularly women , the stigma attached to addiction , and the criminalization of drug possession in iran , we chose our samples from an hiv triangulation center and a dic for women . the triangular hiv center was established in kerman in 2004 to provide specific services exclusively to people living with hiv . these services include counseling , treatment , and methadone maintenance therapy . as the sample of women at the hiv triangulation center who had a history of drug use this center is the only one to provide services to vulnerable women who are either drug - addicted , sex workers or both ; it provides specific harm reduction services such as methadone therapy , distributing free condoms , serving free meals , and counseling . in this qualitative study , a convenience sample of participants ( men and women ) were recruited between september and november 2011 . we prepared an interview guide on the main areas of the study , exploring the high risk behaviours associated with hiv infection , ( e.g. , risky drug use and sexual practices ) . regarding drug use , we asked the participants to share their history of using drugs . we probed the experiences of the participants , their route of drug use and the influential people who encouraged them to use drugs for the first time . data were collected through in - depth interviews that took approximately 45 to 60 minutes to complete . interviews were transcribed on the day of the interview and and data were analyzed using thematic analysis ( 10 ) . to analyses the data , initial codes were extracted and categorized to form the main categories and themes of the study . the participants were given information about the aim of our study and how our results would be disseminated . data were coded and kept on a password - secured desktop computer , and all audio files were destroyed two weeks after the final analysis . due to the hard - to - reach nature of pwud , particularly women , the stigma attached to addiction , and the criminalization of drug possession in iran , we chose our samples from an hiv triangulation center and a dic for women . the triangular hiv center was established in kerman in 2004 to provide specific services exclusively to people living with hiv . these services include counseling , treatment , and methadone maintenance therapy . as the sample of women at the hiv triangulation center who had a history of drug use this center is the only one to provide services to vulnerable women who are either drug - addicted , sex workers or both ; it provides specific harm reduction services such as methadone therapy , distributing free condoms , serving free meals , and counseling . in this qualitative study , a convenience sample of participants ( men and women ) were recruited between september and november 2011 . we prepared an interview guide on the main areas of the study , exploring the high risk behaviours associated with hiv infection , ( e.g. , risky drug use and sexual practices ) . regarding drug use , we asked the participants to share their history of using drugs . we probed the experiences of the participants , their route of drug use and the influential people who encouraged them to use drugs for the first time . data were collected through in - depth interviews that took approximately 45 to 60 minutes to complete . interviews were transcribed on the day of the interview and and data were analyzed using thematic analysis ( 10 ) . to analyses the data , initial codes were extracted and categorized to form the main categories and themes of the study . the proposal of this study was reviewed and approved by the ethics committee of kerman university of medical sciences . the participants were given information about the aim of our study and how our results would be disseminated . data were coded and kept on a password - secured desktop computer , and all audio files were destroyed two weeks after the final analysis . at the time of the interviews , the majority of the participants were living on their own or staying with friends . opium , crystal meth , and heroin were the most frequently reported drugs and most participants had started drugs when they were young . most men ( 13 out of 15 ) in the study were single and had started smoking cigarettes before using drugs . conversely , most women were married ( 13 out of 14 ) when they started using drugs , and had started drug use with opium . only one single woman had begun drug use with heroin . on the other hand , men reported a different pattern ; they had started with a light substance ( i.e. , smoking cigarettes ) , and ended up using chemical ( e.g. , crystal meth ) and heavy substances ( e.g. a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . a number of participants used drugs or smoked to cope with their painful emotions . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . my leg was painful , and my husband told me that opium is good for leg pain and back pain . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . some participants however , reported higher work performance as a motive to start drug use . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . for some women , drawing attention from others was the main cause for drug use initiation . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . at the time of the interviews , the majority of the participants were living on their own or staying with friends . opium , crystal meth , and heroin were the most frequently reported drugs and most participants had started drugs when they were young . most men ( 13 out of 15 ) in the study were single and had started smoking cigarettes before using drugs . conversely , most women were married ( 13 out of 14 ) when they started using drugs , and had started drug use with opium . only one single woman had begun drug use with heroin . on the other hand , men reported a different pattern ; they had started with a light substance ( i.e. , smoking cigarettes ) , and ended up using chemical ( e.g. a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . men s reasons for drug use initiation ranged from curiosity to peer pressure . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . his brother and my brother were also in the park he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . a number of participants used drugs or smoked to cope with their painful emotions . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . my leg was painful , and my husband told me that opium is good for leg pain and back pain . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . some participants however , reported higher work performance as a motive to start drug use . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . for some women , drawing attention from others was the main cause for drug use initiation . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . a majority of men were encouraged to smoke and use drugs by their friends , and only a few of them had commenced drug use on their own . for example , male interviewee 2 stated : my parents were drug users , and i had frequently seen them smoke . he said " do n't be a sissy " , and teased me , so i tried it . some interviewees reported a lack of power to say no as a major reason of commencing their drug use . a number of participants used drugs or smoked to cope with their painful emotions . the results of our study show that women were encouraged to use drugs through different mechanisms than men . women s friends did not have a considerable role in encouraging them to initiate drug use ; however , they had often been introduced to drug use through their husbands , in - laws , or own families . for example , female interviewee 4 reported physical pain as a reason for her first exposure to drugs : i fell over and hurt myself . moreover , female interviewee 1 talked about how she had started drug use because of her difficult delivery : when my first baby was born , they [ her relatives ] said that doctors are useless and would cause you trouble . my mother - in - law and my sister - in - law called a traditional midwife , and i had a very difficult delivery . some participants however , reported higher work performance as a motive to start drug use . furthermore , similar to a subgroup of men , some women used drugs because they did not have the power to say no to a friend or an intimate partner who had offered them drugs . i was scared , so he helped me and taught me how to use drugs . in addition , emotional pain was another reason for starting drug use among men and women . for some women , drawing attention from others was the main cause for drug use initiation . i told him that if he would not come back to me i would use drugs but he did not seem to care . interestingly , two women who were introduced to drugs by their husband or in - laws reported that they were encouraged to use drugs by their husband to avoid bothering them about their job and financial instabilities . for example , female interviewee 8 stated : it was at my wedding night when my father in - law offered me drugs . once i heard my father in - law told my husband : if she becomes a drug addict , she would neither bug you anymore nor leave you . lastly , female interviewee 9 had gone through a similar situation : my husband encouraged me to use drugs , as i would always fight with him over his unemployment . the results of our study suggest that social roles and stereotypes which define the expected gendered behaviours , could create different drug use patterns and practices across men and women . gender differences are not only observed in drug types amongst men and women , but are also present in their motivations or reasons of drug use initiation . such differences could originate from socio - cultural factors or physiological differences across genders . in the current study , men were mainly single when they started smoking cigarettes or using drugs , however , most women were married . in the conservative and patriarchal context of iran , compared to men , single young women are often subject to more restrictions and supervisions ; reducing their chances of accessing drugs . on the other hand , once they get married , they may be encouraged by their partners or their families , to start using drugs . previous studies support the idea that women s decisions to start drug use are mainly influenced by their relationships with men ( 11 ) . the impact of a man as a husband on a woman s decision regarding drug use could be explained through a complex combination of love , power , and women s status ( 12 ) . nonetheless , family influence can be both negative and positive for women . while some women might start using drugs under pressure from their husbands , others might cease drug use for the sake of their family ( 11 ) . for girls , parental socio - economic status and their own physical appearance matter the most , socialization can affect people s behaviors through different mechanisms , including learning from observation and self - concepts , ( i.e , adopting behaviors consistent with a group identity ) . in our study , women did not report showing off as a motive for drug use debut ; however , other studies have observed showing off to play a role in smoking initiation among young women ( 15 ) . this could also be the case in iran where about 93.5% of pwud had smoked cigarettes before they started illicit drug use ( 17 ) . however , consistent with our findings , the evidence suggest a different gateway to drug use debut among women where most iranian women who use drugs have often begun with opium ( 17 ) . this could stem from different gendered expecatations and socio - cultural norms around smoking cigarettes that disapprove women s cigarette smoking but show a tolerance for men who smoke ( 19 ) . the degree of acceptance of drug use in iran could also be rooted in the belief in the usefulness and benefits of drugs . based on a study conducted in iran , among iranian people , residents of kerman had a high acceptance of opium use , both subjectively and objectively ( 9 ) . this could point to the difference between men s and women s perceptions of risk . men often evaluate the outcome of a risky behavior less critically and enjoy engaging in risky behaviors more than women ( 20 ) . overall , the pathology of drug abuse in iran is a multifaceted problem and requires a multi - dimensional policy across different public health and law enforcement sectors . different experiences of men and women in entry into illicit drug use calls for gender - specific harm reduction approaches and polices . educating families on the adverse health - related effects of drug use could be a starting point in initiating a socio - cultural shift . as men have been at the centre of attention in most drug - related policies , scaling up specific interventions catered towards women who use drugs our participants belonged to low- or middle - income families and often had a history of incarceration and drug injection . therefore , the results of this study in two single centres in kerman have limited generalizability to other demographics of pwud across the country . however , given the hard - to - reach nature of this population , female pwud in particular , the current study does contribute to our understanding of different experiences of entry into drug use across men and women in the context of iran .

periodontosis is a serious gum infection , which affects the supporting structure of teeth such as gums , periodontal ligaments , alveolar bones , and dental cementum . periodontosis is caused by the bacteria that stick to the surface of tooth and multiply . toxins produced by these bacteria in plaque , formed below the gum line , irritate the gums and stimulate an inflammatory response . this results in progressive loss of alveolar bone round the teeth and thus forms the pockets ( spaces between the teeth and gums ) that become infected . as the disease progresses , the pockets deepen and more gum tissues and bones are destroyed . this scaling procedure needs the application of local and nerve block / infiltration anesthesia by painful needle therapy . even though topical anesthetic gels are easy to apply , they possess some drawbacks such as tendency to spread in other areas or lower retention in plaque area , thus causing numbness of lips , tongue , and cheeks and chances of swallowing of the gel . to improve the residence time , in situ gels show promising effect . in situ gel stays at application site due to increased viscosity and mucoadhesiveness and shows fast onset of action . lutrol is a triblock polymer , consists of polyoxyethylene - polyoxypropylene - polyoxyethylene units , as shown in figure 1(c ) , and forms micelles at low concentration and clear thermoreversible gel at a high concentration . the concentrated solution of lutrol f127 ( 1630% ) gets transformed from low viscosity transparent solution at 5c to a solid on heating at body temperature . formulation which consisted of lutrol f127 forms a gel in periodontal pocket at a body temperature by modulating the gelation temperature . it is used both internally and externally in various products that are designed for animal and human use . the dental gel can be easily rinsed out with water to stop the anesthetic effect after the treatment . lidocaine hydrochloride ( lh ) ( figure 1(a ) ) is the first amino amide type of local anesthetics and has been in use for many years . in dentistry , it is a drug of choice to temporarily anesthetize the tiny nerve endings located on the surfaces of the oral mucosa . as a local anesthetic , lidocaine is characterized by a rapid onset of action and intermediate duration of efficacy , making it suitable for infiltration and nerve block anesthesia . lidocaine stabilizes the neuronal membrane by inhibiting the ionic fluxes required for the initiation and conduction of impulses , thereby effecting local anesthetic action . tamarind seed polysaccharide ( tsp ) is a high - molecular - weight , branched polysaccharide and consists of celluloselike backbone that carries xylose and galactoxylose substances , as shown in figure 1(b ) . it is insoluble in organic solvents and dispersible in warm water to form a highly viscous mucilaginous gel with a broad ph tolerance and mucoadhesivity . it is a galactoxyloglucan and possesses properties such as mucomimetic , mucoadhesive , and pseudoplastic properties . tsp has been used for development of bioadhesive drug delivery systems owing to their bioadhesive properties . it has been studied earlier for thermoreversible gelation property and also as mucoadhesive component in mucoadhesive buccal patches for controlled release . tsp is used as mucoadhesive polysaccharide polymer for systemic delivery of rizatriptan benzoate through buccal route , formulated in the form of buccal film . the objective of the present study was to develop thermoreversible in situ gel of local anesthetic lh by using xyloglucan based mucoadhesive polymer tsp for insertion into periodontal pocket to have painless treatment . the seeds of tamarindus indica were washed with water to remove the dirt and adhering material . the seeds were slightly roasted in sand and crushed to remove the outer brownish testa . soaked seeds in water ( 24 h ) were boiled for 1 h and kept aside for 2 h to liberate sufficient mucilage into water . mucilage was removed from the marc by squeezing the soaked seeds through the muslin cloth . the mucilage was then isolated with equal quantity of acetone and dried at 50c , powdered , and passed through sieve number 80 to get uniform size fine powder of tsp . accurately weighed , dried , and finely powdered tsp ( 1 g ) was suspended in 75 ml of distilled water for 5 h. volume was made up to 100 ml to produce the concentration of 1% w / v . the mixture was homogenized by mechanical stirrer for 2 h and viscosity was determined at 500 rpm using spindle 61 and 25c using a brookfield viscometer ( lvdve , brookfield engineering ltd . , inc . accurately weighed tsp ( 1 g ) was transferred to 100 ml measuring cylinder and initial volume was noted . measuring cylinder was kept aside for 24 h at room temperature . the change in volume occupied by the swelled polymer swelling index ( si ) was calculated according to the following equation : ( 1)si = s1s0s0100,where s0 is initial volume of the powder in graduated cylindrical and s1 is the volume occupied by swollen gum after 24 h. the preliminary study of gel was performed by performing trial and error of batches varying the concentrations of lutrol f127 ( 12 to 24% ) [ 7 , 8 ] and tsp ( 0.5 to 2.0% ) . the formulations revealed the effect of two factors , lutrol f127 and tsp , on the gel formation . a 3 factorial design was used to get optimized formulation of the in situ gel . using the software design expert ( version 9.0 ) the concentration of lutrol f127 ( x1 ) and tsp ( x2 ) was selected as independent variables ( table 1 ) . the dependent variables evaluated were gelation temperature ( y1 ) and drug release ( y2 ) . mucoadhesive in situ gel was prepared by cold method described by schmolka . in situ gel of lh ( 2% ) was prepared using different concentrations of lutrol f127 and tsp as shown in table 2 . mucoadhesive tsp polymer was slowly added to the water with continuous agitation of the solution . the resulting solution was left at 4c for 24 h to complete the polymer dissolution . finally , benzalkonium chloride ( 0.001% formulations ( f1 to f9 ) were filled in 10 ml vials , capped with rubber plugs , sealed with aluminium caps , and stored in a refrigerator ( 48c ) until further use . the formulations were visually checked for clarity against white and black background and categorized as follows : very clear ( + + + ) , clear ( + + ) , and turbid ( + ) . the ph of each formulation was tested using previously calibrated ph meter ( equiptronics , eq-610 ) . the viscosity study of all the formulations ( f1 to f9 ) of the in situgel was determined by the brookfield viscometer using spindle number 61 ( lvdve , brookfield engineering ltd . , viscosity of the formulation was noted at two different temperatures , 25c and 35c , at 100 rpm shear rate . . 5 ml aliquot of gel was transferred in a test tube and placed in a water bath . with successive increments in temperature of 1c gel was said to have occurred , when the meniscus of the formulation would no longer move , upon tilting through right angle . each preparation was tested thrice to control the repeatability of the measurement . to get the drug content of gel , the formulation was maintained at 10c , throughout the test , to remain in the liquid form . 1 ml of liquid was taken in 100 ml volumetric flask ; 100 ml ph 6.8 phosphate buffer solution was added to it . out of this , 4 ml solution was taken out into a 10 ml volumetric flask and volume was adjusted with ph 6.8 phosphate buffer . absorbance was measured using double beam uv spectrophotometer at 263 nm ( uv-1800 , shimadzu , japan ) . spreadability of the in situ gel was performed using a ct3 texture analyzer ( brookfield engineering lab , inc . , an analytical probe is depressed into the sample at a defined rate to a desired depth , allowing a predefined necessary period , between the end of the first compression cycle and the beginning of second compression cycle . a cone analytical probe sample holder ( ta2/1000 ) ( 30 mm diameter , 60 ) was completely filled with the gel . the tapered cone was forced down into the sample holder at a defined rate of 1 mm / s and to a defined depth of 10 mm . when a trigger force of 10 g was attained , the probe proceeded to pierce the sample at a test speed of 2 mm / s to a depth of 25 mm . when the specified penetration distance was achieved , the probe departed from the sample at the posttest speed of 2 mm / s . the resulting force - time plot provided hardness , cohesiveness , and adhesiveness . the maximum force attained on the graph was a measure of the firmness of the sample at the specified depth ( hardness ) . the maximum negative force was taken as an indication of the stickiness / cohesiveness of the sample . the work required to deform the gel in down movement of probe indicated cohesiveness . the work necessary to overcome the attractive forces between the surface of the sample and the surface of the probe provided adhesiveness of the sample . formulation ( 50 g ) was put in a 100 ml graduated measuring cylinder , which was further placed in thermostatically controlled water bath at 37c . a calibrated weight of 35 g was slowly placed on the surface of the gel . time ( in seconds ) required by the weight to penetrate 5 cm deep into the gel was noted . mucoadhesive strength of gel f7 was performed using a texture analyzer ( ct3 texture analyzer , brookfield engineering lab , inc . a fresh oral gum mucosal tissue of sheep was obtained from local slaughter house , cut ( 20 20 mm ) , and washed with phosphate buffer ph 6.8 . a section of tissue was fixed on the tissue holder , keeping the orifice of the lid open to expose the small section of the tissue . simulated saliva was placed in the 500 ml beaker and put on the thermostatically controlled heater at 37 0.5c . tissue holder was placed in this beaker containing magnetic stirrer and equilibrated for 15 min at physiological temperature . a drop of gel was placed on the tissue through the opening of the holder . the cylinder probe ( ta-5 ) was lowered at a rate of 0.5 mm / s until it touched the membrane . a contact force of 1 n was maintained for 60 s , and the probe was subsequently withdrawn at a rate of 0.5 mm / s to a distance of 15 mm . the maximum force required to separate the probe from the tissue being maximum detachment force in grams ( fmax ) was noted from texturepro ct v1.3 build 14 software and mucoadhesive strength was determined using the following equation : ( 2)mucoadhesive strengthdyne / cm2=fmaxga , where fmax is the maximum detachment force in grams , g is acceleration due to gravity , and a is the area of tissue exposed to the gel . in vitro release study of formulations ( f1 to f9 ) dialysis membrane consisted of cellophane membrane having an average flat width of 24.26 mm , average diameter of 14.3 mm , and capacity of approximately 1.61 ml / cm , utilized for diffusion . prior to the diffusion study , the dialysis membrane was soaked overnight in ph 6.8 phosphate buffer solution . formulation ( 1 ml ) was placed in the dialysis membrane , cut off in the size of 7 cm length , and sealed on both sides . the dialysis tube was then placed in a glass beaker containing 20 ml of ph 6.8 phosphate buffer solution equilibrated at 37 0.5c . 1 ml of aliquot was withdrawn after every 10 min till 2 h to get the amount of drug released through the membrane and entered in the phosphate buffer and replaced with same volume of preheated solution to maintain the sink condition . a fresh gum mucosal tissue was carefully removed from the oral cavity of the sheep , obtained from the local slaughter house . the mucosa was cut off in circular shape of 3.5 cm in diameter and fixed in between the donor and the receptor compartment of the franz diffusion cell , keeping mucosal side up . prior to study , the mucosa was equilibrated by putting in phosphate buffer ph 6.8 for 1 h. the optimized batch gel formulation ( equivalent to 10 mg of lh ) was applied evenly on the mucosal membrane . the receptor compartment was filled with 25 ml of ph 6.8 phosphate buffer solution maintained at temperature 37c . the assembly was put on the magnetic stirrer . at predetermined time periods of 30 min time interval , 1 ml of aliquot was withdrawn from the receptor compartment , replacing the same volume with ph 6.8 phosphate buffer of temperature 37c , for a period of 2 h. after suitable dilution , samples were analyzed uv spectrophotometrically at 263 nm . the optimized batch was packed in amber colour bottle , sealed , kept at 40c , and maintained at 75% relative humidity in stability chamber ( thermolab , india ) for a period of 3 months . samples withdrawn at 1 , 2 , and 3 months were characterized for appearance , drug content , and in vitro drug release . the powder obtained was cream in colour with good swelling index ( 200% ) and viscosity ( aqueous dispersion of 1% w / v was 8.85 cps ) . angle of repose , bulk density , tapped density , and carr 's index revealed good flow properties . mathematical treatment of the nine possible combinations of batches f1f9 is shown in table 2 . the ph of all formulations was found to be in the range of 6.56.8 , which was similar to the normal ph of mouth saliva ( 6.8 to 7.4 ) . scanning of lidocaine hcl solution in ph 6.8 phosphate buffer solution by uv spectrophotometer showed max at 263 nm . at this wavelength the standard curve followed beer - lambert 's law in the concentration range of 5 to 30 g / ml with r = 0.9992 . the drug content of all formulations was found to be in the range of 9399% , thus confirming the uniformity in formulation of the gel ( table 3 ) . as the concentration of the lutrol f127 ( 1822% ) and tsp ( 0.51.5 ) formulations with the higher concentration of tsp ( 0.5 to 1.5% ) at the same amount of lutrol f127 ( 22% ) in f8 , f5 , and f4 showed the increase in viscosity at both temperatures ( 25c and 35c ) . formulations consisted of the increased amount of lutrol f127 ( 14 to 22% ) at the same amount of tsp ( 1.5% ) in f3 , f2 , and f4 and showed the increase in viscosity ( table 3 ) . thus , the viscosity of formulations in gel state was found to be proportionate with the increase in concentration of lutrol f127 and tsp . gelation temperature range suitable for dental gel is 3335c ( table 3 ) , which means a gel should be in liquid form at room temperature and form a gel phase in the buccal cavity . if the gelation temperature of liquid gel is lower than 33c , gelation occurs at room temperature , leading to difficulty in administering the formulation . if the gelation temperature is higher than 35c , the gel remains in a liquid form at physiological temperature , resulting in leakage from the periodontal pocket . the gelation temperature of formulations f1 to f9 , inspected visually , was found to be within the range of 20 to 37c . the data presented in table 3 clearly indicated that the gelation temperature was strongly dependent on the concentration of selected independent variables . a mathematical relationship between factors and levels was studied by response surface regression analysis using design expert ( version 9.0.3.1 ) software . equation ( 3 ) shows the relationship between the variables and response of gelation temperature ( y1 ) . equation ( 3 ) , in the form of coded values , is as follows:(3)y1=+35.148.17x10.83x20.50x1x25.79x12 + 0.21x22,where y1 is the gelation temperature , x1 is concentration of lutrol f127 , and x2 is concentration of tsp . a negative sign before a factor in polynomial equation ( 3 ) indicated that the response has reciprocal effect on both factors . influence of factors x1 and x2 on y1 was best fitted to quadratic model and found to be significant with f value of 345.01 ( p < 0.05 ) . variables x1 and x2 have p value of 0.000404 ( p < 0.05 ) and 0.0061 . the variables , which have p value less than 0.05 , significantly affect the gelation temperature . the predicted r squared value of 0.9828 was in reasonable agreement with the adjusted r squared value of 0.9962 . analysis of variance ( anova ) was applied to determine the significance and the magnitude of the effects of the variables and their interactions . as the temperature of lutrol f127 the gel formation occurs when the concentration is above the micellar concentration ( 50% ) . interestingly , addition of mucoadhesive polymer ( tsp ) lowered the gelation temperature of the gel . these results are in close agreement with the data obtained for in situ gel formulated for periodontal disease using carbopol 934 p and poloxamer 407 . the regression model obtained was used to generate the counter plots for analyzing interactions of the independent factors . gelation temperature of gel decreased with increase in concentration of lutrol f127 but showed much less effect on change in concentration of tsp , which was indicated clearly in vertical axis of counter plot . the combined effect of factors x1 and x2 can be further elucidated with the help of three - dimensional response surface plot as shown in figure 4(b ) . high level of factor x1 showed reduction in gelation temperature and low level showed higher gelation temperature , which indicated that factor x1 has significant negative effect on gelation temperature of gel . factor x2 ( tsp ) was found to have much less reciprocal effect on gelation temperature . formulations f1 to f9 , subjected to in vitro release study , are represented graphically in figure 5 . formulations f1 , f3 , and f9 showed 95% of drug release within 80 min . it was observed that , at less concentration of lutrol f127 ( 14% ) and higher amount of tsp , the initial rate of drug release was very rapid due to incomplete gel formation . further , increase in concentration of lutrol f127 to 18% ( f2 , f6 , and f7 ) delayed the drug release and released more than 95% of the drug within 2 h. however , further more amount of lutrol f127 ( 22% ) in f4 , f5 , and f8 , the drug release was retarded and was not released ( 82%85% ) completely even after 2 h , which is maximal time needed for dental surgery to have anesthetic action . presence of tsp in in situ gel was found to affect the drug release . as the amount of tsp was increased from 0.5% ( f9 , f6 , and f8 ) to 1.0% ( f1 , f7 , and f5 ) and 1.5% ( f3 , f2 , and f4 ) , drug release was found to be increased . hence it was concluded that the concentration of lutrol f127 should not exceed 18% [ 7 , 22 ] . analysis of variance was applied to determine the significance and the magnitude of the effects of the variables and their interactions on drug release . the obtained regression model was used to generate the counter plot for analyzing the interactions of the independent variables on dependent variable . equation ( 4 ) shows the polynomial equation in terms of coded levels obtained for drug release : ( 4)y2=+91.806.47x1 + 1.67x2 . the contour plot and three - dimensional analysis showed that drug release was decreased with increase in concentration of lutrol f127 and increased with increase in concentration of tsp . figure 6 indicates scale from blue colour to red colour . when colour of response surface shifts from blue towards red it indicates increase in percentage of drug release and vice versa . the same effect was reflected in ( 4 ) showing negative sign before x1 and positive sign before x2 . the variable x1 showed higher numerical value than x2 , which confirmed the more negative effect of lutrol f127 compared to the positive effect of tsp on drug release of in situ gel . anova results confirmed the adequacy of the linear model for drug release ( y2 ) and were found to be significant with f value of 9.19 ( p < 0.05 ) . the predicted r squared value of 0.5491 was in reasonable agreement with the adjusted r squared value of 0.6719 . formulations ( f1 to f9 ) showed good gel strength in the range of 40 to 55 s. higher gel strength which was related to viscous polymer verified the retaining capacity of gel in the periodontal pocket . f4 , f5 , and f8 needed more time ( 55 s , 52 s , and 48 s , resp . ) to penetrate the weight in it , compared to the other formulations . spreadability denotes the extent of area to which the gel readily spreads on its application . the greater the viscosity is , the lesser the spreadability is and the more the retention of gel in the dental pocket can be . texture profile analysis spectra of in situ gel of formulation ( f7 ) showed the hardness of 16.3 g , cohesiveness of 0.81 , and adhesiveness of 0.4 mj ( figure 7 ) . these results expressed the applicability of gels to site of application or adhesivity and indicated the retention time of the gel on the site of application . hardness value confirmed the good firmness of gel and cohesiveness value indicated the better consistency of the gel . as the probe returned to its starting position , the initial lifting of the weight of the sample on the upper surface of the disc produced the negative part of the graph . this indicated the cohesiveness and resistance of the sample to be separated ( flow off ) from the disc . gel formulation ( f7 ) showed good mucoadhesive strength ( 1,124 dyne / cm ) to the sheep mucosa , needed to hold the gel in the dental pocket during surgery , to show therapeutic anesthetic action . drug permeation through the oral tissue was performed though the sheep oral mucosa using franz diffusion cell . the liquid gel was immediately converted to solid gel after putting on the oral mucosa , maintained at 37c . initially , faster drug release was observed which was due to the incomplete gel formation . this faster release was actually found to be good to attain faster anesthetic action at the start of dental procedure . as the time progressed , the gelation temperature ( 35.33c ) was achieved and release rate was slowed down . the gel was retained on the mucosa , which confirmed good mucoadhesion using tsp polymer . the formulation exhibited the good release of lh ( 98.05% ) , as shown in figure 5 . the release was found to be in good correlation with in vitro study ( 97.5% ) . accelerated stability study of an optimized batch of in situ gel ( f7 ) was carried out as per ich guidelines . there was no significant change in drug content , ph , viscosity , gelation temperature , and drug diffusion for the selected formulation , f7 , after 90 days at 40c 0.5/75% 5% rh . the drug ( 92.7% ) was diffused through the dialysis membrane within 2 h. the computer optimization technique by the desirability approach was used to produce the optimum formulation . the optimized formula was reached by setting maximum percentage of drug release at 2 h and optimal gelation temperature . formulation f7 was found to be optimized formulation which contained 18% lutrol f127 and 1% tsp . lidocaine hydrochloride loaded periodontal temperature - sensitive in situ gel was successfully developed by cold method using xyloglucan based mucoadhesive polymer tsp for insertion into periodontal pocket to have painless treatment . viscosity study showed the marked increase in the viscosity of gel at 37c due to sol - gel conversion . depicted the rapid onset of drug action , extending till 2 h , to cover period of periodontal treatment . use of natural , less costly , biodegradable , and easily available mucoadhesive tsp polymer as well as avoidance of needle insertions during scaling and root planning of periodontosis helped achieve patient compliance by ultimately reducing the cost of the treatment . tsp ( 1% ) and lutrol f127 ( 18% ) , in combination , imparted viscous behaviour to gel needed to retain the formulation in periodontal pocket . thus , lidocaine hydrochloride thermoreversible in situ gel offered an alternative to painful injection therapy of anesthesia during dental surgery , with rapid onset of anesthetic action lasting throughout the dental procedure .

the present study was aimed at formulating thermoreversible in situ gel of local anesthetic by using xyloglucan based mucoadhesive tamarind seed polysaccharide ( tsp ) into periodontal pocket . temperature - sensitive in situ gel of lidocaine hydrochloride ( lh ) ( 2% w / v ) was formulated by cold method . a full 32 factorial design was employed to study the effect of independent variables concentrations of lutrol f127 and tsp to optimize in situ gel . the dependent variables evaluated were gelation temperature ( y1 ) and drug release ( y2 ) . the results revealed the surface ph of 6.8 , similar to the ph of saliva . viscosity study showed the marked increase in the viscosity of gel at 37c due to sol - gel conversion . tsp was found to act as good mucoadhesive component to retain gel at the site of application in dental pocket . gelation of formulation occurred near to body temperature . in vitro study depicted the fast onset of drug action but lasting the release ( 90% ) till 2 h. formulation f7 was considered as optimized batch , containing 18% lutrol f127 and 1% tamarind seed polysaccharide . thus , lidocaine hydrochloride thermoreversible in situ gel offered an alternative to painful injection therapy of anesthesia during dental surgery , with fast onset of anesthetic action lasting throughout the dental procedure .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

periodontosis is caused by the bacteria that stick to the surface of tooth and multiply . this scaling procedure needs the application of local and nerve block / infiltration anesthesia by painful needle therapy . to improve the residence time , in situ gels show promising effect . in situ gel stays at application site due to increased viscosity and mucoadhesiveness and shows fast onset of action . lutrol is a triblock polymer , consists of polyoxyethylene - polyoxypropylene - polyoxyethylene units , as shown in figure 1(c ) , and forms micelles at low concentration and clear thermoreversible gel at a high concentration . the concentrated solution of lutrol f127 ( 1630% ) gets transformed from low viscosity transparent solution at 5c to a solid on heating at body temperature . formulation which consisted of lutrol f127 forms a gel in periodontal pocket at a body temperature by modulating the gelation temperature . the dental gel can be easily rinsed out with water to stop the anesthetic effect after the treatment . lidocaine hydrochloride ( lh ) ( figure 1(a ) ) is the first amino amide type of local anesthetics and has been in use for many years . as a local anesthetic , lidocaine is characterized by a rapid onset of action and intermediate duration of efficacy , making it suitable for infiltration and nerve block anesthesia . lidocaine stabilizes the neuronal membrane by inhibiting the ionic fluxes required for the initiation and conduction of impulses , thereby effecting local anesthetic action . tamarind seed polysaccharide ( tsp ) is a high - molecular - weight , branched polysaccharide and consists of celluloselike backbone that carries xylose and galactoxylose substances , as shown in figure 1(b ) . it has been studied earlier for thermoreversible gelation property and also as mucoadhesive component in mucoadhesive buccal patches for controlled release . tsp is used as mucoadhesive polysaccharide polymer for systemic delivery of rizatriptan benzoate through buccal route , formulated in the form of buccal film . the objective of the present study was to develop thermoreversible in situ gel of local anesthetic lh by using xyloglucan based mucoadhesive polymer tsp for insertion into periodontal pocket to have painless treatment . soaked seeds in water ( 24 h ) were boiled for 1 h and kept aside for 2 h to liberate sufficient mucilage into water . accurately weighed , dried , and finely powdered tsp ( 1 g ) was suspended in 75 ml of distilled water for 5 h. volume was made up to 100 ml to produce the concentration of 1% w / v . the mixture was homogenized by mechanical stirrer for 2 h and viscosity was determined at 500 rpm using spindle 61 and 25c using a brookfield viscometer ( lvdve , brookfield engineering ltd . accurately weighed tsp ( 1 g ) was transferred to 100 ml measuring cylinder and initial volume was noted . the change in volume occupied by the swelled polymer swelling index ( si ) was calculated according to the following equation : ( 1)si = s1s0s0100,where s0 is initial volume of the powder in graduated cylindrical and s1 is the volume occupied by swollen gum after 24 h. the preliminary study of gel was performed by performing trial and error of batches varying the concentrations of lutrol f127 ( 12 to 24% ) [ 7 , 8 ] and tsp ( 0.5 to 2.0% ) . the formulations revealed the effect of two factors , lutrol f127 and tsp , on the gel formation . a 3 factorial design was used to get optimized formulation of the in situ gel . using the software design expert ( version 9.0 ) the concentration of lutrol f127 ( x1 ) and tsp ( x2 ) was selected as independent variables ( table 1 ) . the dependent variables evaluated were gelation temperature ( y1 ) and drug release ( y2 ) . mucoadhesive in situ gel was prepared by cold method described by schmolka . in situ gel of lh ( 2% ) was prepared using different concentrations of lutrol f127 and tsp as shown in table 2 . mucoadhesive tsp polymer was slowly added to the water with continuous agitation of the solution . the ph of each formulation was tested using previously calibrated ph meter ( equiptronics , eq-610 ) . the viscosity study of all the formulations ( f1 to f9 ) of the in situgel was determined by the brookfield viscometer using spindle number 61 ( lvdve , brookfield engineering ltd . , viscosity of the formulation was noted at two different temperatures , 25c and 35c , at 100 rpm shear rate . 5 ml aliquot of gel was transferred in a test tube and placed in a water bath . to get the drug content of gel , the formulation was maintained at 10c , throughout the test , to remain in the liquid form . 1 ml of liquid was taken in 100 ml volumetric flask ; 100 ml ph 6.8 phosphate buffer solution was added to it . out of this , 4 ml solution was taken out into a 10 ml volumetric flask and volume was adjusted with ph 6.8 phosphate buffer . spreadability of the in situ gel was performed using a ct3 texture analyzer ( brookfield engineering lab , inc . a cone analytical probe sample holder ( ta2/1000 ) ( 30 mm diameter , 60 ) was completely filled with the gel . when the specified penetration distance was achieved , the probe departed from the sample at the posttest speed of 2 mm / s . the maximum force attained on the graph was a measure of the firmness of the sample at the specified depth ( hardness ) . the work necessary to overcome the attractive forces between the surface of the sample and the surface of the probe provided adhesiveness of the sample . formulation ( 50 g ) was put in a 100 ml graduated measuring cylinder , which was further placed in thermostatically controlled water bath at 37c . a calibrated weight of 35 g was slowly placed on the surface of the gel . mucoadhesive strength of gel f7 was performed using a texture analyzer ( ct3 texture analyzer , brookfield engineering lab , inc . a fresh oral gum mucosal tissue of sheep was obtained from local slaughter house , cut ( 20 20 mm ) , and washed with phosphate buffer ph 6.8 . simulated saliva was placed in the 500 ml beaker and put on the thermostatically controlled heater at 37 0.5c . a drop of gel was placed on the tissue through the opening of the holder . the cylinder probe ( ta-5 ) was lowered at a rate of 0.5 mm / s until it touched the membrane . the maximum force required to separate the probe from the tissue being maximum detachment force in grams ( fmax ) was noted from texturepro ct v1.3 build 14 software and mucoadhesive strength was determined using the following equation : ( 2)mucoadhesive strengthdyne / cm2=fmaxga , where fmax is the maximum detachment force in grams , g is acceleration due to gravity , and a is the area of tissue exposed to the gel . in vitro release study of formulations ( f1 to f9 ) dialysis membrane consisted of cellophane membrane having an average flat width of 24.26 mm , average diameter of 14.3 mm , and capacity of approximately 1.61 ml / cm , utilized for diffusion . prior to the diffusion study , the dialysis membrane was soaked overnight in ph 6.8 phosphate buffer solution . formulation ( 1 ml ) was placed in the dialysis membrane , cut off in the size of 7 cm length , and sealed on both sides . the dialysis tube was then placed in a glass beaker containing 20 ml of ph 6.8 phosphate buffer solution equilibrated at 37 0.5c . 1 ml of aliquot was withdrawn after every 10 min till 2 h to get the amount of drug released through the membrane and entered in the phosphate buffer and replaced with same volume of preheated solution to maintain the sink condition . prior to study , the mucosa was equilibrated by putting in phosphate buffer ph 6.8 for 1 h. the optimized batch gel formulation ( equivalent to 10 mg of lh ) was applied evenly on the mucosal membrane . the receptor compartment was filled with 25 ml of ph 6.8 phosphate buffer solution maintained at temperature 37c . at predetermined time periods of 30 min time interval , 1 ml of aliquot was withdrawn from the receptor compartment , replacing the same volume with ph 6.8 phosphate buffer of temperature 37c , for a period of 2 h. after suitable dilution , samples were analyzed uv spectrophotometrically at 263 nm . the optimized batch was packed in amber colour bottle , sealed , kept at 40c , and maintained at 75% relative humidity in stability chamber ( thermolab , india ) for a period of 3 months . samples withdrawn at 1 , 2 , and 3 months were characterized for appearance , drug content , and in vitro drug release . the powder obtained was cream in colour with good swelling index ( 200% ) and viscosity ( aqueous dispersion of 1% w / v was 8.85 cps ) . the ph of all formulations was found to be in the range of 6.56.8 , which was similar to the normal ph of mouth saliva ( 6.8 to 7.4 ) . scanning of lidocaine hcl solution in ph 6.8 phosphate buffer solution by uv spectrophotometer showed max at 263 nm . at this wavelength the standard curve followed beer - lambert 's law in the concentration range of 5 to 30 g / ml with r = 0.9992 . the drug content of all formulations was found to be in the range of 9399% , thus confirming the uniformity in formulation of the gel ( table 3 ) . as the concentration of the lutrol f127 ( 1822% ) and tsp ( 0.51.5 ) formulations with the higher concentration of tsp ( 0.5 to 1.5% ) at the same amount of lutrol f127 ( 22% ) in f8 , f5 , and f4 showed the increase in viscosity at both temperatures ( 25c and 35c ) . formulations consisted of the increased amount of lutrol f127 ( 14 to 22% ) at the same amount of tsp ( 1.5% ) in f3 , f2 , and f4 and showed the increase in viscosity ( table 3 ) . thus , the viscosity of formulations in gel state was found to be proportionate with the increase in concentration of lutrol f127 and tsp . gelation temperature range suitable for dental gel is 3335c ( table 3 ) , which means a gel should be in liquid form at room temperature and form a gel phase in the buccal cavity . if the gelation temperature of liquid gel is lower than 33c , gelation occurs at room temperature , leading to difficulty in administering the formulation . if the gelation temperature is higher than 35c , the gel remains in a liquid form at physiological temperature , resulting in leakage from the periodontal pocket . the gelation temperature of formulations f1 to f9 , inspected visually , was found to be within the range of 20 to 37c . the data presented in table 3 clearly indicated that the gelation temperature was strongly dependent on the concentration of selected independent variables . equation ( 3 ) shows the relationship between the variables and response of gelation temperature ( y1 ) . equation ( 3 ) , in the form of coded values , is as follows:(3)y1=+35.148.17x10.83x20.50x1x25.79x12 + 0.21x22,where y1 is the gelation temperature , x1 is concentration of lutrol f127 , and x2 is concentration of tsp . influence of factors x1 and x2 on y1 was best fitted to quadratic model and found to be significant with f value of 345.01 ( p < 0.05 ) . variables x1 and x2 have p value of 0.000404 ( p < 0.05 ) and 0.0061 . the variables , which have p value less than 0.05 , significantly affect the gelation temperature . analysis of variance ( anova ) was applied to determine the significance and the magnitude of the effects of the variables and their interactions . as the temperature of lutrol f127 the gel formation occurs when the concentration is above the micellar concentration ( 50% ) . interestingly , addition of mucoadhesive polymer ( tsp ) lowered the gelation temperature of the gel . these results are in close agreement with the data obtained for in situ gel formulated for periodontal disease using carbopol 934 p and poloxamer 407 . gelation temperature of gel decreased with increase in concentration of lutrol f127 but showed much less effect on change in concentration of tsp , which was indicated clearly in vertical axis of counter plot . the combined effect of factors x1 and x2 can be further elucidated with the help of three - dimensional response surface plot as shown in figure 4(b ) . high level of factor x1 showed reduction in gelation temperature and low level showed higher gelation temperature , which indicated that factor x1 has significant negative effect on gelation temperature of gel . factor x2 ( tsp ) was found to have much less reciprocal effect on gelation temperature . formulations f1 to f9 , subjected to in vitro release study , are represented graphically in figure 5 . formulations f1 , f3 , and f9 showed 95% of drug release within 80 min . it was observed that , at less concentration of lutrol f127 ( 14% ) and higher amount of tsp , the initial rate of drug release was very rapid due to incomplete gel formation . further , increase in concentration of lutrol f127 to 18% ( f2 , f6 , and f7 ) delayed the drug release and released more than 95% of the drug within 2 h. however , further more amount of lutrol f127 ( 22% ) in f4 , f5 , and f8 , the drug release was retarded and was not released ( 82%85% ) completely even after 2 h , which is maximal time needed for dental surgery to have anesthetic action . presence of tsp in in situ gel was found to affect the drug release . as the amount of tsp was increased from 0.5% ( f9 , f6 , and f8 ) to 1.0% ( f1 , f7 , and f5 ) and 1.5% ( f3 , f2 , and f4 ) , drug release was found to be increased . hence it was concluded that the concentration of lutrol f127 should not exceed 18% [ 7 , 22 ] . analysis of variance was applied to determine the significance and the magnitude of the effects of the variables and their interactions on drug release . the obtained regression model was used to generate the counter plot for analyzing the interactions of the independent variables on dependent variable . equation ( 4 ) shows the polynomial equation in terms of coded levels obtained for drug release : ( 4)y2=+91.806.47x1 + 1.67x2 . the contour plot and three - dimensional analysis showed that drug release was decreased with increase in concentration of lutrol f127 and increased with increase in concentration of tsp . when colour of response surface shifts from blue towards red it indicates increase in percentage of drug release and vice versa . the variable x1 showed higher numerical value than x2 , which confirmed the more negative effect of lutrol f127 compared to the positive effect of tsp on drug release of in situ gel . anova results confirmed the adequacy of the linear model for drug release ( y2 ) and were found to be significant with f value of 9.19 ( p < 0.05 ) . formulations ( f1 to f9 ) showed good gel strength in the range of 40 to 55 s. higher gel strength which was related to viscous polymer verified the retaining capacity of gel in the periodontal pocket . to penetrate the weight in it , compared to the other formulations . the greater the viscosity is , the lesser the spreadability is and the more the retention of gel in the dental pocket can be . texture profile analysis spectra of in situ gel of formulation ( f7 ) showed the hardness of 16.3 g , cohesiveness of 0.81 , and adhesiveness of 0.4 mj ( figure 7 ) . these results expressed the applicability of gels to site of application or adhesivity and indicated the retention time of the gel on the site of application . hardness value confirmed the good firmness of gel and cohesiveness value indicated the better consistency of the gel . gel formulation ( f7 ) showed good mucoadhesive strength ( 1,124 dyne / cm ) to the sheep mucosa , needed to hold the gel in the dental pocket during surgery , to show therapeutic anesthetic action . the liquid gel was immediately converted to solid gel after putting on the oral mucosa , maintained at 37c . initially , faster drug release was observed which was due to the incomplete gel formation . this faster release was actually found to be good to attain faster anesthetic action at the start of dental procedure . as the time progressed , the gelation temperature ( 35.33c ) was achieved and release rate was slowed down . the release was found to be in good correlation with in vitro study ( 97.5% ) . accelerated stability study of an optimized batch of in situ gel ( f7 ) was carried out as per ich guidelines . there was no significant change in drug content , ph , viscosity , gelation temperature , and drug diffusion for the selected formulation , f7 , after 90 days at 40c 0.5/75% 5% rh . the drug ( 92.7% ) was diffused through the dialysis membrane within 2 h. the computer optimization technique by the desirability approach was used to produce the optimum formulation . the optimized formula was reached by setting maximum percentage of drug release at 2 h and optimal gelation temperature . formulation f7 was found to be optimized formulation which contained 18% lutrol f127 and 1% tsp . lidocaine hydrochloride loaded periodontal temperature - sensitive in situ gel was successfully developed by cold method using xyloglucan based mucoadhesive polymer tsp for insertion into periodontal pocket to have painless treatment . viscosity study showed the marked increase in the viscosity of gel at 37c due to sol - gel conversion . depicted the rapid onset of drug action , extending till 2 h , to cover period of periodontal treatment . tsp ( 1% ) and lutrol f127 ( 18% ) , in combination , imparted viscous behaviour to gel needed to retain the formulation in periodontal pocket . thus , lidocaine hydrochloride thermoreversible in situ gel offered an alternative to painful injection therapy of anesthesia during dental surgery , with rapid onset of anesthetic action lasting throughout the dental procedure .

the burden of tuberculosis ( tb ) caused by drug - resistant mycobacterium tuberculosis is increasing . as a result , the standard treatment directly observed therapy , short course ( dots ) , composed of 2 months of a four - drug regimen ( isoniazid [ inh ] , rifampicin [ rif ] , pyrazinamide [ pza ] , and ethambutol [ emb ] ) followed by 4 months of treatment with rif and inh is failing in many settings . while any degree of drug resistance might worsen the prognosis of a tb patient [ 1 , 2 ] , there are two definitions of m. tuberculosis drug resistance that are particularly relevant in the clinic . the first refers to strains resistant to both inh and rif and is termed multidrug - resistant ( mdr ) tb . treatment of patients with mdr - tb takes up to 2 years , and currently relies on fluoroquinolones ( fq ) and injectable aminoglycosides ( ag ) to compensate for the loss of two of the most potent drugs . the acquisition of resistance to these classes of antibiotics defines extensively drug - resistant ( xdr ) tb . xdr - tb requires even longer treatment with drugs that are much more costly and show limited efficacy and increased side effects . the global genetic diversity of drug - resistant m. tuberculosis [ 410 ] indicates that drug resistance evolved on multiple occasions in geographical hotspots characterized by a high incidence of tb and inappropriate drug treatment . the latter is mostly driven by a lack of resources resulting in two important failures : an inability to detect drug resistance and a systemic failure to deploy effective treatments [ 1113 ] . the ongoing evolution of m. tuberculosis in these settings [ 14 , 15 ] , provides an excellent opportunity to explore the genetic determinants of drug resistance in this microbe . unlike most other bacterial pathogens , resistance plasmids and horizontal gene transfer play no role in the acquisition of drug resistance in m. tuberculosis . moreover , efflux mechanisms appear to serve only as a stepping stone to high - level resistance . they allow the bacteria to tolerate higher levels of drug but do not per se result in clinically relevant levels of resistance to multiple antibiotics in m. tuberculosis [ 14 , 1618 ] . instead , the evolution of strains resistant to multiple antibiotics is driven by the sequential acquisition and accumulation of resistance - conferring mutations on the bacterial chromosome . these mutations primarily interfere with drug - target binding ( e.g. rif - rpob , fq - gyra ) , compromise prodrug activation ( e.g. inh - katg , pa-824-fgd ) , or cause over - expression of the target ( inh / eth promoter region of inha ) . the elucidation of the mechanisms of action for many antimycobacterials led to the identification of key determinants of resistance ( see zhang and yew and almeida da silva and palomino for reviews ) , and the realization that the corresponding genetic mutations can be used as reliable molecular markers for drug susceptibility testing ( dst ) . the application of this knowledge to the clinic has resulted in the development of diagnostic tools based on nucleic acid amplification ( naa ) that overcome many of the shortcomings of phenotypic dst ; these include a long turnaround time , outcome variability , and infrastructure requirements [ 2629 ] . recently , one of these tools , known as xpert mtb / rif , has been the subject of a policy update published by the world health organization . their recommendation for xpert mtb / rif to replace microscopy in hiv - positive patients , patients suspected of mdr - tb , and those suspected of tb meningitis follows its successful implementation in many countries [ 31 , 32 ] . the instrument is designed to analyse sputum directly , hence bypassing the need for primary bacterial culture . it simultaneously tests for the presence of m. tuberculosis and rif resistance . even though this technology is having a positive impact on tb control by offering high sensitivity and reducing the time to tb diagnosis , the associated costs and infrastructural requirements ( e.g. a constant power supply , machine maintenance ) remain limiting for many high - burden countries [ 32 , 33 ] . hence , on - going efforts in product development focus on cheaper and simpler so - called point - of - care diagnostics . nonetheless , dst without bacterial culturing will continue to require naa . it should be based on detailed knowledge of the relationship between strain genotype , drug resistance phenotype , and the patient treatment outcome in terms of relapse rate and treatment failure . whole genome sequencing ( wgs ) of clinical drug - resistant strains of m. tuberculosis should be combined with the analysis of clinical information from patients infected by these strains to yield valuable new insights into the biology of drug resistance . in this review we focus specifically on results that shed light on why the acquisition of individual drug resistance - conferring mutations is only part of the problem . we then use specific examples to illustrate how understanding more broadly the evolutionary mechanisms that drive drug resistance can inform the development of improved diagnostic tools as well as better strategies to preserve both existing and new treatment regimens . the evolutionary path leading through drug resistance is strongly influenced by two factors : epistasis and bacterial fitness [ 3538 ] . we define epistasis as a set of genetic interactions where the phenotypic effect of one mutation is determined by the presence of one or more other mutations . for example , resistant strains carrying the same resistance mutations vary in their capacity to transmit from patient to patient [ 3942 ] , showing that the strain genetic background can determine the course of evolution of drug resistance . bacterial fitness , on the other hand , is a function of growth rate , virulence , and transmissibility [ 15 , 43 ] . any mutation that reduces it in relation to the wild - type strain is said to carry a fitness cost. the most immediate way to estimate the relative fitness is to measure the growth rate of bacteria in culture . on average , 3 ) whose magnitude positively correlates with the frequency of different resistance mutations in the clinic ; resistant strains with the smallest fitness defect are most abundant [ 45 , 46 ] . furthermore , the fitness of resistant mutants is not fixed ; evolution is an ongoing process , and a comparison between clinical and laboratory strains carrying the same drug - resistance mutation shows that clinical strains often successfully bypass any fitness cost imposed by resistance . the acquisition of such compensatory mutations is also an example of epistasis and is key in the evolution of transmissible drug - resistant strains that pose the greatest risk to public health [ 6 , 10 , 4751 ] . unfortunately , we do not currently have sufficient knowledge to predict epistatic interactions a priori , so we must rely on detecting them empirically by studying the genetics of drug resistance . a number of recent studies used wgs to address the evolution of drug - resistant m. tuberculosis [ 7 , 9 , 51 , 53 , 54 ] . the authors of these studies used approaches based on phylogeny , molecular epidemiology [ 9 , 51 , 53 ] , and mutation frequency analyses to compare drug - susceptible and drug - resistant clinical strains of m. tuberculosis . the shared aim of these studies was the identification of bacterial genes under positive evolutionary selection by drug pressure . the detailed discussion of the merits and shortcomings of the analytical approaches used in these studies is beyond the scope of this review , but it is important to note that there is considerable overlap in the findings of these studies . in addition to known drug targets , all studies identified novel bacterial genes and intergenic regions whose function may be ancillary to drug - resistance mutations ( see fig . 1 ) . in particular , several genes involved in lipid metabolism , cell wall homeostasis , purine metabolism , and transcriptional control appear to be positively selected in presence of anti - tb drugs [ 7 , 54 ] . however , with the exception of just a few mutations ( e.g. pona1 , and the promoters of thya and thyx [ 7 , 54 ] ) , the actual role of these genes remains unclear , essentially offering an extended list of genes of interest that require further investigation . consequently , it is currently not possible to evaluate the role of these genes in the development of future diagnostics or therapies.fig . key genes in m. tuberculosis drug resistance have been plotted , taking into account their approximate position in the genome . lines denote putative epistatic interactions ; connecting genes involved in the physiology of a drug as well as more broad / indirect mechanisms referred to as ancillary to drug resistance. this categorization is meant to include factors mediating complex aspects of cell physiology , such as cell permeability and mutation - induced physiological changes . bold lines connecting rpob to rpoc and embb to rv3972 refer to in vitro validated compensatory mechanisms . figure based on information from the following references : [ 6 , 7 , 24 , 46 , 47 , 49 , 51 , 54 , 9194 ] . ag aminoglycosides , emb ethambutol , eth ethionamide , fq fluoroquinolones , inh isoniazid , pas para - aminosalicylic acid , pza pyrazinamide , rif rifampicin , str streptomycin . see original papers for a more comprehensive list [ 7 , 54 ] a web of epistasis mediates drug resistance in m. tuberculosis . key genes in m. tuberculosis drug resistance have been plotted , taking into account their approximate position in the genome . lines denote putative epistatic interactions ; connecting genes involved in the physiology of a drug as well as more broad / indirect mechanisms referred to as ancillary to drug resistance. this categorization is meant to include factors mediating complex aspects of cell physiology , such as cell permeability and mutation - induced physiological changes . bold lines connecting rpob to rpoc and embb to rv3972 refer to in vitro validated compensatory mechanisms . figure based on information from the following references : [ 6 , 7 , 24 , 46 , 47 , 49 , 51 , 54 , 9194 ] . ag aminoglycosides , emb ethambutol , eth ethionamide , fq fluoroquinolones , inh isoniazid , pas para - aminosalicylic acid , pza pyrazinamide , rif rifampicin , str streptomycin . see original papers for a more comprehensive list [ 7 , 54 ] in addition to the above genes , there are a growing number of novel genes for which experimental evidence is available to support a role of epistasis in adaptation to resistance . these genes were identified through recent studies aimed at elucidating the evolutionary trajectory of drug - resistant m. tuberculosis in the clinic [ 6 , 47 ] and include rpoc , which mediates the adaptation to rif resistance , and rv3806c , which appears to have a role in emb resistance . rif resistance is caused by mutations of the beta subunit of rna polymerase encoded by rpob . mutated amino acids are normally involved in drug binding and are usually restricted within a short stretch of the protein termed the rifampicin resistance determining region ( rrdr ) . many of these mutations carry a fitness cost [ 45 , 49 ] that appears to be negatively correlated with the activity of the mutant enzyme . the importance of this aspect of rna polymerase physiology is clearly illustrated by the fact that one of the most frequent resistance mutations observed in the clinic , s450l , ( equivalent to s531l in escherichia coli ) also carries the lowest fitness cost . moreover , this mutation appears to be almost ubiquitous among mdr strains of m. tuberculosis [ 6 , 9 , 10 , 48 , 51 ] , and perhaps more importantly , is shown to be strongly associated with the acquisition of compensatory mutations within rna polymerase genes ( rpoa , rpob , and rpoc ) . this combination of mutations is strongly associated with improved transmissibility of strains as evidenced by clonal expansion of m. tuberculosis strains carrying these particular mutations in patient populations [ 9 , 48 , 51 ] . the role of these compensatory mutations ( see koch et al . for a more comprehensive review ) appears to be to restore wild - type function of mutant rna polymerase , probably on the level of enzymatic activity [ 49 , 50 ] . alternatively , these compensatory mutations maybe also restore the baseline gene expression profile of cells . specifically , rpob mutants were shown to have a modified lipid profile as well as a modified expression of many proteins involved in lipid metabolism , particularly phthiocerol dimycocerosates ( pdims ) . interestingly , lipid metabolism genes , including those for pdim metabolism , seem to be under positive selection during the evolution of drug resistance [ 7 , 54 ] . because pdims and other mycobacterial lipids play an important role in virulence [ 59 , 60 ] , this argues that global physiological consequences of drug - resistance mutations could provide a contextual framework , within which the compensatory mechanisms mediated by mutations in ancillary genes can be explored [ 7 , 51 , 54 ] . demonstrate the effect of epistasis in the progressive increase of minimal inhibitory concentration ( mic ) for a drug . focusing their study on emb , they observed that acquisition of high - level resistance to emb is a multistep process . embb m306v they identified nonsynonymous mutations in rv3806c and a synonymous mutation in rv3792 as important contributors to emb resistance in vitro . they proceeded to show that rv3806c is involved in modulating the availability of embb substrates , while the synonymous single nucleotide polymorphism in rv3792 apparently stabilized embc rna , leading to a de facto over - expression of the gene , resulting in reduced susceptibility to emb . investigations into the interaction between mutations resulting in resistance to disparate drugs have suggested that positive epistasis may drive multidrug resistance . our group recently published a report showing that specific resistance mutations in rpob and gyra can compensate for each other s fitness defects , to the point that some strains carrying both mutations are fitter than either single mutant . moreover , the particular combinations of mutations conferring resistance to rif and ofloxacin ( ofx ) associated with the highest overall fitness appear to be positively selected in high - burden settings . the examples listed here are not designed to offer a comprehensive overview of all reported examples of epistasis in drug - resistant m. tuberculosis ; we have tried to sketch a more complete picture in fig . 1 . instead , they were chosen to illustrate important concepts brought to light by recent studies : in the first place , based on current data , it appears that compensatory mutations occur most frequently in strains that already harbor the least costly mutation . second , epistatic interactions occur between specific mutations [ 46 , 47 , 49 ] , and in some cases these can be mutually exclusive ; for example , a strain harboring an rpoa mutation does not then also acquire an rpoc or additional rpob mutations [ 6 , 50 , 51 , 62 ] . continued exposure to a drug seems to impose constraints on evolution that facilitate the acquisition of compensatory mutations in strains that are already resistant [ 36 , 50 ] . once generated , these strains are more likely to be transmitted than strains carrying the resistance mutation alone [ 48 , 51 ] . a further consequence of continued drug exposure is the stepwise accumulation of mutations that result in an increased level of resistance to a drug ; a factor that is already influencing inh resistance in xdr strains and may contribute to higher levels of resistance to fq [ 64 , 65 ] . moreover , epistasis can occur between drug - resistance mutations [ 46 , 61 , 66 ] , implicating individual resistance determinants as drivers of polydrug resistance . combined , these observations clearly indicate that continued inappropriate treatment , in part caused by misdiagnosis of resistance , drives the evolution of more transmissible , increasingly drug - resistant strains [ 10 , 51 ] . a further set of factors , not considered hitherto , is involved in the generation of protein heterogeneity in the cell . the primary source of this is mutations , and mutation rates were shown to vary between different phylogenetic lineages of m. tuberculosis . the beijing family of strains in particular seems to have a higher mutation rate [ 6769 ] . the exact consequences of this remain to be determined , but differences in mutation rate have been used to explain the correlation between this genotype with a higher rate of drug - resistance acquisition . , who argue that protein variability driven by errors in the central information - processing pathway ( dna - rna - protein ) may provide a phenotypic stepping - stone to resistance akin perhaps to other ancillary mechanisms shown in fig . 1 . while intellectually appealing , the clinical relevance of such a scenario remains to be substantiated . the translational potential of the knowledge gained from the above studies lies in the implementation of molecular tools in tb control ( see table 1 for a summary ) . wells et al . offer an excellent review of current and future needs for the clinical management of tb , addressing diagnostics , surveillance , and programmatic approaches . it is useful to take advantage of their conceptual framework to discuss the impact of the themes that emerge from evolutionary studies . they place capabilities available to tb control programs onto a continuum that spans from community - based physicians to supranational reference laboratories ( see fig . 2 ) . accordingly , the available resources combined with the specific needs of each level will dictate the contribution that molecular approaches can offer.table 1implications of understanding evolutionary mechanisms of drug resistance for tuberculosis controlexperimental observationphysiological consequenceimplicationsresistant strains gain compensatory mutations that change the basic physiology , e.g. rif - rpoc increased transmissibility , increased propensity to acquire additional resistancefocus surveillance on mutations that correlate with transmissible , highly resistant strainsuse spent biosamples to establish wide catchment area for wgs - based surveillancecontinued exposure to rif directs evolution towards the acquisition of compensatory mutationsmore rapid evolution of compensatory mutationsuse molecular tools to probe the genotype of strains and stop administering ineffective drugs immediatelyepistasis exists between drug - resistance mutations for a single drug : e.g. emb , fq , inh , rifmulti - step acquisition of high level of drug resistancedefine clinical breakpoint concentrations based on specific susceptibility profiles for a drugpositive epistasis between rpob and gyra mutationsstrains with specific combinations of resistance mutations ( e.g. gyra d94 g and rpob h445y ) are fitter than the wild typedrug regimens containing both rif and fq may fail more quickly . assess current clinical trials for evidenceevaluate if the order in which drugs are administered might enhance negative epistatic interactionssome mutations conferring resistance to fq and bedaquiline have no fitness costs attachedno need to compensate for specific resistance mutationsenforce appropriate administration of drugs to avoid adding these antibiotics to failing regimens or use as monotherapyexplore the spectrum of resistance mutations , identify low cost and frequent mutations before releasing the drug into the marketmutation rates vary between m. tuberculosis lineagesbeijing family strains acquire resistance to inh and rif at a higher rate and are 22 times more likely to develop into mdr than the laboratory - adapted strainincrease frequency of phylogeny - based surveillance and focus monitoring on areas with high rates of beijing strains emb ethambutol , fq fluoroquinolones , inh isoniazid , rif rifampicin , wgs whole genome sequencingfig . 2the healthcare continuum . a diagrammatic representation of the current healthcare continuum as described in wells et al . the resources dimension encompasses a breadth of parameters , from access to infrastructure and apparatus , to technical proficiency of staff and financial resources that are available . different diagnostic and dst technologies are shown as bars with the arrows indicating the levels at which we would ideally deploy them in the future . wgs whole genome sequencing , lpa line probe assay , mgit mycobacterial growth indicator tube : phenotypic dst using the bactec mgit 960 instrument , automated nat automated nucleic acid amplification technology implications of understanding evolutionary mechanisms of drug resistance for tuberculosis control emb ethambutol , fq fluoroquinolones , inh isoniazid , rif rifampicin , wgs whole genome sequencing the healthcare continuum . the resources dimension encompasses a breadth of parameters , from access to infrastructure and apparatus , to technical proficiency of staff and financial resources that are available . different diagnostic and dst technologies are shown as bars with the arrows indicating the levels at which we would ideally deploy them in the future . wgs whole genome sequencing , lpa line probe assay , mgit mycobacterial growth indicator tube : phenotypic dst using the bactec mgit 960 instrument , automated nat automated nucleic acid amplification technology in the clinic , the main goal is rapid and effective treatment of infected individuals . this relies on quick and reliable case detection , and , given the need for speed , is likely to depend on molecular diagnostics in the future . in this setting , focusing on drug - resistance mutations offers high sensitivity and specificity for key first - line drugs . including compensatory mutations ( e.g. in rpoc or rpoa , discussed above ) as diagnostic markers is unlikely to provide additional clinical benefit , because mutations in rpob already display a high sensitivity and specificity for detecting mdr - tb in individual patients [ 33 , 72 ] . in contrast , a key area in which evolutionary lessons could be brought to bear in a significant manner is surveillance . consider the reproductive number of a pathogen ( r0 , see fig . two dimensions are paramount to the outcome of transmission cycles ; one is pathogen transmissibility ( t ) and the other is time to effective treatment ( c d ) . strain fitness plays a role in the former , while drug resistance considerably influences the latter . it is in the interest of public health to identify and eradicate transmissible drug - resistant strains quickly . we are currently failing on this front in 2012 , fewer than one - quarter of estimated mdr - tb cases were detected , and only 23 % of those had dst results reported for pza and fq . to this end , there is scope for the implementation of molecular diagnostics for public health. screening for the emergence of compensatory mutations in a population , perhaps by focusing on re - treatment cases to pinpoint areas with evolved highly transmissible strains as they emerge [ 48 , 51 ] , would aid the public health official to prioritize resource allocation and interrupt the spread of these strains . examples of such measures include targeted delivery of individualized , albeit expensive treatments , resource - intensive active case finding , and , in extreme cases , isolation [ 75 , 76 ] . in line with the described differences in mutation rates between m. tuberculosis lineages [ 67 , 68 ] , the public health official may include phylogenetic markers to focus monitoring on areas with a high incidence of beijing strains . mutations conferring intermediate resistance , namely , below that of empirically determined resistance breakpoints , play a potentially pivotal role in the spread of resistance . we have used the fitness - mic space to illustrate these concepts in fig . it is also important to stress at this point , that strains harboring mutations conferring low - level resistance can often be treated effectively with existing drugs , either by increasing the dosage of the same drug , or by using alternative drugs from the same class , as is the case of fq . conversely , the use of ineffective drugs should be stopped immediately to avoid directing the evolution of a strain [ 36 , 50 ] towards a more transmissible phenotype.fig . the relative fitness of strains carrying key drug - resistance mutations grown in the absence of drug was plotted against their contribution to mic to illustrate the relationship between genotype and phenotype for important drug - resistance mutations . lines connecting individual mutations denote strains carrying two mutations , while arrows denote estimates of the fitness of double / triple - mutants ( three different types of arrows are used to illustrate different evolutionary trajectories ) . reproductive number ( r 0 ) defined as the number of secondary cases caused by an infected individual is roughly equal to the product of an organism s transmissibility ( t ) , number of contacts ( c ) , and the duration of infection ( d ) . we were unable to find true relative fitness measurements for katg s315 t and gyra a90v ; these were estimated from pym et al . and poissy et al . [ 47 , 100 , 101 ] , plinke et al . [ 47 , 100 , 101 ] , and starks et al . [ 94 , 102105 ] , duong et al . [ 94 , 102105 ] , and malik et al . bdq bedaquiline , emb ethambutol , fq fluoroquinolones , inh isoniazid , mic minimum inhibitory concentration , rif rifampicin evolutionary trajectories of epistasis - driven resistance . the relative fitness of strains carrying key drug - resistance mutations grown in the absence of drug was plotted against their contribution to mic to illustrate the relationship between genotype and phenotype for important drug - resistance mutations . lines connecting individual mutations denote strains carrying two mutations , while arrows denote estimates of the fitness of double / triple - mutants ( three different types of arrows are used to illustrate different evolutionary trajectories ) . reproductive number ( r 0 ) defined as the number of secondary cases caused by an infected individual is roughly equal to the product of an organism s transmissibility ( t ) , number of contacts ( c ) , and the duration of infection ( d ) . ( emb , m. tuberculosis ) , and huitric et al . ( bdq , m. tuberculosis ) . we were unable to find true relative fitness measurements for katg s315 t and gyra a90v ; these were estimated from pym et al . and poissy et al . safi et al . [ 47 , 100 , 101 ] , plinke et al . [ 47 , 100 , 101 ] , and starks et al . [ 47 , 100 , 101 ] for emb ; aubry et al . [ 94 , 102105 ] , matrat et al . [ 94 , 102105 ] , cheng et al . [ 94 , 102105 ] , duong et al . [ 94 , 102105 ] , and malik et al . [ 94 , 102105 ] for fq ; and huitric et al . for bdq . bdq bedaquiline , emb ethambutol , fq fluoroquinolones , inh isoniazid , mic minimum inhibitory concentration , rif rifampicin in addition to surveillance , knowledge of epistasis should be applied to the design and deployment of future drug regimens . given the genetic interaction between mutations in rpob and gyra , it may be dangerous to administer rif and fq simultaneously , and should perhaps be avoided . a number of current clinical trials are testing an iteration of such a combination , and their outcomes should be carefully scrutinized by using wgs for evidence of epistasis - driven drug resistance [ 74 , 8082 ] . protecting new drug classes is equally important . bedaquiline is the first new antimycobacterial to be approved by the us fda for over 40 years [ 83 , 84 ] . given the availability of bedaquiline resistance mutations with no fitness cost , or even a fitness benefit ( see fig . 3 ) , it is key that the guidelines for the administration of the drug are adhered to [ 86 , 87 ] , especially in light of the fact that no standardized regimen exists for it yet . most importantly , bedaquiline should not be used to rescue a failing regimen , and should always be administered with other effective drugs to minimize the emergence of resistance . consensus is growing that biological parameters such as the frequency of resistance mutations , the occurrence of low- or no - cost drug - resistance mutations , epistasis between resistance mutations , as well as host genetics should be considered when deciding on future treatment protocols . efficient inclusion of all of these factors for drug - resistance surveillance requires broader implementation of wgs . the world health organization is currently analyzing the results of a surveillance effort looking for underlying resistance to pza and fq where phenotypic dst was paired with dna sequencing ( zignol , unpublished ) . the timing of the study is crucial in view of the fact that many of the new treatment regimens under investigation rely on these two drugs , and underlying resistance may severely compromise their success . applying wgs to clinical trials can also contribute important information on the success of a new treatment , as well as sorely needed data for new - in - class antibiotics such as pa-824 , delamanide , bedaquiline , and sq109 . together , this knowledge should be used to build an accurate picture of how the genetic makeup of a strain ultimately determines the success of treatment . due to high costs and logistical requirements , wgs technology might not find its way into many of the most affected and often resource - poor countries in the short term . however , applied at a supranational or national level with a wide - spread community - based catchment area , wgs would allow high - throughput analysis of known but also unknown mutations . healthcare officials would thus obtain surveillance data and essential information for the development of new diagnostic tests adjusted to the prevailing resistance pattern [ 39 , 40 , 42 , 90 ] . for example , one could imagine an approach in which used sputum - microscopy slides from primary tb - diagnostic centers are recycled as a source of m. tuberculosis dna for pooled dna sequencing to measure the frequency of drug - resistance alleles in a particular patient population . similarly , spent dst samples , such as mycobacterial growth indicator tubes ( mgit ) and loewenstein jensen slopes , would provide an excellent source of dna for wgs - based surveillance . the application of wgs and evolutionary principles to drug resistance in m. tuberculosis has been furthering our understanding of the challenges faced in the clinic as well as contributing key data for developing tools and strategies to control drug - resistant tb . as new treatment regimens containing new drugs are implemented , we will have to establish the spectrum of epidemiologically relevant mutations as soon as possible . this will help us track the emergence of strains resistant to these drugs in real - time , thereby prolonging the life span of the new regimens a fundamental concern given how precious new drugs are .

drug - resistant tuberculosis is a growing threat to global public health . recent efforts to understand the evolution of drug resistance have shown that changes in drug target interactions are only the first step in a longer adaptive process . the emergence of transmissible drug - resistant mycobacterium tuberculosis is the result of a multitude of additional genetic mutations , many of which interact , a phenomenon known as epistasis . the varied effects of these epistatic interactions include compensating for the reduction of the biological cost associated with the development of drug resistance , increasing the level of resistance , and possibly accommodating broader changes in the physiology of resistant bacteria . knowledge of these processes and our ability to detect them as they happen informs the development of diagnostic tools and better control strategies . in particular , the use of whole genome sequencing combined with surveillance efforts in the field could provide a powerful instrument to prevent future epidemics of drug - resistant tuberculosis .

Introduction New Genomic Insights into Implications for Diagnosis and Therapy Conclusion

the burden of tuberculosis ( tb ) caused by drug - resistant mycobacterium tuberculosis is increasing . as a result , the standard treatment directly observed therapy , short course ( dots ) , composed of 2 months of a four - drug regimen ( isoniazid [ inh ] , rifampicin [ rif ] , pyrazinamide [ pza ] , and ethambutol [ emb ] ) followed by 4 months of treatment with rif and inh is failing in many settings . while any degree of drug resistance might worsen the prognosis of a tb patient [ 1 , 2 ] , there are two definitions of m. tuberculosis drug resistance that are particularly relevant in the clinic . the first refers to strains resistant to both inh and rif and is termed multidrug - resistant ( mdr ) tb . treatment of patients with mdr - tb takes up to 2 years , and currently relies on fluoroquinolones ( fq ) and injectable aminoglycosides ( ag ) to compensate for the loss of two of the most potent drugs . the acquisition of resistance to these classes of antibiotics defines extensively drug - resistant ( xdr ) tb . the global genetic diversity of drug - resistant m. tuberculosis [ 410 ] indicates that drug resistance evolved on multiple occasions in geographical hotspots characterized by a high incidence of tb and inappropriate drug treatment . the latter is mostly driven by a lack of resources resulting in two important failures : an inability to detect drug resistance and a systemic failure to deploy effective treatments [ 1113 ] . the ongoing evolution of m. tuberculosis in these settings [ 14 , 15 ] , provides an excellent opportunity to explore the genetic determinants of drug resistance in this microbe . unlike most other bacterial pathogens , resistance plasmids and horizontal gene transfer play no role in the acquisition of drug resistance in m. tuberculosis . they allow the bacteria to tolerate higher levels of drug but do not per se result in clinically relevant levels of resistance to multiple antibiotics in m. tuberculosis [ 14 , 1618 ] . instead , the evolution of strains resistant to multiple antibiotics is driven by the sequential acquisition and accumulation of resistance - conferring mutations on the bacterial chromosome . these mutations primarily interfere with drug - target binding ( e.g. the elucidation of the mechanisms of action for many antimycobacterials led to the identification of key determinants of resistance ( see zhang and yew and almeida da silva and palomino for reviews ) , and the realization that the corresponding genetic mutations can be used as reliable molecular markers for drug susceptibility testing ( dst ) . the application of this knowledge to the clinic has resulted in the development of diagnostic tools based on nucleic acid amplification ( naa ) that overcome many of the shortcomings of phenotypic dst ; these include a long turnaround time , outcome variability , and infrastructure requirements [ 2629 ] . recently , one of these tools , known as xpert mtb / rif , has been the subject of a policy update published by the world health organization . even though this technology is having a positive impact on tb control by offering high sensitivity and reducing the time to tb diagnosis , the associated costs and infrastructural requirements ( e.g. hence , on - going efforts in product development focus on cheaper and simpler so - called point - of - care diagnostics . it should be based on detailed knowledge of the relationship between strain genotype , drug resistance phenotype , and the patient treatment outcome in terms of relapse rate and treatment failure . whole genome sequencing ( wgs ) of clinical drug - resistant strains of m. tuberculosis should be combined with the analysis of clinical information from patients infected by these strains to yield valuable new insights into the biology of drug resistance . in this review we focus specifically on results that shed light on why the acquisition of individual drug resistance - conferring mutations is only part of the problem . we then use specific examples to illustrate how understanding more broadly the evolutionary mechanisms that drive drug resistance can inform the development of improved diagnostic tools as well as better strategies to preserve both existing and new treatment regimens . the evolutionary path leading through drug resistance is strongly influenced by two factors : epistasis and bacterial fitness [ 3538 ] . for example , resistant strains carrying the same resistance mutations vary in their capacity to transmit from patient to patient [ 3942 ] , showing that the strain genetic background can determine the course of evolution of drug resistance . bacterial fitness , on the other hand , is a function of growth rate , virulence , and transmissibility [ 15 , 43 ] . on average , 3 ) whose magnitude positively correlates with the frequency of different resistance mutations in the clinic ; resistant strains with the smallest fitness defect are most abundant [ 45 , 46 ] . furthermore , the fitness of resistant mutants is not fixed ; evolution is an ongoing process , and a comparison between clinical and laboratory strains carrying the same drug - resistance mutation shows that clinical strains often successfully bypass any fitness cost imposed by resistance . the acquisition of such compensatory mutations is also an example of epistasis and is key in the evolution of transmissible drug - resistant strains that pose the greatest risk to public health [ 6 , 10 , 4751 ] . unfortunately , we do not currently have sufficient knowledge to predict epistatic interactions a priori , so we must rely on detecting them empirically by studying the genetics of drug resistance . a number of recent studies used wgs to address the evolution of drug - resistant m. tuberculosis [ 7 , 9 , 51 , 53 , 54 ] . the authors of these studies used approaches based on phylogeny , molecular epidemiology [ 9 , 51 , 53 ] , and mutation frequency analyses to compare drug - susceptible and drug - resistant clinical strains of m. tuberculosis . the detailed discussion of the merits and shortcomings of the analytical approaches used in these studies is beyond the scope of this review , but it is important to note that there is considerable overlap in the findings of these studies . in particular , several genes involved in lipid metabolism , cell wall homeostasis , purine metabolism , and transcriptional control appear to be positively selected in presence of anti - tb drugs [ 7 , 54 ] . pona1 , and the promoters of thya and thyx [ 7 , 54 ] ) , the actual role of these genes remains unclear , essentially offering an extended list of genes of interest that require further investigation . consequently , it is currently not possible to evaluate the role of these genes in the development of future diagnostics or therapies.fig . key genes in m. tuberculosis drug resistance have been plotted , taking into account their approximate position in the genome . lines denote putative epistatic interactions ; connecting genes involved in the physiology of a drug as well as more broad / indirect mechanisms referred to as ancillary to drug resistance. key genes in m. tuberculosis drug resistance have been plotted , taking into account their approximate position in the genome . lines denote putative epistatic interactions ; connecting genes involved in the physiology of a drug as well as more broad / indirect mechanisms referred to as ancillary to drug resistance. these genes were identified through recent studies aimed at elucidating the evolutionary trajectory of drug - resistant m. tuberculosis in the clinic [ 6 , 47 ] and include rpoc , which mediates the adaptation to rif resistance , and rv3806c , which appears to have a role in emb resistance . mutated amino acids are normally involved in drug binding and are usually restricted within a short stretch of the protein termed the rifampicin resistance determining region ( rrdr ) . many of these mutations carry a fitness cost [ 45 , 49 ] that appears to be negatively correlated with the activity of the mutant enzyme . the importance of this aspect of rna polymerase physiology is clearly illustrated by the fact that one of the most frequent resistance mutations observed in the clinic , s450l , ( equivalent to s531l in escherichia coli ) also carries the lowest fitness cost . moreover , this mutation appears to be almost ubiquitous among mdr strains of m. tuberculosis [ 6 , 9 , 10 , 48 , 51 ] , and perhaps more importantly , is shown to be strongly associated with the acquisition of compensatory mutations within rna polymerase genes ( rpoa , rpob , and rpoc ) . this combination of mutations is strongly associated with improved transmissibility of strains as evidenced by clonal expansion of m. tuberculosis strains carrying these particular mutations in patient populations [ 9 , 48 , 51 ] . for a more comprehensive review ) appears to be to restore wild - type function of mutant rna polymerase , probably on the level of enzymatic activity [ 49 , 50 ] . interestingly , lipid metabolism genes , including those for pdim metabolism , seem to be under positive selection during the evolution of drug resistance [ 7 , 54 ] . because pdims and other mycobacterial lipids play an important role in virulence [ 59 , 60 ] , this argues that global physiological consequences of drug - resistance mutations could provide a contextual framework , within which the compensatory mechanisms mediated by mutations in ancillary genes can be explored [ 7 , 51 , 54 ] . demonstrate the effect of epistasis in the progressive increase of minimal inhibitory concentration ( mic ) for a drug . moreover , the particular combinations of mutations conferring resistance to rif and ofloxacin ( ofx ) associated with the highest overall fitness appear to be positively selected in high - burden settings . the examples listed here are not designed to offer a comprehensive overview of all reported examples of epistasis in drug - resistant m. tuberculosis ; we have tried to sketch a more complete picture in fig . instead , they were chosen to illustrate important concepts brought to light by recent studies : in the first place , based on current data , it appears that compensatory mutations occur most frequently in strains that already harbor the least costly mutation . second , epistatic interactions occur between specific mutations [ 46 , 47 , 49 ] , and in some cases these can be mutually exclusive ; for example , a strain harboring an rpoa mutation does not then also acquire an rpoc or additional rpob mutations [ 6 , 50 , 51 , 62 ] . a further consequence of continued drug exposure is the stepwise accumulation of mutations that result in an increased level of resistance to a drug ; a factor that is already influencing inh resistance in xdr strains and may contribute to higher levels of resistance to fq [ 64 , 65 ] . moreover , epistasis can occur between drug - resistance mutations [ 46 , 61 , 66 ] , implicating individual resistance determinants as drivers of polydrug resistance . combined , these observations clearly indicate that continued inappropriate treatment , in part caused by misdiagnosis of resistance , drives the evolution of more transmissible , increasingly drug - resistant strains [ 10 , 51 ] . the primary source of this is mutations , and mutation rates were shown to vary between different phylogenetic lineages of m. tuberculosis . the beijing family of strains in particular seems to have a higher mutation rate [ 6769 ] . the exact consequences of this remain to be determined , but differences in mutation rate have been used to explain the correlation between this genotype with a higher rate of drug - resistance acquisition . , who argue that protein variability driven by errors in the central information - processing pathway ( dna - rna - protein ) may provide a phenotypic stepping - stone to resistance akin perhaps to other ancillary mechanisms shown in fig . while intellectually appealing , the clinical relevance of such a scenario remains to be substantiated . the translational potential of the knowledge gained from the above studies lies in the implementation of molecular tools in tb control ( see table 1 for a summary ) . offer an excellent review of current and future needs for the clinical management of tb , addressing diagnostics , surveillance , and programmatic approaches . accordingly , the available resources combined with the specific needs of each level will dictate the contribution that molecular approaches can offer.table 1implications of understanding evolutionary mechanisms of drug resistance for tuberculosis controlexperimental observationphysiological consequenceimplicationsresistant strains gain compensatory mutations that change the basic physiology , e.g. rif - rpoc increased transmissibility , increased propensity to acquire additional resistancefocus surveillance on mutations that correlate with transmissible , highly resistant strainsuse spent biosamples to establish wide catchment area for wgs - based surveillancecontinued exposure to rif directs evolution towards the acquisition of compensatory mutationsmore rapid evolution of compensatory mutationsuse molecular tools to probe the genotype of strains and stop administering ineffective drugs immediatelyepistasis exists between drug - resistance mutations for a single drug : e.g. emb , fq , inh , rifmulti - step acquisition of high level of drug resistancedefine clinical breakpoint concentrations based on specific susceptibility profiles for a drugpositive epistasis between rpob and gyra mutationsstrains with specific combinations of resistance mutations ( e.g. assess current clinical trials for evidenceevaluate if the order in which drugs are administered might enhance negative epistatic interactionssome mutations conferring resistance to fq and bedaquiline have no fitness costs attachedno need to compensate for specific resistance mutationsenforce appropriate administration of drugs to avoid adding these antibiotics to failing regimens or use as monotherapyexplore the spectrum of resistance mutations , identify low cost and frequent mutations before releasing the drug into the marketmutation rates vary between m. tuberculosis lineagesbeijing family strains acquire resistance to inh and rif at a higher rate and are 22 times more likely to develop into mdr than the laboratory - adapted strainincrease frequency of phylogeny - based surveillance and focus monitoring on areas with high rates of beijing strains emb ethambutol , fq fluoroquinolones , inh isoniazid , rif rifampicin , wgs whole genome sequencingfig . different diagnostic and dst technologies are shown as bars with the arrows indicating the levels at which we would ideally deploy them in the future . wgs whole genome sequencing , lpa line probe assay , mgit mycobacterial growth indicator tube : phenotypic dst using the bactec mgit 960 instrument , automated nat automated nucleic acid amplification technology implications of understanding evolutionary mechanisms of drug resistance for tuberculosis control emb ethambutol , fq fluoroquinolones , inh isoniazid , rif rifampicin , wgs whole genome sequencing the healthcare continuum . different diagnostic and dst technologies are shown as bars with the arrows indicating the levels at which we would ideally deploy them in the future . wgs whole genome sequencing , lpa line probe assay , mgit mycobacterial growth indicator tube : phenotypic dst using the bactec mgit 960 instrument , automated nat automated nucleic acid amplification technology in the clinic , the main goal is rapid and effective treatment of infected individuals . this relies on quick and reliable case detection , and , given the need for speed , is likely to depend on molecular diagnostics in the future . in contrast , a key area in which evolutionary lessons could be brought to bear in a significant manner is surveillance . strain fitness plays a role in the former , while drug resistance considerably influences the latter . it is in the interest of public health to identify and eradicate transmissible drug - resistant strains quickly . to this end , there is scope for the implementation of molecular diagnostics for public health. screening for the emergence of compensatory mutations in a population , perhaps by focusing on re - treatment cases to pinpoint areas with evolved highly transmissible strains as they emerge [ 48 , 51 ] , would aid the public health official to prioritize resource allocation and interrupt the spread of these strains . in line with the described differences in mutation rates between m. tuberculosis lineages [ 67 , 68 ] , the public health official may include phylogenetic markers to focus monitoring on areas with a high incidence of beijing strains . mutations conferring intermediate resistance , namely , below that of empirically determined resistance breakpoints , play a potentially pivotal role in the spread of resistance . it is also important to stress at this point , that strains harboring mutations conferring low - level resistance can often be treated effectively with existing drugs , either by increasing the dosage of the same drug , or by using alternative drugs from the same class , as is the case of fq . conversely , the use of ineffective drugs should be stopped immediately to avoid directing the evolution of a strain [ 36 , 50 ] towards a more transmissible phenotype.fig . the relative fitness of strains carrying key drug - resistance mutations grown in the absence of drug was plotted against their contribution to mic to illustrate the relationship between genotype and phenotype for important drug - resistance mutations . lines connecting individual mutations denote strains carrying two mutations , while arrows denote estimates of the fitness of double / triple - mutants ( three different types of arrows are used to illustrate different evolutionary trajectories ) . [ 47 , 100 , 101 ] , and starks et al . [ 94 , 102105 ] , and malik et al . the relative fitness of strains carrying key drug - resistance mutations grown in the absence of drug was plotted against their contribution to mic to illustrate the relationship between genotype and phenotype for important drug - resistance mutations . lines connecting individual mutations denote strains carrying two mutations , while arrows denote estimates of the fitness of double / triple - mutants ( three different types of arrows are used to illustrate different evolutionary trajectories ) . a number of current clinical trials are testing an iteration of such a combination , and their outcomes should be carefully scrutinized by using wgs for evidence of epistasis - driven drug resistance [ 74 , 8082 ] . bedaquiline is the first new antimycobacterial to be approved by the us fda for over 40 years [ 83 , 84 ] . 3 ) , it is key that the guidelines for the administration of the drug are adhered to [ 86 , 87 ] , especially in light of the fact that no standardized regimen exists for it yet . most importantly , bedaquiline should not be used to rescue a failing regimen , and should always be administered with other effective drugs to minimize the emergence of resistance . consensus is growing that biological parameters such as the frequency of resistance mutations , the occurrence of low- or no - cost drug - resistance mutations , epistasis between resistance mutations , as well as host genetics should be considered when deciding on future treatment protocols . efficient inclusion of all of these factors for drug - resistance surveillance requires broader implementation of wgs . the timing of the study is crucial in view of the fact that many of the new treatment regimens under investigation rely on these two drugs , and underlying resistance may severely compromise their success . applying wgs to clinical trials can also contribute important information on the success of a new treatment , as well as sorely needed data for new - in - class antibiotics such as pa-824 , delamanide , bedaquiline , and sq109 . due to high costs and logistical requirements , wgs technology might not find its way into many of the most affected and often resource - poor countries in the short term . healthcare officials would thus obtain surveillance data and essential information for the development of new diagnostic tests adjusted to the prevailing resistance pattern [ 39 , 40 , 42 , 90 ] . for example , one could imagine an approach in which used sputum - microscopy slides from primary tb - diagnostic centers are recycled as a source of m. tuberculosis dna for pooled dna sequencing to measure the frequency of drug - resistance alleles in a particular patient population . the application of wgs and evolutionary principles to drug resistance in m. tuberculosis has been furthering our understanding of the challenges faced in the clinic as well as contributing key data for developing tools and strategies to control drug - resistant tb . this will help us track the emergence of strains resistant to these drugs in real - time , thereby prolonging the life span of the new regimens a fundamental concern given how precious new drugs are .

improving the quality of obstetric care and especially peripartum care , is one of the most critical challenges to achieve the millennium development goals ( 1 ) . evidence - based practice is an effective strategy to improve the quality of obstetric care ( 2 ) . there is a worldwide concern that no evidence - based interventions and practices in labor and birth remain as standard practices ( 3 , 4 ) . the world health organization ( who ) has already emphasized that the ineffective and harmful clinical practices should be replaced with evidence - based clinical ones ( 5 ) . unfortunately , in many developing countries , some ineffective or harmful interventions are used as routine care during labor and delivery , while beneficial practices are not implemented for a lot of mothers ( 6 ) . it is not always an easy process to encourage healthcare providers to change routine interventions in line with evidence based practice ( 7 ) . professional behavior change is a complex process and the barriers to change may vary in different clinical environments , groups of health care providers or clinical practices ( 8 , 9 ) . previous studies reported some barriers to adopting evidence based practice ( ebp ) including the lack of time and resources , and conflicts between healthcare professionals and physical environment ( 10 ) . it is necessary to understand and determine healthcare providers behavior to develop change effective strategies ( 10 ) . in a cochrane systematic review , baker reported that efforts to change professional practice have a lower likelihood of success unless the barriers are identified and taken into account . there is insufficient evidence on the most effective approaches to tailoring , including how barriers should be identified and how interventions should be selected to address the barriers ( 9 ) . the current study aimed to explore the barriers to adopting evidence based normal labor and delivery management of healthcare professional perspectives that provide obstetric care in iran . a descriptive exploratory qualitative study was conducted from december 2013 to april 2014 in fourteen state medical training centers in iran . purposive sampling was used to recruit the subjects ( maximum variation sampling ) . in purposive sampling , the researcher is seeking the people with rich experiences of the phenomenon and possess the ability and willingness to express them ( 11 ) . inclusion criteria were midwives , specialists , and residents of obstetrics and gynecology with a clinical working experience of at least three years , and the desire to express their experience . exclusion criteria included having less than three years of experience , being dissatisfied , a history of psychological disorders , lack of employment , and request for leaving the study . face - to - face semi structured in - depth interviews were used to gather the data . first , the interviews were started by a general question : what are barriers to using ebp during intrapartum care ? then , they were asked to explain their experiences about challenges of ebp application during labor and delivery management . duration of the interviews ranged from 30 minutes to an 105 minutes , and some participants were interviewed twice , if necessity . in general , data saturation happened when other new codes or categories does not appear from the last two interviews . the conventional content analysis introduced by graneheim and landman was the basis of data analysis ( 12 ) . first , after transcribing each interview , the researcher reviewed the text several times until a general impression was received . third , each meaningful unit was summarized to a condensed meaningful unit and then primary codes were obtained . eventually , themes were determined as the expression of the hidden content of the text in the fifth stage . in the current study , to ensure the reliability of data some criteria were considered . long term involvement with the participants to collect information and feedback from participants was employed to increase the credibility . in terms of enhanced transferability , researchers used purposeful sampling and selected participants with various experiences . also , the researchers attempted to describe the study process and performed accurate and consistent activities considering the objectives approach . in order to increase the dependability and confirmability , member checking ( participants confirmed samples of codes ) , and peer debriefing ( approving examples of coded data by skilled and expert qualitative researchers ) were conducted . the current study was confirmed by the ethics committee of the isfahan university of medical sciences ( no : 391206 ) . the purpose of the study was described to all participants , and written informed consent was signed by them according to the helsinki declaration . furthermore , it was explained that the participants could leave the study whenever they wished . they were also told that their information would remain confidential during and after the research . the data were obtained from interviews with 34 participants . the participants comprised seven directors of labor ward , twelve midwives , eight obstetrician , and seven obstetric residents ( table 1 ) . the main categories were barriers related to laboring women , persons providing care , the organization environment , and barriers related to health system ( table 2 ) . a number of barring factors were found in association with the laboring women including : lack of knowledge in women , request for ineffective or injurious care , lack of partnership in women regarding decision making for the type of care . the participants in this study stated that the laboring women did not have the basic knowledge about safety of clinical practices during labor . sometimes women are not aware of the risks and benefits of unnecessary interventions ; therefore they request ineffective or even harmful interventions during the labor ( participant 3 ) . lack of partnership in women regarding decision making for the type of care was another barrier . in iran , the parturient women do not decide on the type of care and treatment , and it completely depends upon the decision made by the midwife or physician . one of the participants reported that : this is the attitude of iranian women ; they accept physician s decisions and rarely disagree several barriers were identified regarding the healthcare providers , including lack of knowledge ; lack of required skills ; lack of motivation to change behavior or adopt new behaviors ; lack of authority to make decisions ; being afraid of legal issues and lack of good communication between health care providers and the patients . it was observed that the healthcare providers sometimes did not have the basic knowledge about several practices during labor care : although i take care of laboring women for several years , i did not know that routine episiotomy is harmful and it should be avoided ( participant 14 ) . some of the subjects reported that their little care - related skill was formed empirically during obstetric care . sometimes i do not know if what i am doing for her is right or wrong ( participant 4 ) . some of the health providers did not change professional performance duo to their individual belief and attitude . i believe that when the family members are in the labor room the women tend to be uncooperative a main concern of midwives was lack of authority to make decisions about performance of practices during labor . a senior midwife said : we do not have enough power to change women care procedures because , director in our ward do not let the implementation of the ebp during labor ( participant 22 ) . the current study specified that legal issues were an important barrier to implementing evidence - based intrapartum care . one executive director explained : since the safety of mother and baby is very momentous for all obstetricians , many decisions are made based on fear of legal issues , not evidence based . another identified barrier was lack of good communication between health care providers and parturient women . a senior midwife said : verbal communication between health care providers and parturient women about the quality of care is very low . good and honest communication with the doctor and midwife is important for many women in the labor process ( participant 9 ) . there were a number of factors regarding the organizational factors , including problems with educational system ; lack of effective communication between obstetricians and midwives ; lack of time in healthcare providers due to lack of human resources ; inappropriate physical structure of birth setting ; lack of equipment in the birth setting and lack of clinical supervision by first - line managers . subjects reported that : inadequate education of health care providers is an essential barrier in the use of evidence based health care . there was a barrier related to lack of effective communication between obstetricians and midwives for labor and delivery management . one of midwives explained that : the healthcare professionals need to work together as a team to apply evidence - based health care during labor and delivery . a number of obstetricians in our hospital do not collaborate with implementing the ebp during labor ( participant 27 ) . a midwife said that : our labor ward is really busy . we are forced to care on two or three laboring women at a time . not possible for us to provide care steadily . other barriers related to the organization were shortage of equipment and inappropriate physical structure of birth setting . therefore , women have no privacy during labor . also , laboring women had a limited space to walk and change their status . on the other hand , there was not enough equipment to implement the ebp during labor ( participant 10 ) . many subjects suggested that lack of clinical supervision by managers is another barrier at the organization level . a midwife believed that : the managers have a vital role in the eight use of ebp or can be moved to after role ; therefore if clinical governance is done properly and regularly incorporated to the managers meetings , the clinical staff , take more accurate care there were several barriers to health system including inappropriate planning , management , monitoring and feedback system , lack of accurate information system , good communication between healthcare providers and policy makers , and lack of good health policy . a number of participants identified the need for good planning , management and monitoring to use ebp during labor . the managers needed more training for appropriate planning , management and monitoring the use of ebp during labor ( participant 13 ) . some respondents identified the need to record and report accurate information about routine practices and quality of care during childbirth . a midwife commented : i think if the information is not accurately recorded , the problems are not correctly identified and thus we can not improve the quality of maternity care services ( participant 30 ) . lack of communication between healthcare providers and policy makers were reported as a barrier to apply ebp during labor . it appears that if obstacles are inquired directly from the staff , policymakers can plan to overcome these barriers better lack of good health policies was reported as a barrier to adapt ebp during labor . i believe that a good policy in the field of maternity care is to use of ebp , which is a really effective instrument . of course , if the policy makers monitor the correct implementation of the policies many subjects thought that lack of financial resources is a major problem in maternity services . a director said : more often , lack of financial resources is a major problem for quality improvement in the health system ; for example , one of the solutions to overcome the shortage of staff and lack of equipment is financial resources a number of barring factors were found in association with the laboring women including : lack of knowledge in women , request for ineffective or injurious care , lack of partnership in women regarding decision making for the type of care . the participants in this study stated that the laboring women did not have the basic knowledge about safety of clinical practices during labor . sometimes women are not aware of the risks and benefits of unnecessary interventions ; therefore they request ineffective or even harmful interventions during the labor ( participant 3 ) . lack of partnership in women regarding decision making for the type of care was another barrier . in iran , the parturient women do not decide on the type of care and treatment , and it completely depends upon the decision made by the midwife or physician . one of the participants reported that : this is the attitude of iranian women ; they accept physician s decisions and rarely disagree several barriers were identified regarding the healthcare providers , including lack of knowledge ; lack of required skills ; lack of motivation to change behavior or adopt new behaviors ; lack of authority to make decisions ; being afraid of legal issues and lack of good communication between health care providers and the patients . it was observed that the healthcare providers sometimes did not have the basic knowledge about several practices during labor care : although i take care of laboring women for several years , i did not know that routine episiotomy is harmful and it should be avoided ( participant 14 ) . some of the subjects reported that their little care - related skill was formed empirically during obstetric care . sometimes i do not know if what i am doing for her is right or wrong ( participant 4 ) . some of the health providers did not change professional performance duo to their individual belief and attitude . i believe that when the family members are in the labor room the women tend to be uncooperative a main concern of midwives was lack of authority to make decisions about performance of practices during labor . a senior midwife said : we do not have enough power to change women care procedures because , director in our ward do not let the implementation of the ebp during labor ( participant 22 ) . the current study specified that legal issues were an important barrier to implementing evidence - based intrapartum care . one executive director explained : since the safety of mother and baby is very momentous for all obstetricians , many decisions are made based on fear of legal issues , not evidence based . another identified barrier was lack of good communication between health care providers and parturient women . a senior midwife said : verbal communication between health care providers and parturient women about the quality of care is very low . good and honest communication with the doctor and midwife is important for many women in the labor process ( participant 9 ) . there were a number of factors regarding the organizational factors , including problems with educational system ; lack of effective communication between obstetricians and midwives ; lack of time in healthcare providers due to lack of human resources ; inappropriate physical structure of birth setting ; lack of equipment in the birth setting and lack of clinical supervision by first - line managers . subjects reported that : inadequate education of health care providers is an essential barrier in the use of evidence based health care . there was a barrier related to lack of effective communication between obstetricians and midwives for labor and delivery management . one of midwives explained that : the healthcare professionals need to work together as a team to apply evidence - based health care during labor and delivery . a number of obstetricians in our hospital do not collaborate with implementing the ebp during labor ( participant 27 ) . a midwife said that : our labor ward is really busy . we are forced to care on two or three laboring women at a time . not possible for us to provide care steadily . other barriers related to the organization were shortage of equipment and inappropriate physical structure of birth setting . therefore , women have no privacy during labor . also , laboring women had a limited space to walk and change their status . on the other hand , there was not enough equipment to implement the ebp during labor ( participant 10 ) . many subjects suggested that lack of clinical supervision by managers is another barrier at the organization level . a midwife believed that : the managers have a vital role in the eight use of ebp or can be moved to after role ; therefore if clinical governance is done properly and regularly incorporated to the managers meetings , the clinical staff , take more accurate care there were several barriers to health system including inappropriate planning , management , monitoring and feedback system , lack of accurate information system , good communication between healthcare providers and policy makers , and lack of good health policy . a number of participants identified the need for good planning , management and monitoring to use ebp during labor . the managers needed more training for appropriate planning , management and monitoring the use of ebp during labor ( participant 13 ) . some respondents identified the need to record and report accurate information about routine practices and quality of care during childbirth . i think if the information is not accurately recorded , the problems are not correctly identified and thus we can not improve the quality of maternity care services ( participant 30 ) . lack of communication between healthcare providers and policy makers were reported as a barrier to apply ebp during labor . it appears that if obstacles are inquired directly from the staff , policymakers can plan to overcome these barriers better lack of good health policies was reported as a barrier to adapt ebp during labor . i believe that a good policy in the field of maternity care is to use of ebp , which is a really effective instrument . of course , if the policy makers monitor the correct implementation of the policies ( participant 11 ) . many subjects thought that lack of financial resources is a major problem in maternity services . a director said : more often , lack of financial resources is a major problem for quality improvement in the health system ; for example , one of the solutions to overcome the shortage of staff and lack of equipment is financial resources the current study identified several important barriers to the implementation of evidence - based maternity care . low awareness of laboring women of the care options during labor was an important barrier . women should receive adequate information to recognize the benefits and risks of each practice ; therefore , they can choose the best care between the different options of treatment . the clinicians often emphasized the fact that parturient women should have the right of choice , but the reality is that most of them do not have the ability to choose ( 13 ) . therefore , increasing women s awareness about benefits and risks of interventions is a good strategy to sensitize women and increase effective communication between health professionals and parturient women ( 14 ) . low women 's participation in decision - making for the type of care was another barrier . although the involvement of women in decision making about their care has better outcomes for them , the physicians and midwives often do not involve them in making such decisions ( 15 ) . second , there were several obstacles at the level of caregivers . based on the finding of the study , lack of awareness , knowledge , and skill in clinical personnel were factors affecting the failure to adopt the best practice . grady reported that the attendance of skilled midwives and essential maternity care are important strategies to reduce maternal and newborn morbidity and mortality . thus , the shortage of sufficiently skilled birth attendants is a key barrier to achieve this thread ( 16 ) . nevertheless , healthcare professionals need to be trained and motivated to adaptation of the newest evidence into their daily practices ( 17 ) . findings of the current study indicated that one of problems and challenges in intrapartum care is the resistance to acceptance and use of ebp by healthcare professionals . the participants believed that health providers may refuse to accept new care because they do not have the needed skills to do new practices , belizan et al . furthermore , changing a complex process requires commitment and strong , relentless , and creative leadership within the institution ( 19 ) . the midwives ability to use evidence based practice is directly related to the power of professional midwives in the maternity care system that give priority to the power of obstetricians ( 20 ) . being afraid of lawsuits was perceived as another barrier to high quality , evidence based care . fear of legal issues in clinical decision - making process by the obstetricians causes them to implement unnecessary interventions to prevent unwanted consequences for mother and newborn ( 13 ) . another barrier at this level was lack of good communication between healthcare providers and women . developing honest and effective communication between midwives and women makes positive childbirth experiences for parturient women ( 21 ) . the problems of training and educating system for physicians and midwives are still potential barriers to use evidence based intrapartum care . smith reported that inadequate training and education of healthcare professionals is a problem to achieve safety in maternity services . he believed that due to changes in medical training , junior clinicians have less skill than they had in the past . he also said : within the medical profession , gynecology is observed as a more prestigious specialty than obstetrics , which could lead to problems in the future . on one hand , he believed that the shift in midwives training from practical training to academic degree has not necessarily improved parturient women s safety ( 22 ) . thus , according to the statement by sicily on ebp , it is essential to include knowledge , attitudes and skills of ebp in the curriculum ( 23 ) . training needs of healthcare providers also need to be promoted by means of more educational programs ( 6 ) . professionals seemed to agree that lack of communication across clinical groups is another barrier to implementing evidence - based intrapartum care . lack of proper communication can be considered as one of the causes of preventable injury and death of mothers and newborns . in contrast , establishing good communication and teamwork in the healthcare team could create a safe and friendly environment for the mothers and newborns during labor and delivery ( 24 ) . the high workload and shortage of time on health care providers due to limited number of staffs were regarded as an inhibiting factor to apply ebp in the clinical settings . for example , increasing the number of midwives would help to provide continuous care to all parturient women during labor . also , midwives could be better deployed to improve safety by reduction of unnecessary interventions ( 22 ) . the physical structure factor and lack of equipment of birth setting the most important element of the physical structure that can be considered as a barrier is using common rooms for mothers during labor and delivery management . also , some subjects reported that using old instruments in the labor ward is a problem in the care process . gale reported that top barriers to ebp were insufficient time , lack of staff , and not having the right equipment and supplies ( 25 ) . there were some barriers to lack of clinical supervision by managers to implement ebp during labor . the study subjects believed that it is critical that the managers support the employment of evidence based practices . middle managers resistance is often described as a major challenge for upper - level administrators seeking to implement complex innovations such as evidence - based protocols or training new skills ( 26 ) . fourth , a number of barriers were identified to the health system . there were some barriers corresponding to inappropriate planning , management and monitoring of evidence - based clinical standards . successful implementation and diffusion of any practice change requires careful strategic planning ( 27 ) . appropriate management is one of the most important strategies to provide safe and high quality care in the health system ( 28 ) . inaccurate or insufficient data inhibits health information exchange , and hinders clinical research , performance improvement , and quality measurement initiatives . accurate data leads to quality information that is required for quality decision making and women s care ( 30 ) . there were some barriers corresponding to lack of good communication between healthcare providers and policy makers to use ebp during childbirth . in the study by uneke , lack of communication between health policy makers and researchers , and the lack of participation of service recipients in health planning were shown as the major challenges to improve evidence - based policy making process and practice ( 31 ) . furthermore , specific strategies to improve communication among healthcare providers and policymakers are necessary to adapt ebp during labor . there were some barriers corresponding to lack of good health policy to apply ebp during childbirth . health policy and health systems are interdependent to achieve improved health . to successfully implement the policy is dependent on the capacity of the health system ; and for the health system , to ensure access to evidence based , high quality care it is dependent on good policies ( 32 ) . thus , the policy makers should manage health policies at the national level and then they need to monitor using ebp in the clinical settings . there were some barriers to the lack of financial resources to use ebp during childbirth . results of a study suggested that the lack of financial resources is a major contributor to stress among the front line staff , which can be an unrecognized barrier by agency leadership ( 33 ) . the study found that the experiences of healthcare professionals working in the field of maternity care during labor are largely congruent with those of the healthcare professionals in various health care settings . however , there were some study limitations that reduced the potential to transfer the study findings . first , since the study was conducted in one country , the findings may not be generalized to other countries . second , the study subjects may not be representative of all healthcare professionals , according to the purposefully selected collaborators . some strong points of the study include : first , to date , within the field of maternity care services for pregnant women during normal labor and delivery management , it was the first iranian study to explore and identify health care professionals viewpoints on barriers to implementation of evidence - based intrapartum care . second , subjects were interviewed by the same interviewer ; nevertheless data analysis by the four authors contributed to the credibility of the findings . despite the advances in medical science to manage complex health problems , nowadays obstetric care in the clinical settings has increased dangers for low risk parturient women and their newborns ( 34 ) . sometimes , evidence based practices in obstetrics and neonatal care significantly reduce morbidity and mortality ( 35 ) . the current study identified a number of potential barriers according to the viewpoints of health professionals who work in the iranian maternity services to improve the implementation of evidence based intrapartum care . the findings of the study indicated that the women should feel free to ask if they are receiving evidence - based care . on the other hand , the reasons why labor ward staff persists in non - evidence - based practices are that many staff are resistant to change ( 37 ) . the presence of strong leadership and adequate financial resources affect healthcare provider attitudes toward ebps and facilitate implementation of these innovations ( 33 ) . the professional relationships between midwives , physicians , managers and policy makers providing obstetrical care is essential to succeed the application of evidence based intrapartum care . finally , developing evidence based national policies and the quality certainty systems will contribute to the reduction of unnecessary obstetric interventions and strongly emphasize on the needs and requests of women during childbirth ( 38 ) . since one of the elements for both evidence - based intrapartum care and woman - centered care is women preferences ( 39 ) . despite the advances in medical science to manage complex health problems , nowadays obstetric care in the clinical settings has increased dangers for low risk parturient women and their newborns ( 34 ) . sometimes , evidence based practices in obstetrics and neonatal care significantly reduce morbidity and mortality ( 35 ) . the current study identified a number of potential barriers according to the viewpoints of health professionals who work in the iranian maternity services to improve the implementation of evidence based intrapartum care . the findings of the study indicated that the women should feel free to ask if they are receiving evidence - based care . on the other hand , the reasons why labor ward staff persists in non - evidence - based practices are that many staff are resistant to change ( 37 ) . the presence of strong leadership and adequate financial resources affect healthcare provider attitudes toward ebps and facilitate implementation of these innovations ( 33 ) . the professional relationships between midwives , physicians , managers and policy makers providing obstetrical care is essential to succeed the application of evidence based intrapartum care . finally , developing evidence based national policies and the quality certainty systems will contribute to the reduction of unnecessary obstetric interventions and strongly emphasize on the needs and requests of women during childbirth ( 38 ) . since one of the elements for both evidence - based intrapartum care and woman - centered care is women preferences ( 39 ) .

background : evidence based practice is an effective strategy to improve the quality of obstetric care . identification of barriers to adaptation of evidence - based intrapartum care is necessary and crucial to deliver high quality care to parturient women.objectives:the current study aimed to explore barriers to adaptation of evidence - based intrapartum care from the perspective of clinical groups that provide obstetric care in iran.materials and methods : this descriptive exploratory qualitative research was conducted from 2013 to 2014 in fourteen state medical training centers in iran . participants were selected from midwives , specialists , and residents of obstetrics and gynecology , with a purposive sample and snowball method . data were collected through face - to - face semi - structured in - depth interviews and analyzed according to conventional content analysis.results:data analysis identified twenty subcategories and four main categories . main categories included barriers were related to laboring women , persons providing care , the organization environment and health system.conclusions:the adoption of evidence based intrapartum care is a complex process . in this regard , identifying potential barriers is the first step to determine and apply effective strategies to encourage the compliance evidence based obstetric care and improves maternity care quality .

1. Background 2. Objectives 3. Materials and Methods 4. Results 4.1. Barriers to Laboring Women 4.2. Barriers to the Person Providing Care 4.3. Barriers to the Organizational Environment 4.4. Barriers to Health System 4.5. Financial Resources 5. Discussion 5.1. Conclusions

improving the quality of obstetric care and especially peripartum care , is one of the most critical challenges to achieve the millennium development goals ( 1 ) . evidence - based practice is an effective strategy to improve the quality of obstetric care ( 2 ) . there is a worldwide concern that no evidence - based interventions and practices in labor and birth remain as standard practices ( 3 , 4 ) . the world health organization ( who ) has already emphasized that the ineffective and harmful clinical practices should be replaced with evidence - based clinical ones ( 5 ) . it is not always an easy process to encourage healthcare providers to change routine interventions in line with evidence based practice ( 7 ) . professional behavior change is a complex process and the barriers to change may vary in different clinical environments , groups of health care providers or clinical practices ( 8 , 9 ) . previous studies reported some barriers to adopting evidence based practice ( ebp ) including the lack of time and resources , and conflicts between healthcare professionals and physical environment ( 10 ) . it is necessary to understand and determine healthcare providers behavior to develop change effective strategies ( 10 ) . the current study aimed to explore the barriers to adopting evidence based normal labor and delivery management of healthcare professional perspectives that provide obstetric care in iran . a descriptive exploratory qualitative study was conducted from december 2013 to april 2014 in fourteen state medical training centers in iran . in purposive sampling , the researcher is seeking the people with rich experiences of the phenomenon and possess the ability and willingness to express them ( 11 ) . inclusion criteria were midwives , specialists , and residents of obstetrics and gynecology with a clinical working experience of at least three years , and the desire to express their experience . exclusion criteria included having less than three years of experience , being dissatisfied , a history of psychological disorders , lack of employment , and request for leaving the study . face - to - face semi structured in - depth interviews were used to gather the data . first , the interviews were started by a general question : what are barriers to using ebp during intrapartum care ? duration of the interviews ranged from 30 minutes to an 105 minutes , and some participants were interviewed twice , if necessity . in general , data saturation happened when other new codes or categories does not appear from the last two interviews . the conventional content analysis introduced by graneheim and landman was the basis of data analysis ( 12 ) . first , after transcribing each interview , the researcher reviewed the text several times until a general impression was received . in the current study , to ensure the reliability of data some criteria were considered . also , the researchers attempted to describe the study process and performed accurate and consistent activities considering the objectives approach . in order to increase the dependability and confirmability , member checking ( participants confirmed samples of codes ) , and peer debriefing ( approving examples of coded data by skilled and expert qualitative researchers ) were conducted . the current study was confirmed by the ethics committee of the isfahan university of medical sciences ( no : 391206 ) . the purpose of the study was described to all participants , and written informed consent was signed by them according to the helsinki declaration . the data were obtained from interviews with 34 participants . the participants comprised seven directors of labor ward , twelve midwives , eight obstetrician , and seven obstetric residents ( table 1 ) . the main categories were barriers related to laboring women , persons providing care , the organization environment , and barriers related to health system ( table 2 ) . a number of barring factors were found in association with the laboring women including : lack of knowledge in women , request for ineffective or injurious care , lack of partnership in women regarding decision making for the type of care . the participants in this study stated that the laboring women did not have the basic knowledge about safety of clinical practices during labor . in iran , the parturient women do not decide on the type of care and treatment , and it completely depends upon the decision made by the midwife or physician . one of the participants reported that : this is the attitude of iranian women ; they accept physician s decisions and rarely disagree several barriers were identified regarding the healthcare providers , including lack of knowledge ; lack of required skills ; lack of motivation to change behavior or adopt new behaviors ; lack of authority to make decisions ; being afraid of legal issues and lack of good communication between health care providers and the patients . it was observed that the healthcare providers sometimes did not have the basic knowledge about several practices during labor care : although i take care of laboring women for several years , i did not know that routine episiotomy is harmful and it should be avoided ( participant 14 ) . some of the subjects reported that their little care - related skill was formed empirically during obstetric care . the current study specified that legal issues were an important barrier to implementing evidence - based intrapartum care . one executive director explained : since the safety of mother and baby is very momentous for all obstetricians , many decisions are made based on fear of legal issues , not evidence based . another identified barrier was lack of good communication between health care providers and parturient women . a senior midwife said : verbal communication between health care providers and parturient women about the quality of care is very low . there were a number of factors regarding the organizational factors , including problems with educational system ; lack of effective communication between obstetricians and midwives ; lack of time in healthcare providers due to lack of human resources ; inappropriate physical structure of birth setting ; lack of equipment in the birth setting and lack of clinical supervision by first - line managers . subjects reported that : inadequate education of health care providers is an essential barrier in the use of evidence based health care . there was a barrier related to lack of effective communication between obstetricians and midwives for labor and delivery management . one of midwives explained that : the healthcare professionals need to work together as a team to apply evidence - based health care during labor and delivery . we are forced to care on two or three laboring women at a time . other barriers related to the organization were shortage of equipment and inappropriate physical structure of birth setting . also , laboring women had a limited space to walk and change their status . many subjects suggested that lack of clinical supervision by managers is another barrier at the organization level . a midwife believed that : the managers have a vital role in the eight use of ebp or can be moved to after role ; therefore if clinical governance is done properly and regularly incorporated to the managers meetings , the clinical staff , take more accurate care there were several barriers to health system including inappropriate planning , management , monitoring and feedback system , lack of accurate information system , good communication between healthcare providers and policy makers , and lack of good health policy . some respondents identified the need to record and report accurate information about routine practices and quality of care during childbirth . a midwife commented : i think if the information is not accurately recorded , the problems are not correctly identified and thus we can not improve the quality of maternity care services ( participant 30 ) . it appears that if obstacles are inquired directly from the staff , policymakers can plan to overcome these barriers better lack of good health policies was reported as a barrier to adapt ebp during labor . i believe that a good policy in the field of maternity care is to use of ebp , which is a really effective instrument . of course , if the policy makers monitor the correct implementation of the policies many subjects thought that lack of financial resources is a major problem in maternity services . a director said : more often , lack of financial resources is a major problem for quality improvement in the health system ; for example , one of the solutions to overcome the shortage of staff and lack of equipment is financial resources a number of barring factors were found in association with the laboring women including : lack of knowledge in women , request for ineffective or injurious care , lack of partnership in women regarding decision making for the type of care . the participants in this study stated that the laboring women did not have the basic knowledge about safety of clinical practices during labor . in iran , the parturient women do not decide on the type of care and treatment , and it completely depends upon the decision made by the midwife or physician . one of the participants reported that : this is the attitude of iranian women ; they accept physician s decisions and rarely disagree several barriers were identified regarding the healthcare providers , including lack of knowledge ; lack of required skills ; lack of motivation to change behavior or adopt new behaviors ; lack of authority to make decisions ; being afraid of legal issues and lack of good communication between health care providers and the patients . it was observed that the healthcare providers sometimes did not have the basic knowledge about several practices during labor care : although i take care of laboring women for several years , i did not know that routine episiotomy is harmful and it should be avoided ( participant 14 ) . some of the subjects reported that their little care - related skill was formed empirically during obstetric care . the current study specified that legal issues were an important barrier to implementing evidence - based intrapartum care . one executive director explained : since the safety of mother and baby is very momentous for all obstetricians , many decisions are made based on fear of legal issues , not evidence based . another identified barrier was lack of good communication between health care providers and parturient women . a senior midwife said : verbal communication between health care providers and parturient women about the quality of care is very low . there were a number of factors regarding the organizational factors , including problems with educational system ; lack of effective communication between obstetricians and midwives ; lack of time in healthcare providers due to lack of human resources ; inappropriate physical structure of birth setting ; lack of equipment in the birth setting and lack of clinical supervision by first - line managers . subjects reported that : inadequate education of health care providers is an essential barrier in the use of evidence based health care . there was a barrier related to lack of effective communication between obstetricians and midwives for labor and delivery management . one of midwives explained that : the healthcare professionals need to work together as a team to apply evidence - based health care during labor and delivery . we are forced to care on two or three laboring women at a time . other barriers related to the organization were shortage of equipment and inappropriate physical structure of birth setting . also , laboring women had a limited space to walk and change their status . many subjects suggested that lack of clinical supervision by managers is another barrier at the organization level . a midwife believed that : the managers have a vital role in the eight use of ebp or can be moved to after role ; therefore if clinical governance is done properly and regularly incorporated to the managers meetings , the clinical staff , take more accurate care there were several barriers to health system including inappropriate planning , management , monitoring and feedback system , lack of accurate information system , good communication between healthcare providers and policy makers , and lack of good health policy . the managers needed more training for appropriate planning , management and monitoring the use of ebp during labor ( participant 13 ) . some respondents identified the need to record and report accurate information about routine practices and quality of care during childbirth . i think if the information is not accurately recorded , the problems are not correctly identified and thus we can not improve the quality of maternity care services ( participant 30 ) . it appears that if obstacles are inquired directly from the staff , policymakers can plan to overcome these barriers better lack of good health policies was reported as a barrier to adapt ebp during labor . i believe that a good policy in the field of maternity care is to use of ebp , which is a really effective instrument . many subjects thought that lack of financial resources is a major problem in maternity services . a director said : more often , lack of financial resources is a major problem for quality improvement in the health system ; for example , one of the solutions to overcome the shortage of staff and lack of equipment is financial resources the current study identified several important barriers to the implementation of evidence - based maternity care . low awareness of laboring women of the care options during labor was an important barrier . the clinicians often emphasized the fact that parturient women should have the right of choice , but the reality is that most of them do not have the ability to choose ( 13 ) . therefore , increasing women s awareness about benefits and risks of interventions is a good strategy to sensitize women and increase effective communication between health professionals and parturient women ( 14 ) . although the involvement of women in decision making about their care has better outcomes for them , the physicians and midwives often do not involve them in making such decisions ( 15 ) . based on the finding of the study , lack of awareness , knowledge , and skill in clinical personnel were factors affecting the failure to adopt the best practice . grady reported that the attendance of skilled midwives and essential maternity care are important strategies to reduce maternal and newborn morbidity and mortality . thus , the shortage of sufficiently skilled birth attendants is a key barrier to achieve this thread ( 16 ) . nevertheless , healthcare professionals need to be trained and motivated to adaptation of the newest evidence into their daily practices ( 17 ) . findings of the current study indicated that one of problems and challenges in intrapartum care is the resistance to acceptance and use of ebp by healthcare professionals . furthermore , changing a complex process requires commitment and strong , relentless , and creative leadership within the institution ( 19 ) . the midwives ability to use evidence based practice is directly related to the power of professional midwives in the maternity care system that give priority to the power of obstetricians ( 20 ) . being afraid of lawsuits was perceived as another barrier to high quality , evidence based care . developing honest and effective communication between midwives and women makes positive childbirth experiences for parturient women ( 21 ) . the problems of training and educating system for physicians and midwives are still potential barriers to use evidence based intrapartum care . smith reported that inadequate training and education of healthcare professionals is a problem to achieve safety in maternity services . he believed that due to changes in medical training , junior clinicians have less skill than they had in the past . on one hand , he believed that the shift in midwives training from practical training to academic degree has not necessarily improved parturient women s safety ( 22 ) . thus , according to the statement by sicily on ebp , it is essential to include knowledge , attitudes and skills of ebp in the curriculum ( 23 ) . professionals seemed to agree that lack of communication across clinical groups is another barrier to implementing evidence - based intrapartum care . for example , increasing the number of midwives would help to provide continuous care to all parturient women during labor . also , midwives could be better deployed to improve safety by reduction of unnecessary interventions ( 22 ) . also , some subjects reported that using old instruments in the labor ward is a problem in the care process . gale reported that top barriers to ebp were insufficient time , lack of staff , and not having the right equipment and supplies ( 25 ) . there were some barriers to lack of clinical supervision by managers to implement ebp during labor . the study subjects believed that it is critical that the managers support the employment of evidence based practices . middle managers resistance is often described as a major challenge for upper - level administrators seeking to implement complex innovations such as evidence - based protocols or training new skills ( 26 ) . fourth , a number of barriers were identified to the health system . there were some barriers corresponding to inappropriate planning , management and monitoring of evidence - based clinical standards . appropriate management is one of the most important strategies to provide safe and high quality care in the health system ( 28 ) . inaccurate or insufficient data inhibits health information exchange , and hinders clinical research , performance improvement , and quality measurement initiatives . in the study by uneke , lack of communication between health policy makers and researchers , and the lack of participation of service recipients in health planning were shown as the major challenges to improve evidence - based policy making process and practice ( 31 ) . furthermore , specific strategies to improve communication among healthcare providers and policymakers are necessary to adapt ebp during labor . health policy and health systems are interdependent to achieve improved health . to successfully implement the policy is dependent on the capacity of the health system ; and for the health system , to ensure access to evidence based , high quality care it is dependent on good policies ( 32 ) . thus , the policy makers should manage health policies at the national level and then they need to monitor using ebp in the clinical settings . there were some barriers to the lack of financial resources to use ebp during childbirth . results of a study suggested that the lack of financial resources is a major contributor to stress among the front line staff , which can be an unrecognized barrier by agency leadership ( 33 ) . the study found that the experiences of healthcare professionals working in the field of maternity care during labor are largely congruent with those of the healthcare professionals in various health care settings . first , since the study was conducted in one country , the findings may not be generalized to other countries . second , the study subjects may not be representative of all healthcare professionals , according to the purposefully selected collaborators . some strong points of the study include : first , to date , within the field of maternity care services for pregnant women during normal labor and delivery management , it was the first iranian study to explore and identify health care professionals viewpoints on barriers to implementation of evidence - based intrapartum care . despite the advances in medical science to manage complex health problems , nowadays obstetric care in the clinical settings has increased dangers for low risk parturient women and their newborns ( 34 ) . sometimes , evidence based practices in obstetrics and neonatal care significantly reduce morbidity and mortality ( 35 ) . the current study identified a number of potential barriers according to the viewpoints of health professionals who work in the iranian maternity services to improve the implementation of evidence based intrapartum care . the findings of the study indicated that the women should feel free to ask if they are receiving evidence - based care . on the other hand , the reasons why labor ward staff persists in non - evidence - based practices are that many staff are resistant to change ( 37 ) . the professional relationships between midwives , physicians , managers and policy makers providing obstetrical care is essential to succeed the application of evidence based intrapartum care . finally , developing evidence based national policies and the quality certainty systems will contribute to the reduction of unnecessary obstetric interventions and strongly emphasize on the needs and requests of women during childbirth ( 38 ) . since one of the elements for both evidence - based intrapartum care and woman - centered care is women preferences ( 39 ) . despite the advances in medical science to manage complex health problems , nowadays obstetric care in the clinical settings has increased dangers for low risk parturient women and their newborns ( 34 ) . sometimes , evidence based practices in obstetrics and neonatal care significantly reduce morbidity and mortality ( 35 ) . the current study identified a number of potential barriers according to the viewpoints of health professionals who work in the iranian maternity services to improve the implementation of evidence based intrapartum care . the findings of the study indicated that the women should feel free to ask if they are receiving evidence - based care . on the other hand , the reasons why labor ward staff persists in non - evidence - based practices are that many staff are resistant to change ( 37 ) . the professional relationships between midwives , physicians , managers and policy makers providing obstetrical care is essential to succeed the application of evidence based intrapartum care . finally , developing evidence based national policies and the quality certainty systems will contribute to the reduction of unnecessary obstetric interventions and strongly emphasize on the needs and requests of women during childbirth ( 38 ) . since one of the elements for both evidence - based intrapartum care and woman - centered care is women preferences ( 39 ) .

cell cultures : fo mouse myeloma cells ( atcc crl-1646 ) and wehi-164 murine sarcoma cells ( atcc crl1751 ) were obtained from the american type culture collection . fo cells and hybridoma mab 24 cell producing the antibody to feline tnf - alpha were maintained in dulbecco s modified eagle s minimum essential medium supplemented with 10% fcs and antibiotics . wehi-164 cells were maintained in rpmi 1640 growth medium supplemented with 10% fcs , antibiotics , 50 m 2-mercaptoethanol and 2 g / ml of polybrene . cloning of variable regions of heavy chain and light chain of mab 24 and constant regions of heavy chain and light chain of feline immunoglobulin : rna isolation from cells and cdna preparation were performed employing the method of . the variable region genes of the mab 24 ( vh and vl ) were amplified by pcr from cdna of hybridoma mab 24 mrna . the constant region gene of feline immunoglobulin heavy chain ( ch ) was amplified from cdna of feline peripheral blood mononuclear cell mrna . the constant region gene of feline immunoglobulin light chain ( cl ) was artificially synthesized by life technologies ( carlsbad , ca , u.s.a . ) based on a published nucleotide sequence ( genbank af198257.1 ) and inserted into the pma - t plasmid . sequence of pcr primers for variable region of mab 2 - 4 and constant region of feline iggorientationsequencehvforward5-aagcttgccgccaccatggcctggctgtggaactt-3reverse5-ggatccactcacctgaggagacggtgaccgt-3lvforward5-aagcttgccgccaccatggccccgtctattcagtt-3reverse5-ggatccacttattgatttccagctt-3chforward5-ggtgagtggatccagaccacggccccatcg-3reverse5-tgctcatttacccggaga-3clforward5-aagtggatccagagtgatgctcagccatct-3reverse5-ctactctctctgacactcgctcct-3. vh , vl , ch and cl were individually cloned in pcr - blunt ii - topo vectors using the zero blunt topo pcr cloning kit ( life technologies ) . construction and expression of chimeric mab 24 : the ch and cl genes and vh and vl fragments inserted in the pcr - bluntii - topo vectors were connected using a bam hi linker to prepare chimeric h and l chains ( fig . the chimeric h chain fragment was inserted into the ecori site of the pcdna3.1 ( + ) /neo expression vector . the chimeric l chain fragment was inserted into the hindiii / ecorv site of the pcdna3.1 ( + ) /hygro expression vector . fo cells were co - transfected with the h- and l - chain expression vectors with lipofectamine 2000 ( life technologies ) . the transfected fo cells were cultured in medium containing g418 ( roche diagnostics , basel , switzerland ) and hygromycyn b ( roche diagnostics ) , to acquire a stably expressing cell line ( focm24 ) . purification of mouse mab 24 and chimeric mab 24 : mouse mab 24 was purified from the hybridoma mab 24 culture supernatant with protein g sepharose ( ge healthcare , chicago , il , u.s.a . ) according to the product manual . chimeric mab 24 was purified from the focm24 culture supernatant with protein a sepharose ( ge healthcare ) . after purification , the buffer of mabs was exchanged to pbs ( ph 7.4 ) by amicon ultra-15 centrifugal filter devices ( nmwl 30,000 ; millipore , billerica , ma , u.s.a . ) . western immunoblotting assay : purified mouse mab 24 and chimeric mab 24 were incubated without 2-mercaptoethanol at rt for 5 min ( non - reducing condition ) . mabs were incubated with 2-mercaptoethanol at 100c for 10 min ( reducing condition ) . mabs were run using 15% sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and a transferred nitrocellulose membrane . the blot was blocked with 5% nonfat dry milk powder in tbst ( 20 mm tris - hcl , ph 8.0 , 0.88% nacl and 0.05% tween-20 ) for 1 hr at 37c . following washing , the membrane was incubated with horseradish peroxidase conjugated goat anti - mouse igg ( h+l chain specific ) ( southern biotechnology associates inc . , birmingham , al , u.s.a . ) or horseradish peroxidase conjugated goat anti - feline igg ( whole molecular ) ( mp biomedicals , santa ana , ca , u.s.a . ) for 1 hr at 37c and then visualized in the substrate for 10 min . neutralization test of mouse mab 24 and chimeric mab 24 against feline tnf - alpha using wehi-164 cells : neutralization test was performed as described previously . briefly , wehi-164 cells were suspended at 1 10 cells / ml in the dilution medium containing 1 g / ml of actinomycin d ( sigma aldrich , st . louis , mo , u.s.a . ) and pre - incubated at 37c for 3 hr . serially diluted mouse mab 24 , chimeric mab 24 or anti feline apn mab ( mab r - g-4 , as a control for mab 24 ) was mixed with 40 ng / ml recombinant ftnf - alpha ( r&d systems , minneapolis , mn , u.s.a . , 75% cytotoxic activity against wehi-164 cells ) or ascites of cats with fip that were used as natural feline tnf - alpha samples ( final concentration of 1:8 , 80% cytotoxic activity against wehi-164 cells ) . pre - incubated cells were seeded in a volume of 50 l in the wells of a 96-well plate . after incubation at 37c for 24 hr , 10 l of wst-8 solution ( wst-8 cell proliferation assay kit ; kishida chemical co. , ltd . , osaka , japan ) was added , and the cells were returned to the incubator for 1 hr . the absorbance of formazan produced was measured at 450 nm with a 96-well spectrophotometric plate reader , as described by the manufacturer . the percent neutralization was calculated by the following formula : neutralization ( % ) = ( o.d . of wells containing mab and samples o.d . repeated - dose test in cats : the mab repeated - dose test was performed referring to the method reported by umehashi et al . . purified mouse mab 24 , chimeric mab 24 or pbs as a control was administered to 5 specific pathogen free ( spf ) cats aged 2 months . after sedation with medetomidine ( domitor , orion corporation , espoo , finland ) , the spf cats received low- ( 1 mg / kg ) or high - dose ( 5 mg / kg ) mab injection into the cervical vein 5 times at 2- or 4-week intervals . blood pressure and pulse were measured at the forearm or root of the tail before mab administration and 10 min after administration , using a fully automatic electronic sphygmomanometer ( pettrust , aster electric co. , yokohama , japan ) . this animal experiment was performed in accordance with the guidelines for animal experiments of kitasato university ( the number of approval is 14045 ) . changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum : immulon 4hbx elisa plates ( thermo fisher scientific inc . , waltham , ma , u.s.a . ) were coated overnight at 4c with purified mouse mab 24 ( 500 ng/100 l / well ) diluted with carbonate buffer ( 0.05 m , ph 9.6 ) . after washing with phosphate buffered saline ( pbs ) containing 0.02% tween-20 , the plates were blocked with a blocking buffer containing 0.5% skim milk in pbs at 37c for 60 min . each well of the plates then received 100 l of 200-fold diluted serum collected from mab treated cats . after 60 min incubation at 37c , the plates were washed , and horseradish peroxidase conjugated goat anti - feline igg ( whole molecular ) was diluted to the optimal concentrations , and then , 100 l of the dilution was added to each well of the plates . after incubation at 37c for 30 min , the plates were washed , and each well received 100 l of substrate solution and was incubated at 25c for 10 min in the dark . the substrate solution was prepared by dissolving o - phenylenediamine dihydrochloride at a concentration of 0.4 mg / ml in 0.1 m citric acid and 0.2 m na2hpo4 buffer ( ph 4.8 ) and adding 0.2 l / ml of 30% h2o2 . the reaction was stopped with 3 n h2so4 solution , and the optical density ( od ) at 492 nm was determined . changes in neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24- or chimeric mab 24-injected cat serum : sera collected from the mab - treated cats were diluted 10-fold with medium . the diluted sera were reacted for one hour with mouse mab 24 or chimeric mab 24 at the minimum concentration necessary for 80% or higher neutralization of 10 ng / ml recombinant ftnf - alpha . the reactant was then combined with recombinant ftnf - alpha ( final concentration : 10 ng / ml ) . cloning of the variable regions of the heavy and light chains of mab 24 and the constant regions of heavy and light chains of feline immunoglobulin : the vh ( 414 bp ) and vl ( 378 bp ) genes were amplified from cdna derived from hybridoma mab 24 ( fig . s2 ) , and the ch gene ( 1005 bp ) was amplified from cdna derived from feline pbmcs . the cl gene ( 330 bp ) was amplified using a plasmid containing a known cl gene as a template . each pcr product was cloned into the pcr - blunt ii - topo vector and sequenced . the vh and ch genes were ligated , inserted into the pcdna 3.1 ( + ) /neo expression vector and sequenced ( fig . the amino acid sequence deduced from the base sequence was confirmed to show the characteristics of the variable region of mab 24 and the constant region of the feline immunoglobulin heavy chain . similarly , the amino acid sequences of the vl and cl genes were deduced from the base sequences , and it was confirmed that the recombinant protein had the characteristics of the variable region of mab 24 and the constant region of the feline immunoglobulin kappa light chain ( fig . western immunoblotting assay of mouse mab 24 and chimeric mab 24 : fo cells harboring the expression vector were cultured , and chimeric mab 24 in the culture supernatant was collected . chimeric mab 24 and mouse mab 24 were individually purified using a protein a or protein g column . the purified mabs were subjected to sds - page , and their purities were confirmed by cbb staining . no protein band other than those of mabs was detected by sds - page . when non - reduced mouse mab 24 and chimeric mab 24 were electrophoresed , smear - like bands were detected at 135-kda and higher ( fig . mouse mab 24 and chimeric mab 24 were electrophoresed on 12.5% polyacrylamide gel under reducing and non - reducing conditions . mouse mab 24 and chimeric mab 24 were detected by western blotting with anti - mouse igg antibody and anti - feline igg antibody . lanes 1 and 4 ; marker , lanes 2 and 5 ; mouse mab 24 , lanes 3 and 6 ; chimeric mab 24 . ( b ) reducing condition . ) . when the blot was reacted with anti - mouse igg antibody , bands were detected in the lane applied with mouse mab 24 , but no band was detected in the lane applied with chimeric mab 24 . when the blot was reacted with anti - feline igg antibody , bands were detected in the lane applied with chimeric mab 24 , but not in the lane with mouse mab 24 . mouse mab 24 and chimeric mab 24 were electrophoresed on 12.5% polyacrylamide gel under reducing and non - reducing conditions . mouse mab 24 and chimeric mab 24 were detected by western blotting with anti - mouse igg antibody and anti - feline igg antibody . lanes 1 and 4 ; marker , lanes 2 and 5 ; mouse mab 24 , lanes 3 and 6 ; chimeric mab 24 . ( b ) reducing condition . when reduced mouse mab 24 and chimeric mab 24 were electrophoresed , approximately 25-kda and 50-kda bands were visible ( fig . when the blot was reacted with anti - mouse igg antibody , 50- and 25-kda bands were detected in the lane applied with mouse mab 24 , but no band was observed in the lane with chimeric mab 24 . when reacted with anti - feline igg antibody , a band approximately 25-kda was detected in the lane with mouse mab 24 , and 50- and 25-kda bands were detected in the lane with chimeric mab 24 . neutralizing activity of mabs against feline tnf - alpha : mouse mab 24 and chimeric mab 24 were tested for their neutralization activity against recombinant ftnf- using the wehi-164 cytotoxicity assay system . mouse mab 24 and chimeric mab 24 neutralized the cytotoxic activity of recombinant ftnf - alpha on wehi-164 cells in a concentration - dependent manner ( fig . 2.neutralization dose - response curve of mab 24 and chimeric mab 24 against ftnf - alpha . the neutralizing activities of mabs against ftnf - alpha - induced cytotoxicity on wehi-164 cells were measured . wehi-164 cells were treated with mixtures of serial dilutions of mabs and ftnf - alpha , and the level of tnf - alpha - induced cytotoxicity was measured after 24 hr . experiments were performed in triplicate , and the figure shows mean standard errors . ( b ) the neutralizing activities of mabs against natural ftnf - alpha . ) . similarly , the neutralization activity of mouse mab 24 and chimeric mab 24 against endogenous ftnf - alpha contained in ascites of cats with fip was confirmed . mouse mab 24 and chimeric mab 24 neutralized the cytotoxic activity of ascites from cats with fip on wehi-164 cells in a concentration - dependent manner ( fig . mab r - g-4 ( as a control for mab 24 ) did not neutralize the cytotoxic activity of both recombinant ftnf - alpha and ascites of cats with fip ( fig . neutralization dose - response curve of mab 24 and chimeric mab 24 against ftnf - alpha . the neutralizing activities of mabs against ftnf - alpha - induced cytotoxicity on wehi-164 cells were measured . wehi-164 cells were treated with mixtures of serial dilutions of mabs and ftnf - alpha , and the level of tnf - alpha - induced cytotoxicity was measured after 24 hr . experiments were performed in triplicate , and the figure shows mean standard errors . repeated - dose test in cats : purified mouse mab 24 and chimeric mab 24 were repeatedly administered to spf cats at 2- or 4-week intervals , and the blood pressure and pulse were measured before and after administration ( table 2table 2.blood pressure , body tempreture and heart rate of mab injected catsdose of mab(mg / ml)parameterfirst injection(day 0)second injection(day 14)third injection(day 28)fourth injection(day 42)fifth injection(day 72)pbs ( control)0blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection182.0/162.7162.3/129.7169.3/140.7179.7/152.0163.7/140.0post injection164.0/146.3156.0/142.0156.3/139.0158.3/131.7209.7/139.7body tempreture ( c)pre injection39.238.638.938.839.0post injection38.238.038.038.337.4heart rate ( bpm)pre injection138.0130.0151.0115.3121.0post injection136.3140.7140.7113.7114.7mouse mab 241blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection192.3/162.7156.0/130.7151.3/117.0179.0/165.0169.3/113.0post injection156.0/126.7168.0/116.7143.0/121.0208.7/168.7156.7/114.0body tempreture ( c)pre injection39.338.738.238.839.0post injection38.036.936.137.538.9heart rate ( bpm)pre injection128.7135.3125.0114.0136.3post injection129.0115.7127.0114.3128.05blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection160.7/134.0161.3/116.3159.7/135.0189.7/156.0153.3/95.0post injection148.7/128.7170.7/152.0145.3/93.3126.3/98.3165.0/93.3body tempreture ( c)pre injection38.637.737.338.638.8post injection38.037.736.938.338.2heart rate ( bpm)pre injection141.0142.0127.0146.0145.0post injection144.7131.3131.7140.7138.7chimeric mab 241blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection160.7/132.3186.7/161.7196.7/146.0188.0/157.3168.7/136.0post injection161.3/113.0149.3/128.3140.7/120.3161.7/127.3142.7/120.3body tempreture ( c)pre injection38.638.938.338.538.6post injection37.537.936.837.937.9heart rate ( bpm)pre injection105.0141.3141.7108.0115.0post injection86.7138.0132.0120.3113.05blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection172.7/136.0175.0/138.7164.7/140.3160.7/152.0178.3/140.3post injection149.3/103.3163.3/143.3149.0/113.3175.3/147.0148.3/99.3body tempreture ( c)pre injection39.838.739.139.439.2post injection38.138.438.238.238.7heart rate ( bpm)pre injection146.3153.0139.0116.3131.3post injection145.0148.0134.7115.7135.7a ) immediately before administration of pbs or mab . body temperature tended to decrease after administration of pbs , mouse mab 24 and chimeric mab 24 throughout the period with 5 administrations , but no significant difference was observed among the 3 groups . neither were there any differences in blood pressure or pulse between the groups or due to dosing frequency . changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum : serum was collected at various time points from cats treated with pbs , mouse mab 24 and chimeric mab 24 . the feline anti - mouse antibodies in mab - injected cat serum were detected using elisa against mouse mab 24 ( fig . 3fig . 3.changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum . after incubation , the feline anti - mouse antibody was detected with anti - feline igg antibody . ) . changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum . after incubation , the feline anti - mouse antibody was detected with anti - feline igg antibody . to investigate whether there were neutralizing antibodies against mouse and chimeric mab 24 in mab treated cat serum , the collected sera were reacted with mouse mab 24 or chimeric mab 24 , and changes in the ftnf - alpha neutralization activities of these antibodies were measured . when mouse mab 24 was reacted with sera from cats treated with 1 or 5 mg / kg mouse mab 24 , 4.the changes of neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24 or chimeric mab 24 injected cat serum . sera were collected from mab - treated cats at various time points and reacted for one hour with mouse mab 24 or chimeric mab 24 at the minimum concentration neutralizing 80% or more of 10 each reactant was then combined with recombinant ftnf - alpha ( final concentration : 10 ng / ml ) . the level of tnf - alpha - induced cytotoxicity was measured after 24 hr . experiments were performed in triplicate , and the figure shows mean standard errors . ( a ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or mab 24 following reaction with mab 24 . ( b ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or chimeric mab 2 - 4 following reaction with chimeric mab 24 . ) , but when it was reacted with serum from pbs - treated cats , no decrease in the neutralizing activity was observed . the changes of neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24 or chimeric mab 24 injected cat serum . sera were collected from mab - treated cats at various time points and reacted for one hour with mouse mab 24 or chimeric mab 24 at the minimum concentration neutralizing 80% or more of 10 each reactant was then combined with recombinant ftnf - alpha ( final concentration : 10 ng / ml ) . the level of tnf - alpha - induced cytotoxicity was measured after 24 hr . experiments were performed in triplicate , and the figure shows mean standard errors . ( a ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or mab 24 following reaction with mab 24 . ( b ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or chimeric mab 2 - 4 following reaction with chimeric mab 24 . when chimeric mab 24 was reacted with serum from cats treated with 1 mg / kg chimeric mab 24 , no decrease in the neutralizing activity of chimeric mab 24 was observed , similarly to that observed when reacted with serum of pbs - treated cats ( fig . 4b ) . regarding serum from cats treated with 5 mg / kg of chimeric mab 24 , the neutralizing activity of chimeric mab 24 decreased when it was reacted with sera collected after the 3rd administration and thereafter ( 28 days after the initial administration ) ( fig . treatment of fip - induced systemic inflammation with anti - inflammatory drugs , such as steroids , has been investigated , but existing anti - inflammatory drugs only transiently improves fip symptoms , and the survival time and quality of life remain unable to be improved . we previously reported that progression to the fip was prevented by inhibition of the physiological activity of an inflammatory cytokine , tnf - alpha . when anti - ftnf - alpha mouse mab was administered to cats with fip we prepared mouse - feline chimeric mab ( chimeric mab 24 ) by modifying the mouse anti - ftnf - alpha mab 24 and investigated its ftnf - alpha neutralization activity . in addition , changes in the feline anti - mouse antibody response - inducing ability induced by repeated administration of purified mouse mab 24 and chimeric mab 24 in cats were investigated . chimeric mab 24 was prepared by fusion of the variable region of anti - ftnf - alpha mouse mab 24 and the feline antibody constant region and expressed in fo cells . anti - mouse igg antibody reacted with mouse mab 24 but did not react with chimeric mab 24 , whereas anti - feline igg antibody did not react with mouse mab 24 but reacted with chimeric mab 24 , confirming that the antigenicity of chimeric mab 24 is similar to that of feline igg . when neutralization of ftnf - alpha by purified chimeric mab 24 was investigated , chimeric mab 24 neutralized recombinant and natural ftnf - alpha , similarly to mouse mab 24 . based on these findings , chimeric mab 24 possesses ftnf - alpha neutralization activity similar to that of mouse mab 24 while maintaining the characteristics of feline igg . five administrations of the mab did not cause any anaphylactic reaction in either group . umehashi et al . reported the relationship between repeated administration of mouse mab and mouse - feline chimeric mab and the induction of anaphylactic reactions , in which several administrations of 10 mg / kg mouse mab or chimeric mab did not cause any anaphylactic reaction in cats , whereas 50 mg / kg mouse mab did . based on this finding , the antibody dose and dosing frequency adopted in our study were within the range shown not to induce anaphylactic reaction . because a human - mouse chimeric mab , infliximab , is repeatedly administered to humans at doses of 3 or 5 mg / kg , and mouse mab 24 exhibited a therapeutic effect at 3 mg / kg in fip cats , the doses used in the present study were set based on these findings [ 8 , 10 , 11 ] . experiments to confirm whether or not an anaphylactic reaction is induced by high - dose repeated administration are warranted . single administration of mouse mab 24 improved the survival time and quality of life of cats with fip ; however , single administration did not exhibit a therapeutic effect in some cats . it is known that single administration of an anti - ftnf - alpha drug is unlikely to exhibit a therapeutic effect on cats with fip ; thus , several administrations of mab 24 are necessary for treatment . however , several administrations of mouse mab may induce anaphylactic reactions , as described above . moreover , mouse mab is a xenogeneic protein for cats , suggesting that a feline anti - mouse antibody response is induced in cats treated with mouse mab 24 , decreasing the reactivity against ftnf - alpha and thus reducing the therapeutic effect . the possibility of feline anti - chimeric antibody production similar to the production of anti - mouse mab 24 antibody can not be ruled out due to the variable region of chimeric mab 24 being derived from mouse protein . thus , the induction of feline anti - mouse antibodies in cats treated with mouse and chimeric mab was investigated . as the feline anti - mouse antibody was increased in serum , the ftnf - alpha neutralization effect of mab 24 was reduced in cats treated with mouse mab 24 and 5 mg / kg chimeric mab 24 . that is , no neutralizing antibody against chimeric mab 24 induction was observed in cats treated with 1 mg / kg chimeric mab 24 , suggesting that feline anti - mouse antibody induction was prevented by modifying mouse mab 24 to chimeric mab 24 . accordingly , repeated administration of chimeric mab 24 may not decrease the therapeutic effect in cats . however , in cats receiving 5 mg / kg chimeric mab 24 administration , the neutralizing antibody against chimeric mab 24 was induced after the 2nd administration , strongly suggesting that chimeric mab 24 also induces production of neutralizing antibody against chimeric mab 24 when administered at a high dose . a decrease in the therapeutic effect of infliximab due to production of neutralizing antibody against chimeric mab however , neutralizing antibody against chimeric mab production can be inhibited by concomitant administration of an anti - inflammatory drug , methotrexate ( mtx ) . mtx suppresses the humoral immune response and is thought to reduce production of antibody against chimeric mab after infliximab administration [ 10 , 11 ] . concomitant mtx administration with chimeric mab 24 may also inhibit production of neutralizing antibody against chimeric mab . future studies are necessary to investigate the applicability of concomitant mtx administration with chimeric mab 24 in cats with fip . in conclusion , we prepared mouse - feline chimeric mab 24 by fusing the variable region of anti - ftnf - alpha mab 24 to the feline antibody constant region and confirmed that it maintained ftnf - alpha neutralization activity . when the chimeric mab 24 was administered to cats , the induction of feline anti - mouse antibody response was decreased compared to that after mouse mab 24 administration . these results warrant further investigation on the dose and dosing frequency of chimeric mab 24 appropriate for effective treatment of cats with fip .

feline infectious peritonitis ( fip ) is a fatal inflammatory disease caused by fip virus infection . feline tumor necrosis factor ( ftnf)-alpha is closely involved in the aggravation of fip pathology . we previously described the preparation of neutralizing mouse anti - ftnf - alpha monoclonal antibody ( mab 24 ) and clarified its role in the clinical condition of cats with fip using in vitro systems . however , administration of mouse mab 24 to cat may lead to a production of feline anti - mouse antibodies . in the present study , we prepared a mouse - feline chimeric mab ( chimeric mab 24 ) by fusing the variable region of mouse mab 24 to the constant region of feline antibody . the chimeric mab 24 was confirmed to have ftnf - alpha neutralization activity . purified mouse mab 24 and chimeric mab 24 were repeatedly administered to cats , and the changes in the ability to induce feline anti - mouse antibody response were investigated . in the serum of cats treated with mouse mab 24 , feline anti - mouse antibody production was induced , and the ftnf - alpha neutralization effect of mouse mab 24 was reduced . in contrast , in cats treated with chimeric mab 24 , the feline anti - mouse antibody response was decreased compared to that of mouse mab 24-treated cats .

MATERIALS AND METHODS RESULTS DISCUSSION Supplementary Material

purification of mouse mab 24 and chimeric mab 24 : mouse mab 24 was purified from the hybridoma mab 24 culture supernatant with protein g sepharose ( ge healthcare , chicago , il , u.s.a . ) western immunoblotting assay : purified mouse mab 24 and chimeric mab 24 were incubated without 2-mercaptoethanol at rt for 5 min ( non - reducing condition ) . neutralization test of mouse mab 24 and chimeric mab 24 against feline tnf - alpha using wehi-164 cells : neutralization test was performed as described previously . serially diluted mouse mab 24 , chimeric mab 24 or anti feline apn mab ( mab r - g-4 , as a control for mab 24 ) was mixed with 40 ng / ml recombinant ftnf - alpha ( r&d systems , minneapolis , mn , u.s.a . purified mouse mab 24 , chimeric mab 24 or pbs as a control was administered to 5 specific pathogen free ( spf ) cats aged 2 months . changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum : immulon 4hbx elisa plates ( thermo fisher scientific inc . changes in neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24- or chimeric mab 24-injected cat serum : sera collected from the mab - treated cats were diluted 10-fold with medium . the diluted sera were reacted for one hour with mouse mab 24 or chimeric mab 24 at the minimum concentration necessary for 80% or higher neutralization of 10 ng / ml recombinant ftnf - alpha . cloning of the variable regions of the heavy and light chains of mab 24 and the constant regions of heavy and light chains of feline immunoglobulin : the vh ( 414 bp ) and vl ( 378 bp ) genes were amplified from cdna derived from hybridoma mab 24 ( fig . the amino acid sequence deduced from the base sequence was confirmed to show the characteristics of the variable region of mab 24 and the constant region of the feline immunoglobulin heavy chain . similarly , the amino acid sequences of the vl and cl genes were deduced from the base sequences , and it was confirmed that the recombinant protein had the characteristics of the variable region of mab 24 and the constant region of the feline immunoglobulin kappa light chain ( fig . western immunoblotting assay of mouse mab 24 and chimeric mab 24 : fo cells harboring the expression vector were cultured , and chimeric mab 24 in the culture supernatant was collected . mouse mab 24 and chimeric mab 24 were electrophoresed on 12.5% polyacrylamide gel under reducing and non - reducing conditions . mouse mab 24 and chimeric mab 24 were detected by western blotting with anti - mouse igg antibody and anti - feline igg antibody . when the blot was reacted with anti - mouse igg antibody , bands were detected in the lane applied with mouse mab 24 , but no band was detected in the lane applied with chimeric mab 24 . when the blot was reacted with anti - feline igg antibody , bands were detected in the lane applied with chimeric mab 24 , but not in the lane with mouse mab 24 . mouse mab 24 and chimeric mab 24 were electrophoresed on 12.5% polyacrylamide gel under reducing and non - reducing conditions . mouse mab 24 and chimeric mab 24 were detected by western blotting with anti - mouse igg antibody and anti - feline igg antibody . when reduced mouse mab 24 and chimeric mab 24 were electrophoresed , approximately 25-kda and 50-kda bands were visible ( fig . when the blot was reacted with anti - mouse igg antibody , 50- and 25-kda bands were detected in the lane applied with mouse mab 24 , but no band was observed in the lane with chimeric mab 24 . when reacted with anti - feline igg antibody , a band approximately 25-kda was detected in the lane with mouse mab 24 , and 50- and 25-kda bands were detected in the lane with chimeric mab 24 . neutralizing activity of mabs against feline tnf - alpha : mouse mab 24 and chimeric mab 24 were tested for their neutralization activity against recombinant ftnf- using the wehi-164 cytotoxicity assay system . mouse mab 24 and chimeric mab 24 neutralized the cytotoxic activity of recombinant ftnf - alpha on wehi-164 cells in a concentration - dependent manner ( fig . 2.neutralization dose - response curve of mab 24 and chimeric mab 24 against ftnf - alpha . similarly , the neutralization activity of mouse mab 24 and chimeric mab 24 against endogenous ftnf - alpha contained in ascites of cats with fip was confirmed . mouse mab 24 and chimeric mab 24 neutralized the cytotoxic activity of ascites from cats with fip on wehi-164 cells in a concentration - dependent manner ( fig . mab r - g-4 ( as a control for mab 24 ) did not neutralize the cytotoxic activity of both recombinant ftnf - alpha and ascites of cats with fip ( fig . neutralization dose - response curve of mab 24 and chimeric mab 24 against ftnf - alpha . repeated - dose test in cats : purified mouse mab 24 and chimeric mab 24 were repeatedly administered to spf cats at 2- or 4-week intervals , and the blood pressure and pulse were measured before and after administration ( table 2table 2.blood pressure , body tempreture and heart rate of mab injected catsdose of mab(mg / ml)parameterfirst injection(day 0)second injection(day 14)third injection(day 28)fourth injection(day 42)fifth injection(day 72)pbs ( control)0blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection182.0/162.7162.3/129.7169.3/140.7179.7/152.0163.7/140.0post injection164.0/146.3156.0/142.0156.3/139.0158.3/131.7209.7/139.7body tempreture ( c)pre injection39.238.638.938.839.0post injection38.238.038.038.337.4heart rate ( bpm)pre injection138.0130.0151.0115.3121.0post injection136.3140.7140.7113.7114.7mouse mab 241blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection192.3/162.7156.0/130.7151.3/117.0179.0/165.0169.3/113.0post injection156.0/126.7168.0/116.7143.0/121.0208.7/168.7156.7/114.0body tempreture ( c)pre injection39.338.738.238.839.0post injection38.036.936.137.538.9heart rate ( bpm)pre injection128.7135.3125.0114.0136.3post injection129.0115.7127.0114.3128.05blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection160.7/134.0161.3/116.3159.7/135.0189.7/156.0153.3/95.0post injection148.7/128.7170.7/152.0145.3/93.3126.3/98.3165.0/93.3body tempreture ( c)pre injection38.637.737.338.638.8post injection38.037.736.938.338.2heart rate ( bpm)pre injection141.0142.0127.0146.0145.0post injection144.7131.3131.7140.7138.7chimeric mab 241blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection160.7/132.3186.7/161.7196.7/146.0188.0/157.3168.7/136.0post injection161.3/113.0149.3/128.3140.7/120.3161.7/127.3142.7/120.3body tempreture ( c)pre injection38.638.938.338.538.6post injection37.537.936.837.937.9heart rate ( bpm)pre injection105.0141.3141.7108.0115.0post injection86.7138.0132.0120.3113.05blood pressure ( systolic blood pressure / diastolic blood pressure , mmhg)pre injection172.7/136.0175.0/138.7164.7/140.3160.7/152.0178.3/140.3post injection149.3/103.3163.3/143.3149.0/113.3175.3/147.0148.3/99.3body tempreture ( c)pre injection39.838.739.139.439.2post injection38.138.438.238.238.7heart rate ( bpm)pre injection146.3153.0139.0116.3131.3post injection145.0148.0134.7115.7135.7a ) immediately before administration of pbs or mab . body temperature tended to decrease after administration of pbs , mouse mab 24 and chimeric mab 24 throughout the period with 5 administrations , but no significant difference was observed among the 3 groups . changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum : serum was collected at various time points from cats treated with pbs , mouse mab 24 and chimeric mab 24 . the feline anti - mouse antibodies in mab - injected cat serum were detected using elisa against mouse mab 24 ( fig . 3.changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum . after incubation , the feline anti - mouse antibody was detected with anti - feline igg antibody . ) changes in anti - mouse antibody in mouse mab 24- or chimeric mab 24-injected cat serum . after incubation , the feline anti - mouse antibody was detected with anti - feline igg antibody . to investigate whether there were neutralizing antibodies against mouse and chimeric mab 24 in mab treated cat serum , the collected sera were reacted with mouse mab 24 or chimeric mab 24 , and changes in the ftnf - alpha neutralization activities of these antibodies were measured . when mouse mab 24 was reacted with sera from cats treated with 1 or 5 mg / kg mouse mab 24 , 4.the changes of neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24 or chimeric mab 24 injected cat serum . sera were collected from mab - treated cats at various time points and reacted for one hour with mouse mab 24 or chimeric mab 24 at the minimum concentration neutralizing 80% or more of 10 each reactant was then combined with recombinant ftnf - alpha ( final concentration : 10 ng / ml ) . ( a ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or mab 24 following reaction with mab 24 . ( b ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or chimeric mab 2 - 4 following reaction with chimeric mab 24 . ) the changes of neutralization activity of mouse mab 24 and chimeric mab 24 reacted with mouse mab 24 or chimeric mab 24 injected cat serum . ( a ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or mab 24 following reaction with mab 24 . ( b ) the rates of recombinant ftnf - alpha neutralization by sera from cats treated with pbs or chimeric mab 2 - 4 following reaction with chimeric mab 24 . when chimeric mab 24 was reacted with serum from cats treated with 1 mg / kg chimeric mab 24 , no decrease in the neutralizing activity of chimeric mab 24 was observed , similarly to that observed when reacted with serum of pbs - treated cats ( fig . regarding serum from cats treated with 5 mg / kg of chimeric mab 24 , the neutralizing activity of chimeric mab 24 decreased when it was reacted with sera collected after the 3rd administration and thereafter ( 28 days after the initial administration ) ( fig . when anti - ftnf - alpha mouse mab was administered to cats with fip we prepared mouse - feline chimeric mab ( chimeric mab 24 ) by modifying the mouse anti - ftnf - alpha mab 24 and investigated its ftnf - alpha neutralization activity . in addition , changes in the feline anti - mouse antibody response - inducing ability induced by repeated administration of purified mouse mab 24 and chimeric mab 24 in cats were investigated . chimeric mab 24 was prepared by fusion of the variable region of anti - ftnf - alpha mouse mab 24 and the feline antibody constant region and expressed in fo cells . anti - mouse igg antibody reacted with mouse mab 24 but did not react with chimeric mab 24 , whereas anti - feline igg antibody did not react with mouse mab 24 but reacted with chimeric mab 24 , confirming that the antigenicity of chimeric mab 24 is similar to that of feline igg . when neutralization of ftnf - alpha by purified chimeric mab 24 was investigated , chimeric mab 24 neutralized recombinant and natural ftnf - alpha , similarly to mouse mab 24 . based on these findings , chimeric mab 24 possesses ftnf - alpha neutralization activity similar to that of mouse mab 24 while maintaining the characteristics of feline igg . reported the relationship between repeated administration of mouse mab and mouse - feline chimeric mab and the induction of anaphylactic reactions , in which several administrations of 10 mg / kg mouse mab or chimeric mab did not cause any anaphylactic reaction in cats , whereas 50 mg / kg mouse mab did . because a human - mouse chimeric mab , infliximab , is repeatedly administered to humans at doses of 3 or 5 mg / kg , and mouse mab 24 exhibited a therapeutic effect at 3 mg / kg in fip cats , the doses used in the present study were set based on these findings [ 8 , 10 , 11 ] . single administration of mouse mab 24 improved the survival time and quality of life of cats with fip ; however , single administration did not exhibit a therapeutic effect in some cats . it is known that single administration of an anti - ftnf - alpha drug is unlikely to exhibit a therapeutic effect on cats with fip ; thus , several administrations of mab 24 are necessary for treatment . moreover , mouse mab is a xenogeneic protein for cats , suggesting that a feline anti - mouse antibody response is induced in cats treated with mouse mab 24 , decreasing the reactivity against ftnf - alpha and thus reducing the therapeutic effect . the possibility of feline anti - chimeric antibody production similar to the production of anti - mouse mab 24 antibody can not be ruled out due to the variable region of chimeric mab 24 being derived from mouse protein . thus , the induction of feline anti - mouse antibodies in cats treated with mouse and chimeric mab was investigated . as the feline anti - mouse antibody was increased in serum , the ftnf - alpha neutralization effect of mab 24 was reduced in cats treated with mouse mab 24 and 5 mg / kg chimeric mab 24 . that is , no neutralizing antibody against chimeric mab 24 induction was observed in cats treated with 1 mg / kg chimeric mab 24 , suggesting that feline anti - mouse antibody induction was prevented by modifying mouse mab 24 to chimeric mab 24 . however , in cats receiving 5 mg / kg chimeric mab 24 administration , the neutralizing antibody against chimeric mab 24 was induced after the 2nd administration , strongly suggesting that chimeric mab 24 also induces production of neutralizing antibody against chimeric mab 24 when administered at a high dose . a decrease in the therapeutic effect of infliximab due to production of neutralizing antibody against chimeric mab however , neutralizing antibody against chimeric mab production can be inhibited by concomitant administration of an anti - inflammatory drug , methotrexate ( mtx ) . future studies are necessary to investigate the applicability of concomitant mtx administration with chimeric mab 24 in cats with fip . in conclusion , we prepared mouse - feline chimeric mab 24 by fusing the variable region of anti - ftnf - alpha mab 24 to the feline antibody constant region and confirmed that it maintained ftnf - alpha neutralization activity . when the chimeric mab 24 was administered to cats , the induction of feline anti - mouse antibody response was decreased compared to that after mouse mab 24 administration .

the united nations estimates that approximately 650 million people across the world ( or 10% of the world 's population ) are living with a disability . due to population aging and medical advances research has shown that lifestyle risk factors , such as vigorous physical activity , can have a protective effect against disability . specifically , data shows that exercise , even if not started until later in life , can result in the postponement of disability [ 2 , 3 ] . in addition , researchers cite lifestyle differences as one of the contributors to health inequalities in populations . the prevalence of disability varies across countries , however . these countries are of interest because despite having different socioeconomic , demographic , and epidemiological profiles , they are closely linked geographically , economically , and socially . the united states population started to age earlier , and the process started under more advantageous economic conditions compared to when mexico began to age . predictions show that the mexican population over age 60 is expected to grow from 6% of the total population in 2000 to 15% in 2027 . compared to the united states and other developed countries , this 27-year pace is relatively fast . it will take the united states 70 years to reach a similar percentage in 2013 . in addition to a rapid process , this is also premature in mexico , due to the lack of infrastructure and lagging economic development . the two countries are also in different stages of the epidemiological transition , as indicated by the current morbidity rates and mortality due to noncommunicable and communicable diseases . in mexico , communicable diseases are more prevalent , while the relative importance of chronic conditions such as cardiovascular diseases and cancer is higher in the united states . these contrasts mark different stages of transitions for the two countries that we compare . a recent report finds that the levels of disability prevalence , as measured in terms of limitations in activities of daily living ( adls ) , were lower in mexico than in the united states despite the lower level of socioeconomic development in mexico compared to the usa . one possible explanation for this finding may be that the two countries are at different stages of the epidemiological transition . the current cohort of older adults in mexico experienced higher infant and childhood mortality levels , and it is likely that only the in other words , persons surviving into old age in mexico are relatively more robust than those surviving into old age in the usa and therefore have a lower disability rate . the numbers indicate that while life expectancy is lower in mexico than in the usa at younger ages , beginning at age 75 , the life expectancy actually becomes higher for mexico than in the united states . little is known about how lifestyle risk factors , such as lack of physical exercise , can impact functional limitations in societies with very different demographic , epidemiological , and lifestyle behaviors profiles . it is possible that the impact of lifestyle on disability may be very different in mexico than in the usa in part because the current cohorts of older adults in mexico are more selected than in the united states . this paper therefore explores the impact of physical activity on disability transitions , among older adults in mexico compared to the united states . specifically , we postulate that since the selection of survivors among older adults is currently lower in the usa compared to mexico overall , a healthy lifestyle ( such as physical exercise ) is likely to have greater beneficial effect in the usa than in mexico . this hypothesis is of relevance for global aging because it means that lifestyle changes are likely to have differential impact on health according to the stage of the epidemiological transitions in which societies are . the proposed research takes advantage of the comparability of the mexican health and aging study ( mhas ) and the health and retirement study ( hrs ) . to examine disability transitions across mexico and the usa , we use two waves of the mexican health and aging study ( mhas : 2001 and 2003 ) and the rand version of the health and retirement study ( hrs : 2000 and 2002 ) we examine 13,224 individuals at baseline in the hrs ( 2000 ) and 11,064 persons at baseline in the mhas ( 2001 ) . to determine transitions we observed disability outcomes both at baseline ( 2001 for mhas and 2000 for the hrs ) and two - year followup ( 2003 for mhas and 2002 for the hrs ) . the mexican health and aging study ( mhas ) is a nationally representative prospective panel study of community - dwelling individuals born prior to 1951 . the study was conducted by researchers at the university of pennsylvania , university of maryland , university of wisconsin , and the instituto nacional de estadstica , geografia e informtica ( inegi ) in mexico and funded by the national institutes of health / national institute on aging . the mhas used a multistage cluster sampling methodology that randomly selected households with at least one individual aged 50 or older . approximately 15,000 eligible persons and their spouses were interviewed , with a response rate of 90.1% . a direct interview was conducted when it was possible , and proxy interviews were obtained when the subjects were in poor health or were temporarily absent ( 7.3% of the baseline sample were proxy interviews ) . the health and retirement study ( hrs ) is an ongoing large - scale longitudinal study nationally representative for community - dwelling adults over age 50 . it is conducted by the university of michigan with support from the national institute on aging ( nih / nia ) . the hrs used a multistage national area probability sample of households in the united states and oversampled blacks , hispanics , and persons living in the state of florida . we used the rand version of the hrs dataset , which compiled all waves of the hrs data and used bracketing methods to minimize nonresponse in certain variables ( see rand for more details ) . respondents received direct interviews when possible and proxy interviews were conducted when subjects were unable to respond or were unavailable ( 7.5% of the baseline survey were proxy interviews ) . we compare elders living in the community in mexico to community - dwelling non - hispanic whites who were born in and living in the united states . the mhas survey was only conducted among elders living in the community because most long - term care is provided by families in that country . we addressed this limitation by excluding persons that were institutionalized in the usa from the analyses . this allows for a straightforward comparison by focusing on community - dwelling elders in both countries . additionally , only persons that identified as non - hispanic white and who reported being born in the usa were included in the usa sample . because both the hrs and mhas interviewed age - eligible respondents and their spouses regardless of age , we only include persons aged 51 and older for the hrs and adults 52 years and older for mhas , at baseline . finally , we only included subjects with complete information on each variable in our analyses . because the mhas is highly comparable to the hrs , the measures used in the analyses are very similar . the outcome of adl limitations was measured using five components : bathing , toileting , transferring into / out of bed , walking , and eating . each of the components in the questionnaire was coded disabled if the respondent answered yes or can not do to having difficulty in performing the activity . if the respondent answered do not do then the response was coded as missing . however , if they answered do not do and received help performing the activity , they were coded as disabled . our focal independent variable is whether or not respondents reported vigorous physical activity or exercise ( note that we use the terms physical activity and exercise interchangeably throughout the remainder of the paper ) . both the mhas and the hrs asked respondents whether on average they had participated in vigorous physical activity or exercise three times a week a more . vigorous activity or exercise was defined to include any activities such as sports , heavy housework , or a job that involve physical labor . this is a standard question and has been used extensively in the literature to operationally define self - reported physical activity ( e.g. , [ 4 , 10 , 11 ] ) . research has shown support for using self - reported historical physical activity measures to assess physical activity performance . one such study found that self - reported historical walking , running , and jogging activities had reasonable validity when compared with objective measures , such as treadmill performance . moderate spearman 's correlations between objective and self - report measures were significant for all correlations in the study ( p < .001 ) . , the question asked the respondent to reflect on the average over the last two years , whereas in the usa the respondents were asked to estimate over the past 12 months . because only one measure was available to capture the complex behavior of physical activity , it is important to establish validity of the focal independent variable . in order to establish construct validity ( the ability of the measure to correspond to the theoretical concept under study ) , the physical activity measure must be related to other measures consistent with plausible hypotheses . we assessed construct validity using the following two hypotheses : ( 1 ) respondents reporting problems with basic activities of daily living ( walking , bathing , eating , toileting , and transferring in and out of bed ) would be significantly less likely to report doing physical activity at baseline and ( 2 ) these correlations should remain significant across subgroups ( e.g. , gender and age ) . the correlation coefficients between physical activity and the five adl measures were consistently negative and significant . specifically , the correlations between walking and physical activity were 0.159 in the usa and 0.099 in mexico . for bathing and physical activity , the correlations for the usa sample were 0.168 and 0.088 in the mexican sample , while for eating and physical activity , the correlations were 0.102 in the usa and 0.052 for mexico . the correlations between transferring in / out of bed and physical activity were 0.123 in the usa and 0.059 in mexico . finally , the correlations between toileting and the physical activity measure were 0.119 in the usa and 0.062 in mexico . while the correlation coefficients had a lower magnitude in the mexican sample than in the usa sample , all were significant ( p < .01 ) . we performed the same correlations by gender and age categories ( 5159 , 6069 , 6079 , 80 + ) . moreover , while significantly correlated at all ages , the correlations were stronger for older cohorts than for younger cohorts . research has shown that disability rates vary by whether persons live in rural or urban areas ; thus , we added a control variable for area of residence ( reference category : rural ) . for the united states , the hrs dichotomous variable was created using the 1993 beale rural - urban continuum codes ( see hrs ) . the codes were collapsed into urban area ( population size 250,000 or more ) and rural ( population less than 250,000 ) . for mexico , we used the mhas locality size measurement to code areas with 100,000 or more people as urban . wealth was measured using household 's net worth of homes , businesses , rental properties , capital , vehicles , and other debts and assets . due to high nonresponse rates in these single items , both the hrs and mhas used unfolding brackets to recover the nonresponse ( rand ; wong & espinoza [ 9 , 14 ] , resp . ) . health insurance was coded as a binary variable , where respondents were classified as having health insurance if they reported having at least one health insurance , regardless of type of insurance . finally , additional control socioeconomic and demographic variables included age ( continuous ) , gender ( reference category : male ) , marital status ( categorical : married and union , reference category ; widowed ; single , separated , or divorced ) , and education ( continuous ) . we begin by comparing prevalence of self - reported disability and physical activity among all older adults across the two countries regardless of disability level . for these descriptive results , we show data from the full sample at baseline in the united states and mexico . this allows us to estimate and compare the prevalence across countries cross - sectionally . due to the difference in age structures across the two countries , prevalence of adl limitations and prevalence of physical activity ( table 2 ) is age age - adjusted rates of disability let us compare relative differences across the usa and mexico . we also provide cross - sectional weighted descriptive data for both countries according to the exercise variable . in order to determine if physical activity influences the incidence of disability , we next conduct a series of separate analyses by country across time . for this portion of the analyses , we select only those respondents that did not report any adl limitation at time 1 and examine their status two years later . because our study is based on longitudinal data , we face the issue of attrition and death by the followup period two years later ( unobserved respondents ) . the followup outcome is therefore measured by five categories : remaining nondisabled across the two years , moving from no disability to one disability , from no disability to two or more disabilities , from no disability to death and those that are lost to followup . table 1 shows the sample distribution of the outcome by country . because we do not have data on disability status for those lost to followup both datasets showed a 5% loss to followup over the two years . in a separate analysis ( not shown here ) , we found similar sample characteristics of those lost to followup across both studies . we used multinomial models to determine the likelihood of moving to one of the followup categories over two years ( reference group : remaining nondisabled ) . we then combined the panel surveys from the two countries and included a country indicator ( reference group : united states ) to identify significant differences across countries . the estimators of the multinomial model indicate the relationship of the outcome variable across categories of the independent variables that are interpreted as a relative risk ratio compared to a reference category ( hilbe ) . in order to facilitate the interpretation of results , we also present figures of the estimated probabilities of the outcome variable based on the multivariate models . we estimated the general probabilities of each outcome category at followup by exercise , and we break down these predicted probabilities by age . table 2 shows the age - standardized prevalence of functional limitations by country in table 2(a ) as well as the prevalence of exercise according to functional limitations in table 2(b ) . table 2(a ) shows the prevalence of functional limitations across the two countries at baseline . the data confirmed previous research that overall disability rates were higher in the total usa elderly population than in the mexican elderly population . whereas 11.5% of elders in the usa reported at least one adl limitation , about 10.6% of persons in mexico reported a disability . however , mexico showed higher prevalence rate for each individual adl limitation measured . this somewhat counterintuitive finding suggests a higher degree of overlap of adl limitations among persons in mexico , which is confirmed when examining the number of adl limitations reported . less than one percent of the sample in the usa reported problems with all adl limitations , whereas nearly two percent reported having limitations in all five areas in mexico . table 2(b ) compares the prevalence of physical activity in the usa and mexico by adl limitation categories , age standardized . in general , the prevalence of exercise is higher in the usa than in mexico , where approximately 46% of persons in the usa reported doing vigorous physical activity , compared to less than one - third ( 29% ) of elders in mexico . when prevalence of physical activity is broken down by functional limitations , nearly half ( 44% ) of elders without a disability in the usa reported exercising , compared to 27% of elders without a disability in mexico . however , for persons with one disability or with two or more disabilities , the difference between countries becomes very small . table 3 provides prevalence of vigorous physical activity by main characteristics of the sample separately for each country . in both countries , the percent reporting physical activity is lower in mexico than in the united states . in both countries men are more likely to report exercising than women , although the gender gap is much larger in mexico than in the united states . additionally , in the usa , there appeared to be a gradient in physical activity with assets , where those with higher assets had higher rates of physical activity . in fact , those with lower assets were slightly more likely to exercise . similarly , there was a slight gradient in physical activity with educational attainment in the usa , whereas such a pattern was not found in mexico . while for the usa sample there were not large differences in exercise behavior by area of residence and having health insurance , these showed important differences for the mexican sample . persons living in an urban area were more likely to report exercising than those living in a rural area in mexico . those persons without health insurance in mexico were also more likely to exercise , compared to those that were insured . table 4 presents results for a series of multinomial models predicting going from zero adls at baseline to either one adl , two or more adls , or death at followup . results show that the focal independent variable , physical activity , is significantly protective for each of the negative outcomes in the usa regression model ( model 1 ) . however , in the model examining mexico only ( model 2 ) , physical activity is significantly protective only for the worst outcomes ( 2 + adls and death ) . model 3 combines both countries to create a panel model with a country identifier variable . these results show that the level of disability is significantly different between the united states and mexico . also , the propensity to go from no adl to a worse outcome is smaller in mexico . the results indicate that engaging in physical activity prevents negative health outcomes in both mexico and the united states , although the effect is less protective in mexico than in the united states . this is not the case for death , however , where exercise is protective for death in both countries , and the effect is not significantly different . in other words , the effect of physical activity on death additional models ( results available upon request ) examined a variety of potentially interesting interactions . in order to determine if the effect of physical activity is different across various characteristics , we ran interactions between physical activity and gender , physical activity and age , and physical activity and education . finally , in order to test whether physical exercise was more beneficial across groups within countries , we ran interactions for country and education as well as country and area of residence . figures 1 and 2 show the predicted probabilities of the outcome at time 2 by country and various demographic groups . figure 1 visually charts the previous models predicting the probability of going from no adl limitation at time 1 to one adl limitation , 2 or more limitation , or to death at time 2 , by physical activity and by country . the figure indicates that those who exercise have a lower probability of transitioning to a worse disability status or to death in both countries . figure 2 presents predicted probabilities of outcome at time 2 by age . as expected , the probability of disability or death increased with age in each country , regardless of physical activity . however , the probability curve for transitioning to one adl is nearly the same in mexico for exercisers and nonexercisers . on the other hand , for the united states , there is a clear advantage for those who exercise , with a lower probability of transitioning to one adl , especially at the older ages . a protective effect appears for mexico as well as for the usa when examining the probability of transitioning to two or more adls at time 2 . those who exercise in the usa have similar probability of transition to two or more adls as those who exercise in mexico . the probability curve is lower for those who do not exercise in the united states than for those who do not exercise in the united states . the final graph in figure 2 shows the probability of death at time 2 by age , physical activity , and country . while the probability of death is higher for the usa than in mexico regardless of exercise , the impact of exercise appears to be similar across both countries . it is interesting to note that in each of the figures the differences appear especially pronounced at older ages . this paper compared the impact of physical activity on the incidence of disability among older persons in two countries at different stages of the epidemiologic transition , mexico and the united states . to our knowledge , this is the first paper to examine the importance of physical activity on predicting transitions in disability using a comparative approach across two countries . this supports recent evidence that transitions among older adults towards healthy lifestyle habits , such as avoiding tobacco and binge alcohol drinking , or exercising , appear to be underway in the usa but not yet in mexico . one is that the survey questions were asked in a slightly different way across the two countries , and because physical activity was self - reported , it is possible that the interpretation of the question may have been different across the two samples . another more plausible explanation was posited by wong et al . that the two countries are at different stages of a lifestyle transition , where transitions to healthier lifestyles have progressed further in the usa than in mexico . social and policy changes impacting healthier lifestyle choices have occurred earlier in the usa and have been more extensive , which may in part explain this disparity in levels of exercise between the two countries . we also found differences in physical activity across groups , including gender , which echoed findings by wong et al . . men had nearly twice the prevalence rate of physical activity than women in mexico ( 43% versus 23% , resp . ) . while the gender gap was not as large in the usa , men were still more likely to exercise than women . although there were no large differences in physical activity prevalence by area of residence and health insurance status in the usa , there were notable differences by these groups in mexico . it is possible that those uninsured or those living in an urban environment in mexico may be employed in more physically demanding labor , which would be categorized as vigorous exercise in the surveys . it may also be possible that the logistics of exercising ( e.g. , accessing a gym ) are easier in urban areas or that a culture of exercise has been adapted to a greater extent in urban areas of mexico compared to rural areas of the country . the results also show that not only is the level of disability different across the usa and mexico , but that the effect of physical activity on disability is significantly different across the two countries . overall , we found a beneficial effect of physical activity against onset of disability or death at followup in both countries . however , we also found that the protective effect of physical activity on disability is stronger in the usa than in mexico . this supports our initial hypothesis that physical activity is less protective among the mexican older population since they represent a more selected group of survivors than in the us . in other words , we speculate that older adults in the usa are more disabled at older ages and therefore are more likely to benefit from a lifestyle intervention . this result has important implications for aging in developing countries that are lagging in the epidemiological transition . we should expect that policies that are implemented in the countries towards healthy lifestyles should have lower impact while the countries are in early stages of the transition compared to the likely impact that similar policies may have later on . one limitation of this study is the question used to determine physical activity in the surveys . the question determining physical activity asked about exercise broadly , including everything from sports to physical labor . we are therefore unable to differentiate between those who exercise because of daily occupation - related physical labor and those that train at a gym , for example . it is likely that the various types of exercise may have different impacts on health . for instance , those working dangerous and physically demanding jobs may not see a benefit from their regular physical exertion , whereas those training in a gym may see the benefits of an exercise routine . this may be particularly the case when comparing these two countries with different cultures and at different lifestyle transitions . in rural mexican communities , outdoor sports may be more prevalent , whereas gyms with modern fitness equipment may be more prevalent in urban usa areas . similarly , recommendations by the centers for disease control and prevention and american college of sports medicine ( cdc / acsm ) include an emphasis on the value of moderately intense activity , which we were unable to capture . it was not possible to control for the type of activity or for the intensity of the activity in these surveys , and it is not clear how shorter or more moderate bouts of activity may impact functional limitations . future research should consider including measures such as metabolic equivalent task ( met ) values , which allows the researcher to determine both the intensity and rate of exercise . finally , the wording of the activity question was identical across the two surveys except for the different time frames the respondents are asked to reflect on . respondents in mexico were asked about their average exercise in the last two years , whereas those in the usa were asked about their average over the past year . this is a limitation when comparing physical activity rates across countries , and the results should be interpreted with caution . however , the issue of timing is less of a concern when comparing the effects of physical activity on disability within each country , which is the main focus of this study . as the epidemiological and demographic transitions continue to run their course in developing countries such as mexico , as the results of this study confirm , lifestyle changes such as exercise can help in the avoidance of chronic and disabling conditions . this is particularly important in the design of policies for older adults , since our work has shown that the beneficial effects of physical activity extend to these age cohorts in a developing country such as mexico . however , our work has shown also that the positive effect of physical exercise may be dependent on the stage of the transition that societies are undergoing . this is informative , not only to expect lower impact of exercise on health overall compared to more developed countries , but also to motivate the search for modified or alternative exercise interventions to achieve a higher impact in countries that are early in the epidemiologic transition . nevertheless , policies aimed to encourage physical activity in mexico may play particularly important roles in preventing negative health outcomes . our work has also indicated avenues of further work for future population - based studies , in order to better capture the concept of physical exercise among older adults .

evidence suggests that transitions among older adults towards healthy habits , such as physical activity , appear underway in developed countries such as the usa but not in developing countries such as mexico . however , little is known about the potential benefit of physical activity in preventing disability among elders in countries at different stages of epidemiological transition . we explore the impact of physical activity on the disablement process among elders in mexico compared to the usa . data are from two waves of the mexican health and aging study and the health and retirement study . we examine the impact of exercise on the transition from no disability to adl limitations two years later . findings indicate that exercise is more common in the u.s . than in mexico . there is a positive effect of exercise on negative outcomes in both countries . however , the protective effect of exercise is stronger in the u.s . than in mexico .

1. Introduction/Background 2. Methodology 3. Results 4. Discussion and Conclusions

the united nations estimates that approximately 650 million people across the world ( or 10% of the world 's population ) are living with a disability . due to population aging and medical advances research has shown that lifestyle risk factors , such as vigorous physical activity , can have a protective effect against disability . specifically , data shows that exercise , even if not started until later in life , can result in the postponement of disability [ 2 , 3 ] . the united states population started to age earlier , and the process started under more advantageous economic conditions compared to when mexico began to age . predictions show that the mexican population over age 60 is expected to grow from 6% of the total population in 2000 to 15% in 2027 . compared to the united states and other developed countries , this 27-year pace is relatively fast . in addition to a rapid process , this is also premature in mexico , due to the lack of infrastructure and lagging economic development . the two countries are also in different stages of the epidemiological transition , as indicated by the current morbidity rates and mortality due to noncommunicable and communicable diseases . in mexico , communicable diseases are more prevalent , while the relative importance of chronic conditions such as cardiovascular diseases and cancer is higher in the united states . these contrasts mark different stages of transitions for the two countries that we compare . a recent report finds that the levels of disability prevalence , as measured in terms of limitations in activities of daily living ( adls ) , were lower in mexico than in the united states despite the lower level of socioeconomic development in mexico compared to the usa . one possible explanation for this finding may be that the two countries are at different stages of the epidemiological transition . the current cohort of older adults in mexico experienced higher infant and childhood mortality levels , and it is likely that only the in other words , persons surviving into old age in mexico are relatively more robust than those surviving into old age in the usa and therefore have a lower disability rate . the numbers indicate that while life expectancy is lower in mexico than in the usa at younger ages , beginning at age 75 , the life expectancy actually becomes higher for mexico than in the united states . little is known about how lifestyle risk factors , such as lack of physical exercise , can impact functional limitations in societies with very different demographic , epidemiological , and lifestyle behaviors profiles . it is possible that the impact of lifestyle on disability may be very different in mexico than in the usa in part because the current cohorts of older adults in mexico are more selected than in the united states . this paper therefore explores the impact of physical activity on disability transitions , among older adults in mexico compared to the united states . specifically , we postulate that since the selection of survivors among older adults is currently lower in the usa compared to mexico overall , a healthy lifestyle ( such as physical exercise ) is likely to have greater beneficial effect in the usa than in mexico . this hypothesis is of relevance for global aging because it means that lifestyle changes are likely to have differential impact on health according to the stage of the epidemiological transitions in which societies are . the proposed research takes advantage of the comparability of the mexican health and aging study ( mhas ) and the health and retirement study ( hrs ) . to examine disability transitions across mexico and the usa , we use two waves of the mexican health and aging study ( mhas : 2001 and 2003 ) and the rand version of the health and retirement study ( hrs : 2000 and 2002 ) we examine 13,224 individuals at baseline in the hrs ( 2000 ) and 11,064 persons at baseline in the mhas ( 2001 ) . the mexican health and aging study ( mhas ) is a nationally representative prospective panel study of community - dwelling individuals born prior to 1951 . the study was conducted by researchers at the university of pennsylvania , university of maryland , university of wisconsin , and the instituto nacional de estadstica , geografia e informtica ( inegi ) in mexico and funded by the national institutes of health / national institute on aging . the health and retirement study ( hrs ) is an ongoing large - scale longitudinal study nationally representative for community - dwelling adults over age 50 . we used the rand version of the hrs dataset , which compiled all waves of the hrs data and used bracketing methods to minimize nonresponse in certain variables ( see rand for more details ) . we compare elders living in the community in mexico to community - dwelling non - hispanic whites who were born in and living in the united states . the mhas survey was only conducted among elders living in the community because most long - term care is provided by families in that country . we addressed this limitation by excluding persons that were institutionalized in the usa from the analyses . this allows for a straightforward comparison by focusing on community - dwelling elders in both countries . additionally , only persons that identified as non - hispanic white and who reported being born in the usa were included in the usa sample . because the mhas is highly comparable to the hrs , the measures used in the analyses are very similar . the outcome of adl limitations was measured using five components : bathing , toileting , transferring into / out of bed , walking , and eating . each of the components in the questionnaire was coded disabled if the respondent answered yes or can not do to having difficulty in performing the activity . however , if they answered do not do and received help performing the activity , they were coded as disabled . our focal independent variable is whether or not respondents reported vigorous physical activity or exercise ( note that we use the terms physical activity and exercise interchangeably throughout the remainder of the paper ) . both the mhas and the hrs asked respondents whether on average they had participated in vigorous physical activity or exercise three times a week a more . this is a standard question and has been used extensively in the literature to operationally define self - reported physical activity ( e.g. research has shown support for using self - reported historical physical activity measures to assess physical activity performance . one such study found that self - reported historical walking , running , and jogging activities had reasonable validity when compared with objective measures , such as treadmill performance . , the question asked the respondent to reflect on the average over the last two years , whereas in the usa the respondents were asked to estimate over the past 12 months . because only one measure was available to capture the complex behavior of physical activity , it is important to establish validity of the focal independent variable . in order to establish construct validity ( the ability of the measure to correspond to the theoretical concept under study ) , the physical activity measure must be related to other measures consistent with plausible hypotheses . we assessed construct validity using the following two hypotheses : ( 1 ) respondents reporting problems with basic activities of daily living ( walking , bathing , eating , toileting , and transferring in and out of bed ) would be significantly less likely to report doing physical activity at baseline and ( 2 ) these correlations should remain significant across subgroups ( e.g. the correlation coefficients between physical activity and the five adl measures were consistently negative and significant . specifically , the correlations between walking and physical activity were 0.159 in the usa and 0.099 in mexico . for bathing and physical activity , the correlations for the usa sample were 0.168 and 0.088 in the mexican sample , while for eating and physical activity , the correlations were 0.102 in the usa and 0.052 for mexico . the correlations between transferring in / out of bed and physical activity were 0.123 in the usa and 0.059 in mexico . finally , the correlations between toileting and the physical activity measure were 0.119 in the usa and 0.062 in mexico . while the correlation coefficients had a lower magnitude in the mexican sample than in the usa sample , all were significant ( p < .01 ) . for the united states , the hrs dichotomous variable was created using the 1993 beale rural - urban continuum codes ( see hrs ) . we begin by comparing prevalence of self - reported disability and physical activity among all older adults across the two countries regardless of disability level . due to the difference in age structures across the two countries , prevalence of adl limitations and prevalence of physical activity ( table 2 ) is age age - adjusted rates of disability let us compare relative differences across the usa and mexico . we also provide cross - sectional weighted descriptive data for both countries according to the exercise variable . for this portion of the analyses , we select only those respondents that did not report any adl limitation at time 1 and examine their status two years later . because our study is based on longitudinal data , we face the issue of attrition and death by the followup period two years later ( unobserved respondents ) . the followup outcome is therefore measured by five categories : remaining nondisabled across the two years , moving from no disability to one disability , from no disability to two or more disabilities , from no disability to death and those that are lost to followup . we used multinomial models to determine the likelihood of moving to one of the followup categories over two years ( reference group : remaining nondisabled ) . the estimators of the multinomial model indicate the relationship of the outcome variable across categories of the independent variables that are interpreted as a relative risk ratio compared to a reference category ( hilbe ) . in order to facilitate the interpretation of results , we also present figures of the estimated probabilities of the outcome variable based on the multivariate models . table 2 shows the age - standardized prevalence of functional limitations by country in table 2(a ) as well as the prevalence of exercise according to functional limitations in table 2(b ) . table 2(a ) shows the prevalence of functional limitations across the two countries at baseline . the data confirmed previous research that overall disability rates were higher in the total usa elderly population than in the mexican elderly population . whereas 11.5% of elders in the usa reported at least one adl limitation , about 10.6% of persons in mexico reported a disability . this somewhat counterintuitive finding suggests a higher degree of overlap of adl limitations among persons in mexico , which is confirmed when examining the number of adl limitations reported . less than one percent of the sample in the usa reported problems with all adl limitations , whereas nearly two percent reported having limitations in all five areas in mexico . table 2(b ) compares the prevalence of physical activity in the usa and mexico by adl limitation categories , age standardized . in general , the prevalence of exercise is higher in the usa than in mexico , where approximately 46% of persons in the usa reported doing vigorous physical activity , compared to less than one - third ( 29% ) of elders in mexico . when prevalence of physical activity is broken down by functional limitations , nearly half ( 44% ) of elders without a disability in the usa reported exercising , compared to 27% of elders without a disability in mexico . however , for persons with one disability or with two or more disabilities , the difference between countries becomes very small . table 3 provides prevalence of vigorous physical activity by main characteristics of the sample separately for each country . in both countries , the percent reporting physical activity is lower in mexico than in the united states . in both countries men are more likely to report exercising than women , although the gender gap is much larger in mexico than in the united states . additionally , in the usa , there appeared to be a gradient in physical activity with assets , where those with higher assets had higher rates of physical activity . similarly , there was a slight gradient in physical activity with educational attainment in the usa , whereas such a pattern was not found in mexico . while for the usa sample there were not large differences in exercise behavior by area of residence and having health insurance , these showed important differences for the mexican sample . persons living in an urban area were more likely to report exercising than those living in a rural area in mexico . those persons without health insurance in mexico were also more likely to exercise , compared to those that were insured . results show that the focal independent variable , physical activity , is significantly protective for each of the negative outcomes in the usa regression model ( model 1 ) . however , in the model examining mexico only ( model 2 ) , physical activity is significantly protective only for the worst outcomes ( 2 + adls and death ) . also , the propensity to go from no adl to a worse outcome is smaller in mexico . the results indicate that engaging in physical activity prevents negative health outcomes in both mexico and the united states , although the effect is less protective in mexico than in the united states . this is not the case for death , however , where exercise is protective for death in both countries , and the effect is not significantly different . in other words , the effect of physical activity on death additional models ( results available upon request ) examined a variety of potentially interesting interactions . in order to determine if the effect of physical activity is different across various characteristics , we ran interactions between physical activity and gender , physical activity and age , and physical activity and education . figure 1 visually charts the previous models predicting the probability of going from no adl limitation at time 1 to one adl limitation , 2 or more limitation , or to death at time 2 , by physical activity and by country . the figure indicates that those who exercise have a lower probability of transitioning to a worse disability status or to death in both countries . as expected , the probability of disability or death increased with age in each country , regardless of physical activity . however , the probability curve for transitioning to one adl is nearly the same in mexico for exercisers and nonexercisers . on the other hand , for the united states , there is a clear advantage for those who exercise , with a lower probability of transitioning to one adl , especially at the older ages . a protective effect appears for mexico as well as for the usa when examining the probability of transitioning to two or more adls at time 2 . those who exercise in the usa have similar probability of transition to two or more adls as those who exercise in mexico . the final graph in figure 2 shows the probability of death at time 2 by age , physical activity , and country . while the probability of death is higher for the usa than in mexico regardless of exercise , the impact of exercise appears to be similar across both countries . this paper compared the impact of physical activity on the incidence of disability among older persons in two countries at different stages of the epidemiologic transition , mexico and the united states . to our knowledge , this is the first paper to examine the importance of physical activity on predicting transitions in disability using a comparative approach across two countries . this supports recent evidence that transitions among older adults towards healthy lifestyle habits , such as avoiding tobacco and binge alcohol drinking , or exercising , appear to be underway in the usa but not yet in mexico . one is that the survey questions were asked in a slightly different way across the two countries , and because physical activity was self - reported , it is possible that the interpretation of the question may have been different across the two samples . that the two countries are at different stages of a lifestyle transition , where transitions to healthier lifestyles have progressed further in the usa than in mexico . social and policy changes impacting healthier lifestyle choices have occurred earlier in the usa and have been more extensive , which may in part explain this disparity in levels of exercise between the two countries . men had nearly twice the prevalence rate of physical activity than women in mexico ( 43% versus 23% , resp . ) while the gender gap was not as large in the usa , men were still more likely to exercise than women . although there were no large differences in physical activity prevalence by area of residence and health insurance status in the usa , there were notable differences by these groups in mexico . it is possible that those uninsured or those living in an urban environment in mexico may be employed in more physically demanding labor , which would be categorized as vigorous exercise in the surveys . , accessing a gym ) are easier in urban areas or that a culture of exercise has been adapted to a greater extent in urban areas of mexico compared to rural areas of the country . the results also show that not only is the level of disability different across the usa and mexico , but that the effect of physical activity on disability is significantly different across the two countries . overall , we found a beneficial effect of physical activity against onset of disability or death at followup in both countries . however , we also found that the protective effect of physical activity on disability is stronger in the usa than in mexico . this supports our initial hypothesis that physical activity is less protective among the mexican older population since they represent a more selected group of survivors than in the us . in other words , we speculate that older adults in the usa are more disabled at older ages and therefore are more likely to benefit from a lifestyle intervention . this result has important implications for aging in developing countries that are lagging in the epidemiological transition . we should expect that policies that are implemented in the countries towards healthy lifestyles should have lower impact while the countries are in early stages of the transition compared to the likely impact that similar policies may have later on . one limitation of this study is the question used to determine physical activity in the surveys . it is likely that the various types of exercise may have different impacts on health . this may be particularly the case when comparing these two countries with different cultures and at different lifestyle transitions . similarly , recommendations by the centers for disease control and prevention and american college of sports medicine ( cdc / acsm ) include an emphasis on the value of moderately intense activity , which we were unable to capture . it was not possible to control for the type of activity or for the intensity of the activity in these surveys , and it is not clear how shorter or more moderate bouts of activity may impact functional limitations . future research should consider including measures such as metabolic equivalent task ( met ) values , which allows the researcher to determine both the intensity and rate of exercise . finally , the wording of the activity question was identical across the two surveys except for the different time frames the respondents are asked to reflect on . respondents in mexico were asked about their average exercise in the last two years , whereas those in the usa were asked about their average over the past year . this is a limitation when comparing physical activity rates across countries , and the results should be interpreted with caution . however , the issue of timing is less of a concern when comparing the effects of physical activity on disability within each country , which is the main focus of this study . as the epidemiological and demographic transitions continue to run their course in developing countries such as mexico , as the results of this study confirm , lifestyle changes such as exercise can help in the avoidance of chronic and disabling conditions . this is particularly important in the design of policies for older adults , since our work has shown that the beneficial effects of physical activity extend to these age cohorts in a developing country such as mexico . however , our work has shown also that the positive effect of physical exercise may be dependent on the stage of the transition that societies are undergoing . this is informative , not only to expect lower impact of exercise on health overall compared to more developed countries , but also to motivate the search for modified or alternative exercise interventions to achieve a higher impact in countries that are early in the epidemiologic transition . nevertheless , policies aimed to encourage physical activity in mexico may play particularly important roles in preventing negative health outcomes . our work has also indicated avenues of further work for future population - based studies , in order to better capture the concept of physical exercise among older adults .

translesion dna synthesis ( tls)1 is a significant dna damage tolerance mechanism to overcome replication blocks caused by dna lesions or to seal gaps opposite lesions ( 1,2 ) . in the past decade , numerous dna polymerases ( pols ) have been discovered that possess lesion bypass activity . recently , a new human dna pol has been identified , poln or pol , and falls in the a - family of dna pols ( 3 ) . this family includes escherichia coli ( e. coli ) pol i and human pol . pol is a moderately processive enzyme and is able to carry out strand displacement dna synthesis with much higher efficiency than the klenow fragment , the pol - proficient portion of e. coli pol i. it also replicates nondamaged ( nd ) dna with low fidelity ; specifically , it frequently misincorporates dt opposite template dg with about half the efficiency of incorporation of the correct nucleotide dc opposite template dg ( 4,5 ) . because low fidelity of dna synthesis is common for tls pols and pol lacks 35 exonuclease proofreading activity , a role of pol in the bypass of specific dna lesions has been proposed ( 4,5 ) . indeed , this pol can carry out tls in vitro , being specifically proficient in accurate bypass of thymine glycols , major groove dna lesions ( 5 ) . however , it is completely blocked by a number of other dna modifications , including a cisplatin - induced gpg intrastrand cross - link , an abasic site , a cyclobutane pyrimidine dimer , and a thyminethymine 6 - 4 photoproduct ( 5 ) . 1abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . a function of pol in dna cross - link repair in mammalian cells has been proposed ( 6 ) . in particular , the data suggested that pol is involved in homologous recombination in response to various dna cross - linking agents and potentially interacts with multiple proteins in the fanconi anemia pathway , which are relevant to dna cross - link repair . however , a cellular role for pol in homologous recombination - independent , tls - dependent repair of dna cross - links has not yet been addressed . additionally , as already mentioned , thymine glycol is the only lesion that pol has been reported to be able to bypass in vitro ( 5 ) . to understand the cellular function of pol in tls , it is crucial to identify types of dna lesions that this pol can bypass . because it has been previously established that both the exo and the exo klenow fragment can catalyze tls past certain major groove dna adducts ( 7,8 ) , we hypothesized that in addition to thymine glycols , pol may catalyze replication bypass of additional major groove dna lesions this study was designed to test this hypothesis to facilitate insight into the cellular function of pol in tls . p-6 bio - spin columns were obtained from bio - rad ( hercules , ca ) . uracil dna glycosylase , t4 dna ligase , and t4 polynucleotide kinase were purchased from new england biolabs ( beverly , ma ) . slide - a - lyzer dialysis cassettes with a molecular weight cutoff of 10000 were purchased from thermo scientific ( rockford , il ) . yeast pol and pcna were purified as previously described ( 9 ) and were generous gifts from dr . the lys - trp - lys - lys and lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr peptides were obtained from sigma - genosys ( st . louis , mo ) . ( a ) all nd oligodeoxynucleotides were synthesized by the molecular microbiology and immunology research core facility at oregon health & science university ( portland , or ) . ( b ) oligodeoxynucleotides containing -hydroxypropano - da or -hydroxypropano - dg were synthesized as previously described ( 10 ) . dnas containing dnapeptide cross - links ( pcls ) ( figure 1a , c ) were prepared by the reaction of the -hydroxypropano - da- or -hydroxypropano - dg - containing oligodeoxynucleotides with either 20 nmol of lys - trp - lys - lys or 4 nmol of lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr in the presence of 50 mm sodium cyanoborohydride according to the previously published procedure ( 11 ) . the sequences of oligodeoxynucleotides containing a n - da pcl and a n - dg pcl are shown in table 1 , column 2 , rows 1 and 2 , respectively . structures of dna adducts . the pcls consist of either a tetra ( lys - trp - lys - lys ) or dodecyl ( lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr ) peptide attached via an acrolein moiety at the n position of da ( a ) or n position of dg ( c ) . the resulting pcls are referred to as n - da pcl4 , n - da pcl12 , n - dg pcl4 , and n - dg pcl12 . ( f ) structure of ( + ) -trans - anti - bpde - da . a 3-glycerol unit ( gl ) or a dideoxycytidine ( dd ) were incorporated to prevent dna synthesis off of the shorter strand of the icl - containing oligodeoxynucleotides . the number in the parentheses in front of the primer sequence indicates the position of the primer 3-oh . ( c ) the synthesis of the oligodeoxynucleotides containing interstrand dna cross - link ( icl ) between n - das of complementary strands ( figure 1b ) is described in the supporting information . the sequences of a n - da icl1 and a n - da icl2 are shown in table 1 , column 2 , rows 3 and 5 , respectively . the n - da icl2 oligodeoxynucleotide was generated from a precursor oligodeoxynucleotide ( pre - n - da icl2 , table 1 , column 2 , row 4 ) as described previously ( 12 ) . ( d ) oligodeoxynucleotides containing an icl between n - dgs ( figure 1d ) were synthesized as previously described ( 1214 ) , and the sequences of the oligodeoxynucleotides containing each n - dgn - dg icl ( n - dg icl1 , n - dg icl2 , n - dg icl3 , and n - dg icl4 ) are shown in table 1 , column 2 , rows 69 . a detailed description of each type of n - dg icls has been reported previously ( 12 ) . ( e ) an oligodeoxynucleotide containing 1,n - etheno - da ( da ) ( figure 1e and table 1 , column 2 , row 10 ) was synthesized by the molecular microbiology and immunology research core facility at oregon health & science university ( portland , or ) . ( f ) an oligodeoxynucleotide ( 5-ctctcacttcc-3 ) containing ( + ) -trans - anti - benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide - da [ ( + ) -bpde - da ] ( figure 1f ) at the underlined nucleotide was synthesized as previously described ( 15 ) and was a generous gift from dr . this oligodeoxynucleotide was ligated to 16-mer oligodeoxynucleotide ( 5-attccgtatccatttt-3 ) using 800 units of t4 dna ligase at 12 c overnight in the presence of 23-mer scaffold oligodeoxynucleotide ( 5-tggatacggaatggaagtgagag-3 ) . the ligation reaction was terminated by the addition of an equal volume of formamide dye solution [ 95% ( v / v ) formamide , 10 mm edta , 0.03% ( w / v ) xylene cyanol , and 0.03% ( w / v ) bromphenol blue ] , and the products were separated on a 10% acrylamide gel containing 8 m urea . dna was eluted from the gel with 0.5 m ammonium acetate/10 mm magnesium acetate solution , dialyzed against 10 mm tris - hcl ( ph 7.4 ) buffer containing 1 mm edta in slide - a - lyzer dialysis cassette ( mwco of 10000 ) , and further purified through a p-6 bio - spin column . the resulting 27-mer was used for dna replication assays ( table 1 , column 2 , row 11 ) . for replication assays , primers were designed for reactions under a running or standing start condition . in the former case , the primer 3-oh was positioned several nucleotides upstream of the lesion site . in latter case , the primer 3-oh was positioned immediately prior to the lesion site ( 1 primer ) . dna replication assays were carried out in the following buffers : for pol , the reaction mixture contained 25 mm tris - hcl ( ph 7.5 ) , 8 mm mgcl2 , 10% glycerol , 100 g / ml bovine serum albumin , and 5 mm dithiothreitol . for reaction with pol with n - dgn - dg icls , mg - acetate was used instead of mgcl2 . for pol , the reaction mixture contained 40 mm hepes - koh ( ph 6.8 ) , 10% glycerol , 200 g / ml bovine serum albumin , 1 mm dithiothreitol , and 8 mm mgcl2 . dna substrate , pol , and dntp(s ) concentrations , as well as reaction times , are given in figure legends . reaction products were resolved through 15% acrylamide gel containing 8 m urea and visualized using a phosphorimager screen . the percentage of primer extended in extension reactions was determined by taking the ratio of extended primer beyond the adducted base to the total amount of primer ( unextended primer + extended primer up to and opposite the lesion + extended primer beyond the lesion ) . the percentage of primer extended in single nucleotide incorporations was determined by taking the ratio of extended primer to the total amount of primer ( unextended primer + extended primer ) . to address the hypothesis that pol can catalyze replication bypass of additional major groove dna lesions , we utilized n - da acrolein - mediated pcls as a model for the bulky major groove dna adducts ( 11 ) . specifically , tetra- and dodecylpeptide cross - links ( figure 1a , n - da pcl4 and n - da pcl12 , respectively ) were used . the molecular masses of these pcl - modified bases were 763 da for n - da pcl4 and 1738 da for n - da pcl12 versus 135 da for unmodified adenine . these lesions were positioned 15 nucleotides from the 5 end of the 30-mer template and annealed with a 16-mer radiolabeled primer in which the 3-oh was three nucleotides upstream of the lesion site . data in figure 2a revealed that pol could fully extend primers annealed to these adducted templates ; under the conditions used , very minimal blockage to dna synthesis was observed one nucleotide prior to the lesion . to assess the identity of the nucleotides incorporated opposite the lesion , single nucleotide incorporations were conducted . because n - da pcl12 is bulkier than n - da pcl4 , further assays used only substrate containing this lesion . data showed that pol incorporated only the correct nucleotide , dt , opposite n - da pcl12 ( figure 2b ) , suggesting that it catalyzed error - free bypass of this adduct . it is of interest to note that two nucleotides were incorporated for both the nd and the n - da pcl12 substrates when only dttp was present . it has been reported that pol catalyzes error - prone insertion of dt opposite dg ( 4,5 ) . because the templating nucleotide immediately downstream of the adducted base is a dg , this specific sequence context likely accounts for the observed incorporation of the second dt . to quantitatively evaluate the extent of the blockage posed by n - da pcl12 for the pol -catalyzed nucleotide insertion , dttp titration assays ( 6.4 nm to 100 m dttp at 5-fold incrementally increased concentrations , left to right ) these results demonstrated that pol could incorporate approximately an equal amount of dt opposite the adducted base at concentrations only 5-fold higher relative to nd da ; specifically , 20% of primers were extended at 800 nm dttp using nd substrate and at 4 m dttp using damaged substrate . replication bypass of n - da peptide cross - links by human pol . ( a ) primer extensions were catalyzed by 50 nm human pol under running start conditions ( 3 primer ) in the presence of 5 nm primer template containing nd , n - da pcl4 , or n - da pcl12 for 30 min at 37 c . ( b ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) in the presence of 2 nm primer template containing nd or n - da pcl12 for 15 min at 37 c . reactions shown in lanes 16 and lanes 712 were conducted side - by - side , and products were separated on the same gel . the reactions shown in panels a and b were carried out in the presence of 100 m individual or all dntps . ( c ) dttp titration assays with 2 nm primer template were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) for 10 min at 37 c using the same substrates as described in panel b. dttp concentration ranges from 6.4 nm to 100 m ( 5-fold incremental increase ) . reactions shown in lanes 18 and lanes 916 were conducted side - by - side , and products were separated on the same gel . having demonstrated that pol can readily bypass n - da pcls , we hypothesized that it may also be able to carry out tls past other n - da major groove adducts , including n - dan - da icls ( figure 1b ) . although icls had been previously thought to pose an insurmountable block to replication , our laboratory has recently demonstrated that human dna pol and e. coli dna pol iv can catalyze replication bypass of an icl between the n - positions of dgs , which models the icl formed by acrolein in a cpg sequence context ( 12,16 ) . related icls were constructed between the n - positions of da placing the cross - link in the major groove ( figure 1b and table 1 , rows 3 and 5 ) . an n - da icl1 was designed to model for a potential intermediate of icl processing that could be generated by exo / endonucleolytic removal of dna patch 3 to the icl site . even though n - da icl1 contains an eight nucleotide duplex region 5 to the icl site , it was anticipated that pol would efficiently catalyze strand displacement dna synthesis due to its intrinsic high strand displacement activity ( 5 ) , a property that is useful for replicating this type of dna lesion . a n - da icl2 was constructed to model another potential intermediate of icl processing ; this could be generated by removal of dna patches both 3 and 5 to the icl site . when these major groove icl - containing dnas were used in primer extension reactions , pol could extend primers and generate full - length products ( figure 3a , b ) . for both types of icls , the amount of primers extended beyond the adducted site was less as compared to nd substrate ; specifically , a 10- and 4-fold decrease was observed for n - da - icl1 and n - da - icl2 , respectively . as expected , pol catalyzed strand displacement synthesis on the n - da icl1 template ( figure 3a ) . when the identity of nucleotides incorporated opposite the cross - linked site was examined by single nucleotide incorporations using the n - da icl2 template , the data indicated that pol preferentially incorporated the correct nucleotide , dt , opposite the n - modified da ( figure 3c ) . replication bypass of n - dan - da interstrand cross - links by human pol . primer extensions were catalyzed by 10 nm human pol under running start conditions ( 9 primer ) using nd , n - da icl1 ( a ) , or n - da icl2 ( b ) . reactions shown in lanes 12 and lanes 34 were conducted side - by - side , and products were separated on the same gel . ( c ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) using nd and n - da icl2 . all reactions were carried out in the presence of 5 nm primer template and 100 m individual or all dntps for 15 min at 37 c . given that pol was able to bypass the extremely bulky major groove n - da pcls and icls , we tested whether it could replicate past chemically identical pcl and icl lesions except situated in the minor groove through a linkage with the n position of dg ( figure 1c , d ) . primer extensions using these n - dg linked pcls revealed that pol was strongly blocked one nucleotide prior to both pcls and was completely blocked at the site opposite the lesion , with no further primer extension observed ( figure 4a ) . the identity of the nucleotide(s ) incorporated opposite the pcls was not determined due to the limited nucleotide incorporation . even though pol could not replicate past the n - dg pcls , suggesting an inability to replicate past minor groove dna adducts , we tested whether pol could catalyze tls past n - dgn - dg icls ( figure 1d and table 1 , rows 69 ) . no incorporation opposite the lesion site was observed for these icl - containing dnas ( figure 4b ) . thus , similar to the observations made using pcl - containing dnas , pol has much higher tolerance for the major groove n - da icls than the minor groove n - dg icls . replication bypass of n - dg peptide and interstrand cross - links by human pol . ( a ) primer extensions were catalyzed by 10 nm human pol under running start conditions ( 4 primer ) in the presence of 5 nm primer template containing nd , n - dg pcl4 , or n - dg pcl12 for 30 min at 37 c . ( b ) primer extensions were catalyzed by 25 nm human pol under running start conditions ( 10 primer ) in the presence of 7.5 nm primer template containing nd , n - dg icl1 , n - dg icl2 , n - dg icl3 , or n - dg icl4 for 10 min at 37 c . all reactions were carried out in the presence of 100 m dntps . because pol was able to accurately replicate past major groove n - da pcl and icl lesions , we wanted to further explore the substrate range of pol and address the question concerning pol ability to bypass da adducts in which both n1 and n sites are blocked by modification . thus , the ability of pol to bypass an da ( figure 1e ) was examined . da is a promutagenic lesion that can be formed by exposure to industrial toxicants such as vinyl chloride and by the reaction of dna with bis - electrophiles produced by lipid peroxidation ( 17 ) . the running start primer extensions demonstrated that pol was severely blocked one nucleotide prior to the da adduct , with only a very small amount of full - length product formed ( figure 5a ) . however , the nucleotide incorporation opposite the lesion appeared to be quite accurate . under the conditions used , the primers were extended in the presence of dttp but not other dntps ( figure 5b ) . on the basis of these data , we conclude that relatively nonbulky modifications at both n1 and n of da are sufficient to inhibit pol -catalyzed tls . replication bypass of 1,n - etheno - da and ( + ) -trans - anti - bpde - da by human pol . ( a ) primer extensions were catalyzed by 10 nm human pol under running start conditions ( 3 primer ) in the presence of 5 nm primer template containing nd or da . ( b ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) in the presence of 5 nm primer template using the same substrates as described in panel a. ( c ) primer extensions were catalyzed by 10 nm human pol under running start conditions ( 6 primer ) in the presence of 2 nm primer template containing nd or ( + ) -bpde - da . ( d ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) in the presence of 2 nm primer template using the same substrates as described in panel c. all reactions were carried out in the presence of 100 m individual or all dntps for 15 min at 37 c . we also investigated the ability of pol to carry out tls past ( + ) -bpde - da ( figure 1f ) , one of the adducts formed by the active metabolite of the prevalent environmental pollutant , benzo[a]pyrene ( 18 ) . to construct an oligodeoxynucleotide of sufficient length for tls analyses , an 11-mer oligodeoxynucleotide containing the ( + ) -bpde - da was p - labeled , ligated to a 16-mer oligodeoxynucleotide , and gel - purified . residual radioactive signals of the resulting 27-mer template are evident in all lanes [ figure 5c , d , ( + ) -bpde - da-27-mer template ] . primer extensions revealed that pol could not fully extend the 6 primer and was severely blocked one nucleotide prior to the adducted site ( figure 5c ) . in addition , pol displayed error - prone patterns of nucleotide incorporation opposite the lesion ( figure 5d ) ; in the presence of datp , 16% of primers were extended , while only 1% of primers were extended in the presence of the correct dttp . a limited primer extension ( < 1% ) was also observed in the presence of dgtp . pol -catalyzed tls past ( + ) -bpde - da was inefficient , incomplete , and error - prone . although very significant progress has been made in both structural analyses of many of the tls pols and the range of damaged dna substrates that these pols can bypass , in the majority of cases , the biological role of these enzymes can only be inferred by changes in efficiency and mutagenic rates of tls in their absence . little data have been gathered concerning the role of these enzymes in developmental biology , their tissue - specific expression and steady - state distribution in adult organisms , the mechanism(s ) for recruitment to sites of dna damage , and the processes that regulate their expressions in response to environmental toxicant exposures and/or endogenous stresses . in the case of pol , the initial characterizations have demonstrated a very limited repertoire of lesions that pol can efficiently bypass ( 5 ) . although a cellular role of pol in tls has not yet been established , its high frequency of misincorporation of dt opposite dg suggests that its expression would be tightly regulated . thus , understanding the potential substrate specificity of pol may help to guide future investigations of its biological role in tls and circumstances for induction or activation . in this regard , we demonstrated that although pol could carry out tls past bulky major groove dna lesions such as n - da pcls and n - dan - da icls , it failed to replicate dnas containing chemically similar minor groove dna lesions . thus , the location of the lesion within dna is one important structural determinant that influences the ability of pol to bypass the lesion . we also discovered that pol could not efficiently bypass da , and we speculate that this may be either due to pol requirement for the watsoncrick edge for efficient da templating or structural heterogeneity of the lesion within the active site . regarding the former possibility , pol may fail to catalyze efficient tls past da because the watsoncrick edge of this adduct is blocked by the modification . the majority of dna pols utilize the geometry of the canonical watsoncrick pairs for the nucleotide selection ( 19 ) , but some tls pols utilize alternative mechanisms . for example , pol induces a syn conformation on template purines , which results in a hoogsteen base pairing with the correctly matched incoming nucleotide and enables bypass of lesions that disrupt the watsoncrick edge but not the hoogsteen edge ( 20 ) . the diminished capability of pol to bypass da strongly suggests that the accessibility of the watsoncrick edge of the template da is important for the efficient replication by this pol ; thus , it is likely that pol -catalyzed polymerization involves the recognition of the watsoncrick geometry . with regard to structural heterogeneity of da adduct , nmr analyses have revealed that orientation of the base around the glycosydic bond is affected by identity of nucleotide opposite the lesion ( 21,22 ) . when paired with dt , the da is in a normal anti conformation ; however , the da base prefers the syn conformation when placed opposite a mispaired dg . additionally , the cocrystal structure of dna containing an da bound to pol also revealed that the lesion adopted a syn conformation ( 20 ) . thus , da can adopt a syn or anti conformation depending on the base it is paired with , as well as interactions with the pol . there is a possibility that similar to pot , the adducted base is in a syn conformation in the pol active site . however , it is unclear whether either orientation of the -modified base can be efficiently utilized for nucleotide insertion . intriguingly , pol also could not fully bypass a ( + ) -bpde - da . the nmr solution structure of this lesion opposite dt in duplex dna showed complex conformational heterogeneity ( 23 ) . computational studies using an nmr structure of duplex dna where dt was placed opposite the modified da have revealed that the glycosydic bond of ( + ) -bpde - da was in syn - anti equilibrium ; therefore , the modified da has a diminished capability to participate in watsoncrick base pairing ( 24 ) . it is possible that the glycosydic bond of modified da rotates and adopts a syn conformation at the primer - template junction and therefore can not participate in watsoncrick base pairing . an alternative explanation may be that the structure of the active site of pol is significantly distorted by bulky aromatic ring system of this lesion . it has been shown that the bpde moiety of this lesion is intercalated into the double helix , on the 3 side of the adducted da , and thus causes significant structural distortions at the active site of t7 pol ( 25 ) . because the t7 pol is also a member of the a - family pols , the inability of pol to bypass this lesion may be due to the active site distortions , similar to those shown for t7 pol . with regard to the low fidelity synthesis past this lesion by pol , it has been shown that the adducted da is displaced from the active site of t7 pol as a result of steric hindrance between the bpde moiety and the primer template . among the four dntps , datp fits best into the dntp binding pocket that is enlarged due to the displacement of adducted da ( 25 ) . this structural change provides a likely explanation for the predominant misincorporation of da by pol . recently , we reported a comparative study of the mutagenic consequences of identical lesions linked in the major groove via n - da versus the minor groove linkage via n - dg during the replication of single - stranded pms2 shuttle vectors in cos-7 cells ( 11 ) ; these lesions were the same site - specific pcl4s substrates used in this investigation . these data revealed that dna containing either lesion could be replicated , with the n - dg pcl4 being moderately mutagenic , while the n - da pcl4 showed very low mutagenic potential . although it is anticipated that the extremely bulky nature of the n - da pcls lesions would block replicative pols , we carried out in vitro replication assays using pol ( figure 6 ) . the primer extensions and single nucleotide incorporations showed some limited bypass of n - da pcl12 at very high dntp concentrations ( 100 m ) ( figure 6a ) , while the titration experiment with varying concentrations of dttp ( from 6.4 nm to 100 m ) revealed the severity of the blockage posed by this lesion for the pol -catalyzed nucleotide insertion ( figure 6b ) . specifically , pol could incorporate dt opposite the nd da at concentrations as low as 6.4 nm with 8% of primers being extended , while even at 100 m dttp , only 2% of primers were extended on the n - da pcl12-containing substrate . these results suggest that although in vitro conditions could be manipulated to observe bypass of n - da pcls by pol , the block to replication is likely to occur when pol encounters such bulky lesions ; therefore , specialized pols may be recruited to carry out tls . the data presented herein suggest that pol is a likely candidate to perform tls past a variety of major groove lesions . at present , the structural basis is unknown why pol could efficiently bypass pcl and icl as large as 3025 da but fails to bypass less bulky bpde adduct . however , we speculate that the lesions that are readily bypassed by pol may have significant conformational flexibility and thus , can be accommodated without distorting dnaenzyme interactions within the pol active site . overall , our data suggest that since pol is capable of bypassing extremely large major ( but not minor ) groove lesions , it may play a role in tls following toxicant challenges that result in the formation of such lesions in the cells . replication bypass of n - da peptide cross - link by yeast pol . ( a ) single nucleotide incorporations and primer extensions were catalyzed by 0.5 nm yeast pol with 2 nm primer template containing nd or n - da pcl12 under standing start conditions ( 1 primer ) in the presence or absence of 5 nm pcna for 20 min at 37 c . ( b ) dttp titration assays with 2 nm primer template were catalyzed by 0.5 nm yeast pol under standing start conditions ( 1 primer ) in the absence of pcna for 10 min at 37 c using the same substrates as described in panel a. dttp concentration ranges from 6.4 nm to 100 m ( 5-fold incremental increase ) .

dna polymerase ( poln or pol ) is a newly discovered a family polymerase that generates a high error rate when incorporating nucleotides opposite dg ; its translesion dna synthesis ( tls ) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions . in the current investigation , we describe a novel tls substrate specificity of pol , demonstrating that it is able to bypass exceptionally large dna lesions whose linkages are through the dna major groove . specifically , pol catalyzed efficient and high fidelity tls past peptides linked to n6-da via a reduced schiff base linkage with a -hydroxypropano - da . additionally , pol could bypass dna interstrand cross - links with linkage between n6-das in complementary dna strands . however , the chemically identical dnapeptide and dna interstrand cross - links completely blocked pol when they were located in the minor groove via a n2-dg linkage . furthermore , we showed that pol incorporated a nucleotide opposite the 1,n6-etheno - da ( da ) in an error - free manner and ( + ) -trans - anti - benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide - da [ ( + ) -bpde - da ] in an error - prone manner , albeit with a greatly reduced capability . collectively , these data suggest that although pol bypass capacity can not be generalized to all major groove dna adducts , this polymerase could be involved in tls when genomic replication is blocked by extremely large major groove dna lesions . in view of the recent observation that pol may have a role in cellular tolerance to dna cross - linking agents , our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some dnaprotein and dnadna cross - links .

Introduction Experimental Procedures Results Discussion

translesion dna synthesis ( tls)1 is a significant dna damage tolerance mechanism to overcome replication blocks caused by dna lesions or to seal gaps opposite lesions ( 1,2 ) . recently , a new human dna pol has been identified , poln or pol , and falls in the a - family of dna pols ( 3 ) . pol is a moderately processive enzyme and is able to carry out strand displacement dna synthesis with much higher efficiency than the klenow fragment , the pol - proficient portion of e. coli pol i. it also replicates nondamaged ( nd ) dna with low fidelity ; specifically , it frequently misincorporates dt opposite template dg with about half the efficiency of incorporation of the correct nucleotide dc opposite template dg ( 4,5 ) . because low fidelity of dna synthesis is common for tls pols and pol lacks 35 exonuclease proofreading activity , a role of pol in the bypass of specific dna lesions has been proposed ( 4,5 ) . indeed , this pol can carry out tls in vitro , being specifically proficient in accurate bypass of thymine glycols , major groove dna lesions ( 5 ) . however , it is completely blocked by a number of other dna modifications , including a cisplatin - induced gpg intrastrand cross - link , an abasic site , a cyclobutane pyrimidine dimer , and a thyminethymine 6 - 4 photoproduct ( 5 ) . 1abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . a function of pol in dna cross - link repair in mammalian cells has been proposed ( 6 ) . in particular , the data suggested that pol is involved in homologous recombination in response to various dna cross - linking agents and potentially interacts with multiple proteins in the fanconi anemia pathway , which are relevant to dna cross - link repair . however , a cellular role for pol in homologous recombination - independent , tls - dependent repair of dna cross - links has not yet been addressed . additionally , as already mentioned , thymine glycol is the only lesion that pol has been reported to be able to bypass in vitro ( 5 ) . to understand the cellular function of pol in tls , it is crucial to identify types of dna lesions that this pol can bypass . because it has been previously established that both the exo and the exo klenow fragment can catalyze tls past certain major groove dna adducts ( 7,8 ) , we hypothesized that in addition to thymine glycols , pol may catalyze replication bypass of additional major groove dna lesions this study was designed to test this hypothesis to facilitate insight into the cellular function of pol in tls . dnas containing dnapeptide cross - links ( pcls ) ( figure 1a , c ) were prepared by the reaction of the -hydroxypropano - da- or -hydroxypropano - dg - containing oligodeoxynucleotides with either 20 nmol of lys - trp - lys - lys or 4 nmol of lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr in the presence of 50 mm sodium cyanoborohydride according to the previously published procedure ( 11 ) . ( f ) structure of ( + ) -trans - anti - bpde - da . ( c ) the synthesis of the oligodeoxynucleotides containing interstrand dna cross - link ( icl ) between n - das of complementary strands ( figure 1b ) is described in the supporting information . ( e ) an oligodeoxynucleotide containing 1,n - etheno - da ( da ) ( figure 1e and table 1 , column 2 , row 10 ) was synthesized by the molecular microbiology and immunology research core facility at oregon health & science university ( portland , or ) . ( f ) an oligodeoxynucleotide ( 5-ctctcacttcc-3 ) containing ( + ) -trans - anti - benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide - da [ ( + ) -bpde - da ] ( figure 1f ) at the underlined nucleotide was synthesized as previously described ( 15 ) and was a generous gift from dr . in the former case , the primer 3-oh was positioned several nucleotides upstream of the lesion site . dna replication assays were carried out in the following buffers : for pol , the reaction mixture contained 25 mm tris - hcl ( ph 7.5 ) , 8 mm mgcl2 , 10% glycerol , 100 g / ml bovine serum albumin , and 5 mm dithiothreitol . to address the hypothesis that pol can catalyze replication bypass of additional major groove dna lesions , we utilized n - da acrolein - mediated pcls as a model for the bulky major groove dna adducts ( 11 ) . specifically , tetra- and dodecylpeptide cross - links ( figure 1a , n - da pcl4 and n - da pcl12 , respectively ) were used . data in figure 2a revealed that pol could fully extend primers annealed to these adducted templates ; under the conditions used , very minimal blockage to dna synthesis was observed one nucleotide prior to the lesion . data showed that pol incorporated only the correct nucleotide , dt , opposite n - da pcl12 ( figure 2b ) , suggesting that it catalyzed error - free bypass of this adduct . it has been reported that pol catalyzes error - prone insertion of dt opposite dg ( 4,5 ) . because the templating nucleotide immediately downstream of the adducted base is a dg , this specific sequence context likely accounts for the observed incorporation of the second dt . to quantitatively evaluate the extent of the blockage posed by n - da pcl12 for the pol -catalyzed nucleotide insertion , dttp titration assays ( 6.4 nm to 100 m dttp at 5-fold incrementally increased concentrations , left to right ) these results demonstrated that pol could incorporate approximately an equal amount of dt opposite the adducted base at concentrations only 5-fold higher relative to nd da ; specifically , 20% of primers were extended at 800 nm dttp using nd substrate and at 4 m dttp using damaged substrate . replication bypass of n - da peptide cross - links by human pol . ( a ) primer extensions were catalyzed by 50 nm human pol under running start conditions ( 3 primer ) in the presence of 5 nm primer template containing nd , n - da pcl4 , or n - da pcl12 for 30 min at 37 c . ( b ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol under standing start conditions ( 1 primer ) in the presence of 2 nm primer template containing nd or n - da pcl12 for 15 min at 37 c . having demonstrated that pol can readily bypass n - da pcls , we hypothesized that it may also be able to carry out tls past other n - da major groove adducts , including n - dan - da icls ( figure 1b ) . although icls had been previously thought to pose an insurmountable block to replication , our laboratory has recently demonstrated that human dna pol and e. coli dna pol iv can catalyze replication bypass of an icl between the n - positions of dgs , which models the icl formed by acrolein in a cpg sequence context ( 12,16 ) . related icls were constructed between the n - positions of da placing the cross - link in the major groove ( figure 1b and table 1 , rows 3 and 5 ) . even though n - da icl1 contains an eight nucleotide duplex region 5 to the icl site , it was anticipated that pol would efficiently catalyze strand displacement dna synthesis due to its intrinsic high strand displacement activity ( 5 ) , a property that is useful for replicating this type of dna lesion . when these major groove icl - containing dnas were used in primer extension reactions , pol could extend primers and generate full - length products ( figure 3a , b ) . for both types of icls , the amount of primers extended beyond the adducted site was less as compared to nd substrate ; specifically , a 10- and 4-fold decrease was observed for n - da - icl1 and n - da - icl2 , respectively . as expected , pol catalyzed strand displacement synthesis on the n - da icl1 template ( figure 3a ) . when the identity of nucleotides incorporated opposite the cross - linked site was examined by single nucleotide incorporations using the n - da icl2 template , the data indicated that pol preferentially incorporated the correct nucleotide , dt , opposite the n - modified da ( figure 3c ) . replication bypass of n - dan - da interstrand cross - links by human pol . given that pol was able to bypass the extremely bulky major groove n - da pcls and icls , we tested whether it could replicate past chemically identical pcl and icl lesions except situated in the minor groove through a linkage with the n position of dg ( figure 1c , d ) . primer extensions using these n - dg linked pcls revealed that pol was strongly blocked one nucleotide prior to both pcls and was completely blocked at the site opposite the lesion , with no further primer extension observed ( figure 4a ) . even though pol could not replicate past the n - dg pcls , suggesting an inability to replicate past minor groove dna adducts , we tested whether pol could catalyze tls past n - dgn - dg icls ( figure 1d and table 1 , rows 69 ) . thus , similar to the observations made using pcl - containing dnas , pol has much higher tolerance for the major groove n - da icls than the minor groove n - dg icls . replication bypass of n - dg peptide and interstrand cross - links by human pol . because pol was able to accurately replicate past major groove n - da pcl and icl lesions , we wanted to further explore the substrate range of pol and address the question concerning pol ability to bypass da adducts in which both n1 and n sites are blocked by modification . thus , the ability of pol to bypass an da ( figure 1e ) was examined . however , the nucleotide incorporation opposite the lesion appeared to be quite accurate . under the conditions used , the primers were extended in the presence of dttp but not other dntps ( figure 5b ) . replication bypass of 1,n - etheno - da and ( + ) -trans - anti - bpde - da by human pol . ( c ) primer extensions were catalyzed by 10 nm human pol under running start conditions ( 6 primer ) in the presence of 2 nm primer template containing nd or ( + ) -bpde - da . we also investigated the ability of pol to carry out tls past ( + ) -bpde - da ( figure 1f ) , one of the adducts formed by the active metabolite of the prevalent environmental pollutant , benzo[a]pyrene ( 18 ) . to construct an oligodeoxynucleotide of sufficient length for tls analyses , an 11-mer oligodeoxynucleotide containing the ( + ) -bpde - da was p - labeled , ligated to a 16-mer oligodeoxynucleotide , and gel - purified . residual radioactive signals of the resulting 27-mer template are evident in all lanes [ figure 5c , d , ( + ) -bpde - da-27-mer template ] . in addition , pol displayed error - prone patterns of nucleotide incorporation opposite the lesion ( figure 5d ) ; in the presence of datp , 16% of primers were extended , while only 1% of primers were extended in the presence of the correct dttp . pol -catalyzed tls past ( + ) -bpde - da was inefficient , incomplete , and error - prone . although very significant progress has been made in both structural analyses of many of the tls pols and the range of damaged dna substrates that these pols can bypass , in the majority of cases , the biological role of these enzymes can only be inferred by changes in efficiency and mutagenic rates of tls in their absence . in the case of pol , the initial characterizations have demonstrated a very limited repertoire of lesions that pol can efficiently bypass ( 5 ) . although a cellular role of pol in tls has not yet been established , its high frequency of misincorporation of dt opposite dg suggests that its expression would be tightly regulated . thus , understanding the potential substrate specificity of pol may help to guide future investigations of its biological role in tls and circumstances for induction or activation . in this regard , we demonstrated that although pol could carry out tls past bulky major groove dna lesions such as n - da pcls and n - dan - da icls , it failed to replicate dnas containing chemically similar minor groove dna lesions . thus , the location of the lesion within dna is one important structural determinant that influences the ability of pol to bypass the lesion . we also discovered that pol could not efficiently bypass da , and we speculate that this may be either due to pol requirement for the watsoncrick edge for efficient da templating or structural heterogeneity of the lesion within the active site . regarding the former possibility , pol may fail to catalyze efficient tls past da because the watsoncrick edge of this adduct is blocked by the modification . the diminished capability of pol to bypass da strongly suggests that the accessibility of the watsoncrick edge of the template da is important for the efficient replication by this pol ; thus , it is likely that pol -catalyzed polymerization involves the recognition of the watsoncrick geometry . with regard to structural heterogeneity of da adduct , nmr analyses have revealed that orientation of the base around the glycosydic bond is affected by identity of nucleotide opposite the lesion ( 21,22 ) . there is a possibility that similar to pot , the adducted base is in a syn conformation in the pol active site . however , it is unclear whether either orientation of the -modified base can be efficiently utilized for nucleotide insertion . intriguingly , pol also could not fully bypass a ( + ) -bpde - da . computational studies using an nmr structure of duplex dna where dt was placed opposite the modified da have revealed that the glycosydic bond of ( + ) -bpde - da was in syn - anti equilibrium ; therefore , the modified da has a diminished capability to participate in watsoncrick base pairing ( 24 ) . it is possible that the glycosydic bond of modified da rotates and adopts a syn conformation at the primer - template junction and therefore can not participate in watsoncrick base pairing . an alternative explanation may be that the structure of the active site of pol is significantly distorted by bulky aromatic ring system of this lesion . because the t7 pol is also a member of the a - family pols , the inability of pol to bypass this lesion may be due to the active site distortions , similar to those shown for t7 pol . recently , we reported a comparative study of the mutagenic consequences of identical lesions linked in the major groove via n - da versus the minor groove linkage via n - dg during the replication of single - stranded pms2 shuttle vectors in cos-7 cells ( 11 ) ; these lesions were the same site - specific pcl4s substrates used in this investigation . these data revealed that dna containing either lesion could be replicated , with the n - dg pcl4 being moderately mutagenic , while the n - da pcl4 showed very low mutagenic potential . although it is anticipated that the extremely bulky nature of the n - da pcls lesions would block replicative pols , we carried out in vitro replication assays using pol ( figure 6 ) . the primer extensions and single nucleotide incorporations showed some limited bypass of n - da pcl12 at very high dntp concentrations ( 100 m ) ( figure 6a ) , while the titration experiment with varying concentrations of dttp ( from 6.4 nm to 100 m ) revealed the severity of the blockage posed by this lesion for the pol -catalyzed nucleotide insertion ( figure 6b ) . specifically , pol could incorporate dt opposite the nd da at concentrations as low as 6.4 nm with 8% of primers being extended , while even at 100 m dttp , only 2% of primers were extended on the n - da pcl12-containing substrate . these results suggest that although in vitro conditions could be manipulated to observe bypass of n - da pcls by pol , the block to replication is likely to occur when pol encounters such bulky lesions ; therefore , specialized pols may be recruited to carry out tls . the data presented herein suggest that pol is a likely candidate to perform tls past a variety of major groove lesions . at present , the structural basis is unknown why pol could efficiently bypass pcl and icl as large as 3025 da but fails to bypass less bulky bpde adduct . however , we speculate that the lesions that are readily bypassed by pol may have significant conformational flexibility and thus , can be accommodated without distorting dnaenzyme interactions within the pol active site . overall , our data suggest that since pol is capable of bypassing extremely large major ( but not minor ) groove lesions , it may play a role in tls following toxicant challenges that result in the formation of such lesions in the cells . replication bypass of n - da peptide cross - link by yeast pol . ( a ) single nucleotide incorporations and primer extensions were catalyzed by 0.5 nm yeast pol with 2 nm primer template containing nd or n - da pcl12 under standing start conditions ( 1 primer ) in the presence or absence of 5 nm pcna for 20 min at 37 c .

the standard therapy for patients with a primary epithelial ovarian cancer is cytoreductive surgery followed by chemotherapy . despite significant advances in primary treatment , 60% of patients with an advanced stage epithelial ovarian cancer ultimately suffer disease recurrence . as most cases of recurrent ovarian cancer are multifocal and the prognoses of such patients are rarely curative , the standard management for these patients with recurrent disease has been chemotherapy . in general , second - line chemotherapy in patients with recurrent ovarian cancer will lead to response rates of 5060% , 2030% and 10% in platinum - sensitive , platinum - resistant and platinum - refractory patients , respectively . in the 1980s and 1990s , the use of salvage whole abdominopelvic radiation therapy ( rt ) after cis - platinum failure was investigated [ 46 ] . however , the consensus of these studies was that salvage whole abdominopelvic rt was minimally effective and too toxic in this situation . recent data have indicated that complete cytoreduction , with appropriate surgical selection criteria ( prolonged disease - free interval > 6 months , largest dimension of the recurrent tumor 10 cm , high performance status , and no symptoms due to recurrence ) , for recurrent ovarian cancer is associated with a significant prolongation of survival . thus , in a subset of patients , secondary cytoreductive surgery might significantly improve survival . on the other hand , modern three - dimensional conformal rt planning techniques allow the administration of a conformal dose distribution around the tumor , potentially minimizing the dose of radiation administered to adjacent critical structures , and permitting escalated dose delivery to the tumor . recently , several studies have shown that curative - intent rt using modern techniques in patients with limited recurrent or oligometastatic disease , including breast cancer , colorectal cancer , lung cancer and pancreatic cancer , resulted in good local tumor control without severe toxicity [ 811 ] . in this context , we hypothesized that definitive rt for the recurrence of epithelial ovarian cancer limited to one or two regions , after complete remission had been achieved with aggressive front - line therapy , may improve survival . however , there have been only a few reports of definitive rt for tumor control of recurrent ovarian cancer [ 1214 ] . the purpose of this study was to assess the efficacy and toxicity of definitive rt for the limited recurrence of epithelial ovarian cancer after complete remission had been achieved with aggressive front - line therapy , and to identify the predictors of long - term survival . from september 1983 to january 2010 , 48 patients with ovarian cancer were treated with rt in the division of therapeutic radiology at our university hospital . there were consecutive 27 patients ( 56% ) with one or two gross recurrent lesions who were treated with definitive rt and were retrospectively analyzed . all 27 patients satisfied the following requirements and were included in this retrospective study : patients had a pathologically confirmed epithelial ovarian carcinoma , had achieved a complete remission after aggressive front - line therapy , and had one or two gross recurrent lesions at the start of the rt . written informed consent for treatment was obtained from all patients . the study was approved by the institutional review board of the university of occupational and environmental health . the characteristics and treatments of the patients are listed in tables 1and 2 . for aggressive front - line therapy , total abdominal hysterectomy ( tah ) , bilateral salpingo - oophorectomy ( bso ) and omentectomy were performed in 10 patients ; tah , bso , omentectomy and appendectomy in seven ; tah , left salpingo - oophorectomy ( lso ) , omentectomy , and appendectomy in four ; supracervical hystectomy , bso and omentectomy in three ; bso in one ; right salpingo - oophorectomy and omentectomy in one ; and lso in one patient . table 1.patient characteristicsvariablen(%)age median ( range)52 ( 3279)performance status 04 ( 15 ) 118 ( 66 ) 25 ( 19)histological type serous adenocarcinoma16 ( 59 ) non - classified adenocarcinoma6 ( 22 ) clear cell carcinoma5 ( 19)postoperative stage i3 ( 11 ) ii4 ( 15 ) iii18 ( 66 ) iv2 ( 7)site(s ) of the limited recurrence one site19 ( 70 ) paraaortic ln 8 ( 36 ) douglas ' pouch 5 ( 19 ) vagina 4 ( 15 ) iliac ln 2 ( 7 ) two sites8 ( 30 ) iliac and paraaortic ln 4 ( 15 ) douglas ' pouch and inguinal ln 1 ( 4 ) douglas ' pouch + paraaortic ln 1 ( 4 ) vagina + liver 1 ( 4 ) douglas ' pouch and spleen 1 ( 4)recurrent tumor size ( cm ) median ( range)3.0 ( 1.06.1)ln = lymph node.at the start of the radiotherapy . table 2.treatment methodsvariablen(%)radiotherapy external irradiation alone25 ( 93 ) median total dose ( gy , range ) 60.0 , 50.061.2 median daily dose ( gy , range ) 2.0 , 1.82.0 external irradiation plus brachytherapy1 ( 4 ) brachytherapy alone1 ( 4)chemotherapy for the recurrent tumor before rt20 ( 74 ) number of the regimens for the recurrent tumor before rt 115 22 33 response to chemotherapy immediately before rt cr0 pr5 nc9 pd6concurrent chemotherapy during rt6 ( 22)adjuvant chemotherapy after rt3 ( 11)hyperthermia during rt7 ( 26)rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . the tumor / node / metastasis ( tnm ) stages ( international union against cancer tnm classification , 6th edition ) were pathologically evaluated at the initial surgery : t1bn0m0 in one patient , t1cn0m0 in two patients , t2cn0m0 in four , t3bn0m0 in three , t3cn0m0 in 13 , t3cn1m0 in two , t3cn0m1 in one patient and t3cn1m1 in one . the time between front - line surgery and rt for the limited recurrence ranged from 658 months ( median 22 months ) . the period between completion of adjuvant chemotherapy and recurrence ranged from 146 months ( median 8 months ) . the median recurrent tumor size was 3.0 cm ( range 1.06.1 cm ) . the eastern cooperative oncology group performance status was evaluated at the start of the rt . all patients were diagnosed as having recurrent disease based on longitudinal computed tomography ( ct ) scans and tumor marker levels , and , in some cases , f - fluorodeoxyglucose positron emission tomography / ct and/or magnetic resonance imaging were also used . after initial surgery , all patients received adjuvant chemotherapy as follows : paclitaxel in combination with carboplatin in 15 patients , and a combination of cyclophosphamide , adriamycin and cisplatin in 12 patients . twenty ( 74% ) of the 27 patients received systemic chemotherapy for the limited recurrent tumor followed by definitive rt as follows : paclitaxel in combination with carboplatinin in 10 patients , docetaxel in combination with carboplatin in six , irinotecan in combination with mitomycin c in three , cyclophophamide and adriamycin in combination with cisplatin in two , cyclophosphamide in combination with adriamycin in one , etoposide in combination with cisplatin in one , etoposide in combination with carboplatin in one , irinotecan in combination with cisplatin in one , irinotecan in combination with carboplatin in one , cisplatin in one and tegafur - uracil in one patient . although no specific chemotherapy protocol existed , six ( 22% ) of the 27 patients were treated with concomitant systemic chemotherapy during the course of rt as follows : paclitaxel in combination with carboplatin in three patients , carboplatin in two and cisplatin in one . three ( 11% ) patients received adjuvant chemotherapy using paclitaxel in combination with carboplatin after rt . seven ( 26% ) of the 27 patients were also treated with whole abdominal ( n= 2 ) or pelvic ( n= 5 ) regional hyperthermia during rt . an 8-mhz radiofrequency ( rf)-capacitive regional hyperthermia system ( thermotron rf-8 ; yamamoto vinita , osaka , japan ) was used . using this system , the patient is placed between two electrodes connected to a high power rf generator [ 1516 ] . the heating duration was adjusted from 4060 min based on the patient 's tolerance ( median 50 min ) . the number of hyperthermia treatments during the rt ranged from 2 to 11 ( median 5 ) . the rf - output power was increased to the maximum level tolerated by the patients , and was maintained with the goal of 42c based on the correlative data between the rf - output power and the deep regional temperature . twenty - five ( 93% ) of the 27 patients were treated with external rt , one patient with external rt plus brachytherapy and the remaining patient with brachytherapy alone ( table 2 ) . the total radiation dose of external rt , using a 4- , 6- or 10-mv linear accelerator , ranged from 50.061.2 gy ( median 60.0 gy ) , and the daily dose was 1.82.0 gy ( median 2.0 gy ) . ct - assisted three - dimensional treatment planning ( xio or focus ; cms japan , tokyo , japan ) was used to determine the radiation fields in 23 ( 85% ) of the 27 patients between october 1995 and january 2010 . prophylactic nodal irradiation for paraaortic lymph node ( ln ) lesions was administered in all 14 patients with paraaortic ln metastases ; the clinical target volume ( ctv ) was defined as the gross tumor volume ( gtv ) and the paraaortic ln area ( the upper margin of the field was at the th11th12 inter - vertebral space , and the lower margin was at the l5s1 inter - vertebral space ) plus a 0.5-cm margin . prophylactic irradiation for the whole pelvic region was also performed in five ( 26% ) of 19 patients with limited recurrence in the pelvic lesion . the planning target volume ( ptv ) included the ctv plus a 1.02.0-cm margin for daily set - up variation . normally , the initial field area covered the ptv with a four - field box technique , and the field was then shrunk to the gtv ( limited recurrent tumor ) with 0.51.5 cm margins at a dose of 4050 gy for the boost doses of 1020 gy using a four - field beam arrangement or conformational therapy . the remaining patients were treated without the prophylactic irradiation and with rt using a four - field box technique or conformational therapy ; the ctv was defined as the gross tumor volume plus 0.5 cm , and the ptv was the ctv plus 0.51.5 cm for the daily setup variation and respiratory movement . two patients with recurrence in the vagina underwent brachytherapy delivered to the limited recurrent tumor of the vagina at a high dose rate ( co ) using a vaginal cylinder , which was prescribed to 1 cm below the mucosa at a dose of 30 gy in six fractions or 9 gy in three fractions immediately after external rt doses of 50.4 gy in 28 fractions . the biologically effective dose ( bed ) can be used to compare the efficacy of various dose - fractionation regimens in providing tumor control . the bed ( total dose ) ( 1 + daily dose/[a / b ] ) using a linear quadratic model with a / b ratios of ranged from 45.0 to 87.2 gy10(median 72.0gy10 ) . the objective tumor response was evaluated by measuring the tumor size by ct before and after rt , and follow - up evaluations were performed by ct every 16 months . the treatment response was evaluated according to the world health organization criteria . a complete response ( cr ) was defined as the complete disappearance of all clinically detectable tumors for at least 4 weeks . a partial response ( pr ) required at least a 50% reduction in the sum of the products of the longest perpendicular diameters of all measurable lesions . progressive disease required either a 25% increase in measurable lesions or the appearance of any new measurable or non - measurable lesions . patients who did not meet the definitions of response or progression were classified as having no change . the overall , progression - free and local control ( defined as failure to have a recurrence within the radiation field ) survival rates were calculated from the start of rt using the kaplan meier method . the statistical significance of the difference between the actuarial curves was assessed using the log - rank test . to identify prognostic factors for overall survival , disease progression - free survival and the local control rates , univariate analyses were performed using the performance status , tumor size , number of recurrent lesions , period between front - line therapy and rt , total radiation dose ( bed ) , objective tumor response , concurrent chemotherapy , hyperthermia and response to chemotherapy immediately before rt . multivariate analyses using the cox proportional - hazards model were performed to determine the overall and progression - free survival rates related to such factors as the tumor size , period between front - line therapy and rt and the objective tumor response . the national cancer institute common toxicity criteria version 3 ( ctcae ) was used to score the patient toxicity . the toxicity was defined as acute ( during therapy and up to 3 months after the combination therapy ) or late ( over 3 months after the completion of the combination therapy ) . from september 1983 to january 2010 , 48 patients with ovarian cancer were treated with rt in the division of therapeutic radiology at our university hospital . there were consecutive 27 patients ( 56% ) with one or two gross recurrent lesions who were treated with definitive rt and were retrospectively analyzed . all 27 patients satisfied the following requirements and were included in this retrospective study : patients had a pathologically confirmed epithelial ovarian carcinoma , had achieved a complete remission after aggressive front - line therapy , and had one or two gross recurrent lesions at the start of the rt . written informed consent for treatment was obtained from all patients . the study was approved by the institutional review board of the university of occupational and environmental health . the characteristics and treatments of the patients are listed in tables 1and 2 . for aggressive front - line therapy , total abdominal hysterectomy ( tah ) , bilateral salpingo - oophorectomy ( bso ) and omentectomy were performed in 10 patients ; tah , bso , omentectomy and appendectomy in seven ; tah , left salpingo - oophorectomy ( lso ) , omentectomy , and appendectomy in four ; supracervical hystectomy , bso and omentectomy in three ; bso in one ; right salpingo - oophorectomy and omentectomy in one ; and lso in one patient . table 1.patient characteristicsvariablen(%)age median ( range)52 ( 3279)performance status 04 ( 15 ) 118 ( 66 ) 25 ( 19)histological type serous adenocarcinoma16 ( 59 ) non - classified adenocarcinoma6 ( 22 ) clear cell carcinoma5 ( 19)postoperative stage i3 ( 11 ) ii4 ( 15 ) iii18 ( 66 ) iv2 ( 7)site(s ) of the limited recurrence one site19 ( 70 ) paraaortic ln 8 ( 36 ) douglas ' pouch 5 ( 19 ) vagina 4 ( 15 ) iliac ln 2 ( 7 ) two sites8 ( 30 ) iliac and paraaortic ln 4 ( 15 ) douglas ' pouch and inguinal ln 1 ( 4 ) douglas ' pouch + paraaortic ln 1 ( 4 ) vagina + liver 1 ( 4 ) douglas ' pouch and spleen 1 ( 4)recurrent tumor size ( cm ) median ( range)3.0 ( 1.06.1)ln = lymph node.at the start of the radiotherapy . table 2.treatment methodsvariablen(%)radiotherapy external irradiation alone25 ( 93 ) median total dose ( gy , range ) 60.0 , 50.061.2 median daily dose ( gy , range ) 2.0 , 1.82.0 external irradiation plus brachytherapy1 ( 4 ) brachytherapy alone1 ( 4)chemotherapy for the recurrent tumor before rt20 ( 74 ) number of the regimens for the recurrent tumor before rt 115 22 33 response to chemotherapy immediately before rt cr0 pr5 nc9 pd6concurrent chemotherapy during rt6 ( 22)adjuvant chemotherapy after rt3 ( 11)hyperthermia during rt7 ( 26)rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . the tumor / node / metastasis ( tnm ) stages ( international union against cancer tnm classification , 6th edition ) were pathologically evaluated at the initial surgery : t1bn0m0 in one patient , t1cn0m0 in two patients , t2cn0m0 in four , t3bn0m0 in three , t3cn0m0 in 13 , t3cn1m0 in two , t3cn0m1 in one patient and t3cn1m1 in one . the time between front - line surgery and rt for the limited recurrence ranged from 658 months ( median 22 months ) . the period between completion of adjuvant chemotherapy and recurrence ranged from 146 months ( median 8 months ) . the median recurrent tumor size was 3.0 cm ( range 1.06.1 cm ) . the eastern cooperative oncology group performance status was evaluated at the start of the rt . all patients were diagnosed as having recurrent disease based on longitudinal computed tomography ( ct ) scans and tumor marker levels , and , in some cases , f - fluorodeoxyglucose positron emission tomography / ct and/or magnetic resonance imaging were also used . after initial surgery , all patients received adjuvant chemotherapy as follows : paclitaxel in combination with carboplatin in 15 patients , and a combination of cyclophosphamide , adriamycin and cisplatin in 12 patients . twenty ( 74% ) of the 27 patients received systemic chemotherapy for the limited recurrent tumor followed by definitive rt as follows : paclitaxel in combination with carboplatinin in 10 patients , docetaxel in combination with carboplatin in six , irinotecan in combination with mitomycin c in three , cyclophophamide and adriamycin in combination with cisplatin in two , cyclophosphamide in combination with adriamycin in one , etoposide in combination with cisplatin in one , etoposide in combination with carboplatin in one , irinotecan in combination with cisplatin in one , irinotecan in combination with carboplatin in one , cisplatin in one and tegafur - uracil in one patient . although no specific chemotherapy protocol existed , six ( 22% ) of the 27 patients were treated with concomitant systemic chemotherapy during the course of rt as follows : paclitaxel in combination with carboplatin in three patients , carboplatin in two and cisplatin in one . three ( 11% ) patients received adjuvant chemotherapy using paclitaxel in combination with carboplatin after rt . seven ( 26% ) of the 27 patients were also treated with whole abdominal ( n= 2 ) or pelvic ( n= 5 ) regional hyperthermia during rt . an 8-mhz radiofrequency ( rf)-capacitive regional hyperthermia system ( thermotron rf-8 ; yamamoto vinita , osaka , japan ) was used . using this system , the patient is placed between two electrodes connected to a high power rf generator [ 1516 ] . the heating duration was adjusted from 4060 min based on the patient 's tolerance ( median 50 min ) . the number of hyperthermia treatments during the rt ranged from 2 to 11 ( median 5 ) . the rf - output power was increased to the maximum level tolerated by the patients , and was maintained with the goal of 42c based on the correlative data between the rf - output power and the deep regional temperature . twenty - five ( 93% ) of the 27 patients were treated with external rt , one patient with external rt plus brachytherapy and the remaining patient with brachytherapy alone ( table 2 ) . the total radiation dose of external rt , using a 4- , 6- or 10-mv linear accelerator , ranged from 50.061.2 gy ( median 60.0 gy ) , and the daily dose was 1.82.0 gy ( median 2.0 gy ) . ct - assisted three - dimensional treatment planning ( xio or focus ; cms japan , tokyo , japan ) was used to determine the radiation fields in 23 ( 85% ) of the 27 patients between october 1995 and january 2010 . prophylactic nodal irradiation for paraaortic lymph node ( ln ) lesions was administered in all 14 patients with paraaortic ln metastases ; the clinical target volume ( ctv ) was defined as the gross tumor volume ( gtv ) and the paraaortic ln area ( the upper margin of the field was at the th11th12 inter - vertebral space , and the lower margin was at the l5s1 inter - vertebral space ) plus a 0.5-cm margin . prophylactic irradiation for the whole pelvic region was also performed in five ( 26% ) of 19 patients with limited recurrence in the pelvic lesion . the planning target volume ( ptv ) included the ctv plus a 1.02.0-cm margin for daily set - up variation . normally , the initial field area covered the ptv with a four - field box technique , and the field was then shrunk to the gtv ( limited recurrent tumor ) with 0.51.5 cm margins at a dose of 4050 gy for the boost doses of 1020 gy using a four - field beam arrangement or conformational therapy . the remaining patients were treated without the prophylactic irradiation and with rt using a four - field box technique or conformational therapy ; the ctv was defined as the gross tumor volume plus 0.5 cm , and the ptv was the ctv plus 0.51.5 cm for the daily setup variation and respiratory movement . two patients with recurrence in the vagina underwent brachytherapy delivered to the limited recurrent tumor of the vagina at a high dose rate ( co ) using a vaginal cylinder , which was prescribed to 1 cm below the mucosa at a dose of 30 gy in six fractions or 9 gy in three fractions immediately after external rt doses of 50.4 gy in 28 fractions . the biologically effective dose ( bed ) can be used to compare the efficacy of various dose - fractionation regimens in providing tumor control . the bed ( total dose ) ( 1 + daily dose/[a / b ] ) using a linear quadratic model with a / b ratios of ranged from 45.0 to 87.2 gy10(median 72.0gy10 ) . the objective tumor response was evaluated by measuring the tumor size by ct before and after rt , and follow - up evaluations were performed by ct every 16 months . the treatment response was evaluated according to the world health organization criteria . a complete response ( cr ) was defined as the complete disappearance of all clinically detectable tumors for at least 4 weeks . a partial response ( pr ) required at least a 50% reduction in the sum of the products of the longest perpendicular diameters of all measurable lesions . progressive disease required either a 25% increase in measurable lesions or the appearance of any new measurable or non - measurable lesions . patients who did not meet the definitions of response or progression were classified as having no change . the overall , progression - free and local control ( defined as failure to have a recurrence within the radiation field ) survival rates were calculated from the start of rt using the kaplan meier method . the statistical significance of the difference between the actuarial curves was assessed using the log - rank test . to identify prognostic factors for overall survival , disease progression - free survival and the local control rates , univariate analyses were performed using the performance status , tumor size , number of recurrent lesions , period between front - line therapy and rt , total radiation dose ( bed ) , objective tumor response , concurrent chemotherapy , hyperthermia and response to chemotherapy immediately before rt . multivariate analyses using the cox proportional - hazards model were performed to determine the overall and progression - free survival rates related to such factors as the tumor size , period between front - line therapy and rt and the objective tumor response . the national cancer institute common toxicity criteria version 3 ( ctcae ) was used to score the patient toxicity . the highest toxicity grade for each patient the toxicity was defined as acute ( during therapy and up to 3 months after the combination therapy ) or late ( over 3 months after the completion of the combination therapy ) . acute toxicities grade 2 occurred in six patients ( 22% ) ; grade 3 leucopenia / neutropenia in one , grade 2 anemia in two , grade 2 thrombocytopenia in one , grade 2 gastritis in one and grade 2 diarrhea in one . the median follow - up for the surviving patients was 25 months ( range 3 to 95 months ) . twenty - two ( 82% ) of the 27 patients experienced an objective response ( cr in 11 patients , pr in 11 , nc in five ) . the first sites of disease progression were local ( in - field ) in two patients ( 7% ) ( paraaortic ln in one patient and peritoneum in one ) , out - field in 13 patients ( peritoneum in six patients , paraaortic ln in four , liver in three , supraclavicular ln in two , lung in two , spleen in one and soft tissue in one ) , and both in - field ( paraaortic ln ) and out - field ( liver and pleura ) in one patient ( 4% ) . the 3-year overall survival , progression - free survival and local ( in - field ) control rates after rt were 46% , 39% and 96% , respectively ( fig . the 2-year overall survival , progression - free survival and local ( in - field ) control rates after rt were 53% , 39% and 96% , respectively . the median survival times ( mst ) with regard to the overall and progression - free survival rates after rt were 25 and 12 months , respectively . univariate analyses showed the tumor size ( < 3 cm ) , period between front - line therapy and rt ( 2 year ) , objective tumor response ( cr ) and response to chemotherapy immediately before rt ( pr ) to be significant prognostic factors of the overall survival rate ( table 3 , fig . 2 ) . the tumor size ( < 3 cm ) and objective tumor response ( cr ) were also significant prognostic factors for the rates of disease - free survival . regarding the overall and progression - free survival rates based on the multivariate analyses , none of these factors ( tumor size < 3 cm , period between front - line therapy and rt 2 years or objective tumor response ( cr ) ) were significant . 1.overall survival ( os ) , progression - free survival ( pfs ) and local ( in - field ) control rates ( lc ) after rt of all patients . 2.(a ) tumor size ( < 3 cm ) was a significant predictor for overall survival rate after rt ( p= 0.0013 ) . ( b ) period between front - line therapy and rt ( 2 years ) was a statistically significant prognostic indicator for overall survival rate after rt ( p= 0.023 ) . ( c ) objective tumor response ( cr ) was a significant predictor for overall survival rate after rt ( p= 0.0044 ) . ( n)overall survival rateprogression - free survival ratelocal ( in - field ) control rate2-year ( % ) p2-year ( % ) p2-year ( % ) pperformance status 0122460.17380.83950.27 258040100tumor size <3 cm11880.0013640.0161000.41 3 cm16292293irradiated lesion one lesion19520.28380.791000.11 two lesions8544386period between start of first - line treatment and rt < 2 years16350.023190.088930.40 2 years118064100total dose of radiation ( bed , gy10 ) < 7213490.72510.11920.68 72145726100concurrent chemotherapy yes600.31nr0.23100nr no21574295hyperthermia yes700.89nr0.73100nr no20564295objective tumor response cr11890.0044670.0131000.079 pr or nc16282193response to chemotherapy immediately before rt pr51000.019800.069100nr nc or pd154734100bed = biologically effective dose ; nr = not reached ; cr = complete response ; pr = partial response ; nc = no change . overall survival ( os ) , progression - free survival ( pfs ) and local ( in - field ) control rates ( lc ) after rt of all patients . ( a ) tumor size ( < 3 cm ) was a significant predictor for overall survival rate after rt ( p= 0.0013 ) . ( b ) period between front - line therapy and rt ( 2 years ) was a statistically significant prognostic indicator for overall survival rate after rt ( p= 0.023 ) . ( c ) objective tumor response ( cr ) was a significant predictor for overall survival rate after rt ( p= 0.0044 ) . the results of the univariate analyses of factors predicting the survival rates bed = biologically effective dose ; nr = not reached ; cr = complete response ; pr = partial response ; nc = no change . the essential treatment for recurrent ovarian cancer is chemotherapy , and in selected patients , secondary cytoreductive surgery can improve survival [ 13 ] . a number of studies have demonstrated that the most important factors for predicting the outcome for recurrent ovarian cancer are the initial response of the tumor recurrence and the length of the disease - free interval prior to tumor recurrence . the role of rt for recurrent ovarian cancer has been considered as a palliative therapy for symptom relief , and whole abdominal radiation therapy has not been recommended due to its severe toxicity . many studies have reported that local external beam irradiation could resolve various symptoms caused by metastatic or recurrent tumors and may be useful for palliative treatment [ 2022 ] . however , in the present study , we delivered rt with curative intent . there have been only a few previous studies of rt with the aim of achieving a cure or prolongation of survival in patients with limited recurrence of epithelial ovarian cancer [ 1214 ] . albuquerque et al.reported treatment results using tumor volume - directed involved field rt ( median dose 50.4 gy ) for localized extraperitoneal recurrence ; of 20 patients , 17 had a cr after rt with acceptable toxicity , and the local recurrence - free and disease - free survival rates at 5 years from the date of rt were 66% and 34% , respectively . firat et al.reported the role of salvage irradiation in 28 patients with vaginal and/or perirectal recurrence or persistence of ovarian carcinoma ; 21 patients were treated with external beam rt alone ( median dose 50.4 gy ) , two patients with brachytherapy alone and five patients with both external beam rt and brachytherapy . they indicated that pelvic local irradiation may be effective for salvage , and that a cure is possible in a subset of patients . the median external rt dose of 60 gy in the current study was relatively higher than that in the previous study . we also found that in the patients with limited recurrence in various sites , definitive rt was feasible , could achieve a better local control rate without severe toxicity , although two patients showed a local recurrence more than 3 years after rt , and many patients died within 2 years . the results justify further evaluation to clarify the optimal radiation dose and treatment fields for the treatment of limited recurrence . recently , a prospective study of curative - intent stereotactic rt in patients with five or fewer oligometastatic lesions of various sites demonstrated that aggressive local therapy for limited metastases can result in prolonged life . the prospective study also implied that aggressive first - line therapy , including systemic chemotherapy , has the potential to downstage some patients to limited recurrence , such as oligometastatic disease , allowing for a prolonged life or cure with aggressive local therapy . kim et al.reported the effects of hyperfractionated rt with concurrent chemotherapy for paraaortic ln recurrence in patients with cervical cancer of the uterus : patients with a latent period > 24 months until paraaortic ln recurrence had a more favorable survival rate than those with a latent period 24 months . traditionally , ovarian cancer is considered to be a disease with a high incidence of widespread metastases . however , we have shown in the current study that patients with limited recurrence , who achieved a complete remission after aggressive front - line therapy , could be successfully salvaged , and further evaluation of the benefits of the definitive rt in the progression - free survivals would therefore be valuable , especially in patients with a smaller recurrent tumor or a longer period between the initial treatment and rt . previous studies have demonstrated the role of secondary cytoreductive surgery for recurrent epithelial ovarian cancer to be limited in selected patients . numerous studies have shown that the median survival time after recurrence ranged from 38 to 61 months in patients who are left with no or minimal residual disease at the time of secondary cytoreductive surgery , and that the survival time is 5 to 27 months in patients who undergo suboptimal cytoreduction . however , secondary cytoreductive surgery can be associated with significant morbidity and occasional mortality ; in a meta - analysis , the rate of major perioperative complications and mortality were 11% and 1.4% , respectively . in the current study for definitive local rt using mainly computed assisted three - dimensional rt in patients with limited recurrence after complete remission , a 3-year local control rate of 96% without severe toxicity is promising . in addition , the period between the front - line therapy and rt , tumor size ( < 3 cm ) and objective tumor response ( cr ) were significant prognostic factors for the overall survival rate , and these factors were comparable with those for secondary cytoreductive surgery . definitive rt may be a promising alternative to surgery in recurrent ovarian cancer patients with the above selection criteria . in addition , our retrospective results justify further evaluations of the role of definitive rt in a larger number of patients with recurrent epithelial ovarian cancer . due to the fact that the current study was a small retrospective case series with heterogeneous treatment , the possibility of some selection bias with regard to the prognostic factors could not be ruled out , although we did perform both univariate and multivariate analyses for the survival rates . a formal prospective trial is consequently needed to determine the efficacy and prognostic factors of this therapy in patients with recurrent epithelial ovarian cancer . in summary , rt for curative intent in ovarian cancer patients with limited recurrence could achieve a better local control rate without severe toxicity , and is a promising treatment that may result in long - term survival in selected patients . at least , these results justify further evaluations with detailed treatment protocols to clarify whether definitive rt could improve survival in selected patients .

the purpose of this study was to assess the efficacy and toxicity of definitive radiotherapy ( rt ) for the recurrence of epithelial ovarian cancer , which is limited to one or two gross regions , after complete remission had been achieved with aggressive front - line therapy . twenty - seven patients were treated with definitive rt and were retrospectively analyzed . their median tumor size was 3.0 cm . twenty - six ( 96% ) patients received external irradiation at a median total dose of 60 gy , and a median daily dose of 2 gy . only two patients received intracavitary brachytherapy . twenty ( 74% ) of the 27 patients received systemic chemotherapy for the treatment of a limited recurrent tumor followed by definitive rt . six ( 22% ) of the patients received concurrent chemotherapy and seven ( 26% ) of the patients also underwent regional hyperthermia during definitive rt . twenty - two ( 82% ) patients had an objective response ( cr : 11 , pr : 11 ) . the 2-year overall survival , progression - free survival and local ( in - field ) control rates after rt were 53% , 39% and 96% , respectively . the toxicities were mild , no grade 3 or higher toxicity was observed in any of the patients . the tumor size ( < 3 cm ) , period between front - line therapy and rt ( 2 year ) and objective tumor response ( cr ) were significant prognostic factors of the overall survival rate . in conclusion , definitive rt for limited recurrence of epithelial ovarian cancer achieves a better local control rate without severe toxicity , and it may therefore be a potentially effective modality for inducing long - term survival in selected patients .

INTRODUCTION MATERIALS AND METHODS Patients Radiotherapy Evaluation and follow-up RESULTS DISCUSSION

in this context , we hypothesized that definitive rt for the recurrence of epithelial ovarian cancer limited to one or two regions , after complete remission had been achieved with aggressive front - line therapy , may improve survival . the purpose of this study was to assess the efficacy and toxicity of definitive rt for the limited recurrence of epithelial ovarian cancer after complete remission had been achieved with aggressive front - line therapy , and to identify the predictors of long - term survival . there were consecutive 27 patients ( 56% ) with one or two gross recurrent lesions who were treated with definitive rt and were retrospectively analyzed . all 27 patients satisfied the following requirements and were included in this retrospective study : patients had a pathologically confirmed epithelial ovarian carcinoma , had achieved a complete remission after aggressive front - line therapy , and had one or two gross recurrent lesions at the start of the rt . for aggressive front - line therapy , total abdominal hysterectomy ( tah ) , bilateral salpingo - oophorectomy ( bso ) and omentectomy were performed in 10 patients ; tah , bso , omentectomy and appendectomy in seven ; tah , left salpingo - oophorectomy ( lso ) , omentectomy , and appendectomy in four ; supracervical hystectomy , bso and omentectomy in three ; bso in one ; right salpingo - oophorectomy and omentectomy in one ; and lso in one patient . table 1.patient characteristicsvariablen(%)age median ( range)52 ( 3279)performance status 04 ( 15 ) 118 ( 66 ) 25 ( 19)histological type serous adenocarcinoma16 ( 59 ) non - classified adenocarcinoma6 ( 22 ) clear cell carcinoma5 ( 19)postoperative stage i3 ( 11 ) ii4 ( 15 ) iii18 ( 66 ) iv2 ( 7)site(s ) of the limited recurrence one site19 ( 70 ) paraaortic ln 8 ( 36 ) douglas ' pouch 5 ( 19 ) vagina 4 ( 15 ) iliac ln 2 ( 7 ) two sites8 ( 30 ) iliac and paraaortic ln 4 ( 15 ) douglas ' pouch and inguinal ln 1 ( 4 ) douglas ' pouch + paraaortic ln 1 ( 4 ) vagina + liver 1 ( 4 ) douglas ' pouch and spleen 1 ( 4)recurrent tumor size ( cm ) median ( range)3.0 ( 1.06.1)ln = lymph node.at the start of the radiotherapy . table 2.treatment methodsvariablen(%)radiotherapy external irradiation alone25 ( 93 ) median total dose ( gy , range ) 60.0 , 50.061.2 median daily dose ( gy , range ) 2.0 , 1.82.0 external irradiation plus brachytherapy1 ( 4 ) brachytherapy alone1 ( 4)chemotherapy for the recurrent tumor before rt20 ( 74 ) number of the regimens for the recurrent tumor before rt 115 22 33 response to chemotherapy immediately before rt cr0 pr5 nc9 pd6concurrent chemotherapy during rt6 ( 22)adjuvant chemotherapy after rt3 ( 11)hyperthermia during rt7 ( 26)rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . the time between front - line surgery and rt for the limited recurrence ranged from 658 months ( median 22 months ) . the median recurrent tumor size was 3.0 cm ( range 1.06.1 cm ) . twenty ( 74% ) of the 27 patients received systemic chemotherapy for the limited recurrent tumor followed by definitive rt as follows : paclitaxel in combination with carboplatinin in 10 patients , docetaxel in combination with carboplatin in six , irinotecan in combination with mitomycin c in three , cyclophophamide and adriamycin in combination with cisplatin in two , cyclophosphamide in combination with adriamycin in one , etoposide in combination with cisplatin in one , etoposide in combination with carboplatin in one , irinotecan in combination with cisplatin in one , irinotecan in combination with carboplatin in one , cisplatin in one and tegafur - uracil in one patient . although no specific chemotherapy protocol existed , six ( 22% ) of the 27 patients were treated with concomitant systemic chemotherapy during the course of rt as follows : paclitaxel in combination with carboplatin in three patients , carboplatin in two and cisplatin in one . seven ( 26% ) of the 27 patients were also treated with whole abdominal ( n= 2 ) or pelvic ( n= 5 ) regional hyperthermia during rt . twenty - five ( 93% ) of the 27 patients were treated with external rt , one patient with external rt plus brachytherapy and the remaining patient with brachytherapy alone ( table 2 ) . normally , the initial field area covered the ptv with a four - field box technique , and the field was then shrunk to the gtv ( limited recurrent tumor ) with 0.51.5 cm margins at a dose of 4050 gy for the boost doses of 1020 gy using a four - field beam arrangement or conformational therapy . two patients with recurrence in the vagina underwent brachytherapy delivered to the limited recurrent tumor of the vagina at a high dose rate ( co ) using a vaginal cylinder , which was prescribed to 1 cm below the mucosa at a dose of 30 gy in six fractions or 9 gy in three fractions immediately after external rt doses of 50.4 gy in 28 fractions . the overall , progression - free and local control ( defined as failure to have a recurrence within the radiation field ) survival rates were calculated from the start of rt using the kaplan meier method . to identify prognostic factors for overall survival , disease progression - free survival and the local control rates , univariate analyses were performed using the performance status , tumor size , number of recurrent lesions , period between front - line therapy and rt , total radiation dose ( bed ) , objective tumor response , concurrent chemotherapy , hyperthermia and response to chemotherapy immediately before rt . multivariate analyses using the cox proportional - hazards model were performed to determine the overall and progression - free survival rates related to such factors as the tumor size , period between front - line therapy and rt and the objective tumor response . there were consecutive 27 patients ( 56% ) with one or two gross recurrent lesions who were treated with definitive rt and were retrospectively analyzed . all 27 patients satisfied the following requirements and were included in this retrospective study : patients had a pathologically confirmed epithelial ovarian carcinoma , had achieved a complete remission after aggressive front - line therapy , and had one or two gross recurrent lesions at the start of the rt . for aggressive front - line therapy , total abdominal hysterectomy ( tah ) , bilateral salpingo - oophorectomy ( bso ) and omentectomy were performed in 10 patients ; tah , bso , omentectomy and appendectomy in seven ; tah , left salpingo - oophorectomy ( lso ) , omentectomy , and appendectomy in four ; supracervical hystectomy , bso and omentectomy in three ; bso in one ; right salpingo - oophorectomy and omentectomy in one ; and lso in one patient . table 1.patient characteristicsvariablen(%)age median ( range)52 ( 3279)performance status 04 ( 15 ) 118 ( 66 ) 25 ( 19)histological type serous adenocarcinoma16 ( 59 ) non - classified adenocarcinoma6 ( 22 ) clear cell carcinoma5 ( 19)postoperative stage i3 ( 11 ) ii4 ( 15 ) iii18 ( 66 ) iv2 ( 7)site(s ) of the limited recurrence one site19 ( 70 ) paraaortic ln 8 ( 36 ) douglas ' pouch 5 ( 19 ) vagina 4 ( 15 ) iliac ln 2 ( 7 ) two sites8 ( 30 ) iliac and paraaortic ln 4 ( 15 ) douglas ' pouch and inguinal ln 1 ( 4 ) douglas ' pouch + paraaortic ln 1 ( 4 ) vagina + liver 1 ( 4 ) douglas ' pouch and spleen 1 ( 4)recurrent tumor size ( cm ) median ( range)3.0 ( 1.06.1)ln = lymph node.at the start of the radiotherapy . table 2.treatment methodsvariablen(%)radiotherapy external irradiation alone25 ( 93 ) median total dose ( gy , range ) 60.0 , 50.061.2 median daily dose ( gy , range ) 2.0 , 1.82.0 external irradiation plus brachytherapy1 ( 4 ) brachytherapy alone1 ( 4)chemotherapy for the recurrent tumor before rt20 ( 74 ) number of the regimens for the recurrent tumor before rt 115 22 33 response to chemotherapy immediately before rt cr0 pr5 nc9 pd6concurrent chemotherapy during rt6 ( 22)adjuvant chemotherapy after rt3 ( 11)hyperthermia during rt7 ( 26)rt = radiotherapy ; cr = complete response ; pr = partial response ; nc = no change . the time between front - line surgery and rt for the limited recurrence ranged from 658 months ( median 22 months ) . the median recurrent tumor size was 3.0 cm ( range 1.06.1 cm ) . twenty ( 74% ) of the 27 patients received systemic chemotherapy for the limited recurrent tumor followed by definitive rt as follows : paclitaxel in combination with carboplatinin in 10 patients , docetaxel in combination with carboplatin in six , irinotecan in combination with mitomycin c in three , cyclophophamide and adriamycin in combination with cisplatin in two , cyclophosphamide in combination with adriamycin in one , etoposide in combination with cisplatin in one , etoposide in combination with carboplatin in one , irinotecan in combination with cisplatin in one , irinotecan in combination with carboplatin in one , cisplatin in one and tegafur - uracil in one patient . although no specific chemotherapy protocol existed , six ( 22% ) of the 27 patients were treated with concomitant systemic chemotherapy during the course of rt as follows : paclitaxel in combination with carboplatin in three patients , carboplatin in two and cisplatin in one . seven ( 26% ) of the 27 patients were also treated with whole abdominal ( n= 2 ) or pelvic ( n= 5 ) regional hyperthermia during rt . twenty - five ( 93% ) of the 27 patients were treated with external rt , one patient with external rt plus brachytherapy and the remaining patient with brachytherapy alone ( table 2 ) . normally , the initial field area covered the ptv with a four - field box technique , and the field was then shrunk to the gtv ( limited recurrent tumor ) with 0.51.5 cm margins at a dose of 4050 gy for the boost doses of 1020 gy using a four - field beam arrangement or conformational therapy . two patients with recurrence in the vagina underwent brachytherapy delivered to the limited recurrent tumor of the vagina at a high dose rate ( co ) using a vaginal cylinder , which was prescribed to 1 cm below the mucosa at a dose of 30 gy in six fractions or 9 gy in three fractions immediately after external rt doses of 50.4 gy in 28 fractions . to identify prognostic factors for overall survival , disease progression - free survival and the local control rates , univariate analyses were performed using the performance status , tumor size , number of recurrent lesions , period between front - line therapy and rt , total radiation dose ( bed ) , objective tumor response , concurrent chemotherapy , hyperthermia and response to chemotherapy immediately before rt . multivariate analyses using the cox proportional - hazards model were performed to determine the overall and progression - free survival rates related to such factors as the tumor size , period between front - line therapy and rt and the objective tumor response . twenty - two ( 82% ) of the 27 patients experienced an objective response ( cr in 11 patients , pr in 11 , nc in five ) . the first sites of disease progression were local ( in - field ) in two patients ( 7% ) ( paraaortic ln in one patient and peritoneum in one ) , out - field in 13 patients ( peritoneum in six patients , paraaortic ln in four , liver in three , supraclavicular ln in two , lung in two , spleen in one and soft tissue in one ) , and both in - field ( paraaortic ln ) and out - field ( liver and pleura ) in one patient ( 4% ) . the 3-year overall survival , progression - free survival and local ( in - field ) control rates after rt were 46% , 39% and 96% , respectively ( fig . the 2-year overall survival , progression - free survival and local ( in - field ) control rates after rt were 53% , 39% and 96% , respectively . the median survival times ( mst ) with regard to the overall and progression - free survival rates after rt were 25 and 12 months , respectively . univariate analyses showed the tumor size ( < 3 cm ) , period between front - line therapy and rt ( 2 year ) , objective tumor response ( cr ) and response to chemotherapy immediately before rt ( pr ) to be significant prognostic factors of the overall survival rate ( table 3 , fig . the tumor size ( < 3 cm ) and objective tumor response ( cr ) were also significant prognostic factors for the rates of disease - free survival . regarding the overall and progression - free survival rates based on the multivariate analyses , none of these factors ( tumor size < 3 cm , period between front - line therapy and rt 2 years or objective tumor response ( cr ) ) were significant . 1.overall survival ( os ) , progression - free survival ( pfs ) and local ( in - field ) control rates ( lc ) after rt of all patients . (a ) tumor size ( < 3 cm ) was a significant predictor for overall survival rate after rt ( p= 0.0013 ) . ( b ) period between front - line therapy and rt ( 2 years ) was a statistically significant prognostic indicator for overall survival rate after rt ( p= 0.023 ) . ( c ) objective tumor response ( cr ) was a significant predictor for overall survival rate after rt ( p= 0.0044 ) . ( n)overall survival rateprogression - free survival ratelocal ( in - field ) control rate2-year ( % ) p2-year ( % ) p2-year ( % ) pperformance status 0122460.17380.83950.27 258040100tumor size <3 cm11880.0013640.0161000.41 3 cm16292293irradiated lesion one lesion19520.28380.791000.11 two lesions8544386period between start of first - line treatment and rt < 2 years16350.023190.088930.40 2 years118064100total dose of radiation ( bed , gy10 ) < 7213490.72510.11920.68 72145726100concurrent chemotherapy yes600.31nr0.23100nr no21574295hyperthermia yes700.89nr0.73100nr no20564295objective tumor response cr11890.0044670.0131000.079 pr or nc16282193response to chemotherapy immediately before rt pr51000.019800.069100nr nc or pd154734100bed = biologically effective dose ; nr = not reached ; cr = complete response ; pr = partial response ; nc = no change . overall survival ( os ) , progression - free survival ( pfs ) and local ( in - field ) control rates ( lc ) after rt of all patients . ( a ) tumor size ( < 3 cm ) was a significant predictor for overall survival rate after rt ( p= 0.0013 ) . ( b ) period between front - line therapy and rt ( 2 years ) was a statistically significant prognostic indicator for overall survival rate after rt ( p= 0.023 ) . ( c ) objective tumor response ( cr ) was a significant predictor for overall survival rate after rt ( p= 0.0044 ) . albuquerque et al.reported treatment results using tumor volume - directed involved field rt ( median dose 50.4 gy ) for localized extraperitoneal recurrence ; of 20 patients , 17 had a cr after rt with acceptable toxicity , and the local recurrence - free and disease - free survival rates at 5 years from the date of rt were 66% and 34% , respectively . we also found that in the patients with limited recurrence in various sites , definitive rt was feasible , could achieve a better local control rate without severe toxicity , although two patients showed a local recurrence more than 3 years after rt , and many patients died within 2 years . however , we have shown in the current study that patients with limited recurrence , who achieved a complete remission after aggressive front - line therapy , could be successfully salvaged , and further evaluation of the benefits of the definitive rt in the progression - free survivals would therefore be valuable , especially in patients with a smaller recurrent tumor or a longer period between the initial treatment and rt . in the current study for definitive local rt using mainly computed assisted three - dimensional rt in patients with limited recurrence after complete remission , a 3-year local control rate of 96% without severe toxicity is promising . in addition , the period between the front - line therapy and rt , tumor size ( < 3 cm ) and objective tumor response ( cr ) were significant prognostic factors for the overall survival rate , and these factors were comparable with those for secondary cytoreductive surgery . a formal prospective trial is consequently needed to determine the efficacy and prognostic factors of this therapy in patients with recurrent epithelial ovarian cancer . in summary , rt for curative intent in ovarian cancer patients with limited recurrence could achieve a better local control rate without severe toxicity , and is a promising treatment that may result in long - term survival in selected patients .

bacterial strains and growth of bacteria strains used in this work are presented in table 1 . e. coli strains were grown in lysogeny broth or on lysogeny broth plates , and b. subtilis strains were grown in nutrient sporulation medium with phosphate ( 15 ) with appropriate antibiotics added as follows : ampicillin , 100 g / ml ; kanamycin , 10 g / ml ( for b. subtilis ) or 50 g / ml ( for e. coli ) ; and chloramphenicol , 3 or 4 g / ml ( for b. subtilis ) and 15 g / ml ( for e. coli ) . liquid cultures were grown in baffled e - flasks on a rotary shaker ( 200 rpm ) at 37 c . table 1strains and plasmids used in this workstrain or plasmidgenotype and/or relevant propertiesaorigin or ref.e . coli bl21 fompt hsdsb(rb mb ) gal dcm novagen b834(de3 ) fompt hsdsb(rb mb ) gal dcm met ( de3 ) novagen mm294 thi , pro , hsdr supe4 45 top10 fmcra [mrr - hsdrms - mcrbc ] f80laczm15 lacx74 reca1 arad139 [ara - leu]7697 galu galk rpsl enda1 nupg invitrogen b. subtilis 1a1 trpc2bacillus genetic stock center , columbus , oh lul20 trpc2 stoaplle39 ; cm 2 lul30 trpc2 (ykvu - stoa)::tet ; tc 2 plasmids pbluescript sk(+ ) cloning vector ; ap stratagene pcr - blunt - ii - topo cloning vector ; km invitrogen pdg148 expression vector ; em km 46 pgex4t1 gst fusion expression vector ; ap ge healthcare plle83 pdg148 derivative containing the stoa gene ; km 2 plmc19 pbadmyc - hisc derivative encoding sstoa ; ap this work plym001 pbluescript sk(+ ) derivative containing stoa on a 2-kb fragment ; ap this work plym004 plym001 derivative encoding c68a stoa ; ap this work plym006 plym001 derivative encoding e71q stoa ; ap this work plym009 plym001 derivative encoding c65a stoa ; ap this work plym012 plle83 variant encoding e71q stoa ; km this work plym013 plle83 variant encoding c68a stoa ; km this work plym015 plle83 variant encoding c65a stoa ; km this work plym025 pcr - blunt - ii - topo containing stoa ; km this work plym028 pgex4t1 derivative encoding gst - sstoa fusion protein ; ap this work plym031 plym028 variant encoding gst - sstoa with c65a mutation ; ap this work plym032 plym028 variant encoding gst - sstoa with c68a mutation ; ap this work plym033 plym028 variant encoding gst - sstoa with e71q mutation ; ap this work aap , cm , em , km , and tc indicate resistance to ampicillin , chloramphenicol , erythromycin , kanamycin , and tetracycline , respectively . strains and plasmids used in this work ap , cm , em , km , and tc indicate resistance to ampicillin , chloramphenicol , erythromycin , kanamycin , and tetracycline , respectively . construction of plasmids encoding sstoa for production of sstoa plasmid plmc19 was constructed by amplifying part of the stoa gene using oligonucleotides le051 and le052 ( supplemental table s1 ) , phusion polymerase ( finnzymes ) , and b. subtilis 1a1 chromosomal dna as template . the pcr product was cloned into pcr-blunt - ii - topo ( invitrogen ) . the insert was cut out from the plasmid using psti and hindiii and ligated into pbadmyc - hisc cut with the same enzymes resulting in plasmid plmc19 . the cloned stoa fragment was verified by dna sequence analysis . for production of a thrombin - cleavable gst - sstoa fusion protein , a fragment of the stoa gene encoding residues 21 - 165 of stoa was first amplified by pcr as above using oligonucleotides ly001 and ly002 as primers and subsequently cloned into pcr - blunt - ii - topo generating plym025 , which was verified by sequencing . plym025 , propagated in e. coli strain mm294 , was digested by bamhi and sali , and the stoa fragment was cloned into pgex4t1 , resulting in plym028 . for the construction of plasmids encoding mutant stoa variants , plle83 was digested by hindiii and bamhi , and the 2-kb fragment containing stoa was cloned in pbluescript sk(+ ) , resulting in plym001 . site - directed mutagenesis was carried out with the quikchange ii kit and protocol ( stratagene ) using plym001 and primers ly003-ly008 to generate plasmids plym009 ( c65a ) , plym004 ( c68a ) , and plym006 ( e71q ) , respectively , which were verified by sequencing . the hindiii / bamhi fragment of each of these three plasmids was subsequently cloned into pdg148 , generating , respectively , plym015 , plym013 , and plym012 , which were used for expression of full - length mutant stoa genes in b. subtilis . plasmids encoding gst - sstoa fusion protein with c65a or c68a in sstoa were obtained by first amplifying stoa as above using primers ly001 and ly002 and plym009 or plym004 plasmid dna , respectively , as template . pcr products were then cloned into pgex4t1 as described above for the wild - type variant , generating plasmids plym031 and plym032 , which were verified by sequencing . purification of sstoa non - tagged sstoa , which was utilized in initial crystallization trials and to generate a stoa antiserum , was purified from e. coli top10/plmc19 as described in the supplemental data . for the production of gst - sstoa fusion protein , e. coli bl21/plym028 was grown in 1-liter portions in 5-liter e - flasks . at a600 = 0.6 - 0.8 expression was induced by addition of 1 mm isopropyl -d - thiogalactoside ( final concentration ) . after incubation for 5 h , cells were collected by centrifugation , washed in pbs ( 140 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 , 1.8 mm kh2po4 , ph 7.3 ) , and stored as pellets at -20 c until required . the cell pellet from 1 liter of culture was suspended in 20 ml of ice - cold pbs containing 1 mm dtt and lysed by passage ( three times ) through a french pressure cell at 18,000 p.s.i . the lysate was centrifuged at 48,000 g for 40 min at 4 c , and the supernatant was centrifuged at 100,000 g for 1 h at 4 c . the final supernatant was mixed with 2 ml of 50% slurry of glutathione - sepharose 4b ( ge healthcare ) , and the gst - sstoa fusion protein was purified according to the resin manufacturer 's instructions . affinity - purified gst - sstoa fusion protein was cleaved by 50 units of thrombin ( ge healthcare ) at room temperature for 5 h and then loaded onto a sephacryl s-100 hr gel filtration column . protein was eluted using 20 mm tris - hcl , ph 8.0 , containing 100 mm nacl and 1 mm dtt . fractions containing sstoa were identified using sds - page and western blot with stoa antiserum , pooled , and concentrated . the n - terminal amino acid residue sequence of the purified protein was verified by edman degradation ( see fig . cysteine variants of sstoa were produced in e. coli bl21 containing plym031 or plym032 and purified as described for wild - type sstoa . for the production of selenomethionine - labeled sstoa , e. coli b834(de3)/plym028 was grown overnight in 10 ml of selenomet medium ( athenaes ) supplemented with 50 g / ml methionine . the overnight culture was used to inoculate 1 liter of selenomet medium containing 50 g/l methionine to an a600 of 0.1 , and the culture was grown until a600 was 0.8 . cells were harvested by centrifugation for 10 min at 10,000 g at 4 c , and the pellet was resuspended in 1 liter of non - supplemented selenomet medium and incubated for 2 h. selenomethionine was then added to a final concentration of 50 g / ml , and the culture was incubated for a further 30 min when production of gst - sstoa was induced by the addition of 1 mm isopropyl -d - thiogalactoside ( final concentration ) . four hours after induction the culture was harvested by centrifugation for 10 min at 10,000 g at 4 c . the cells were washed in cold pbs and stored as a pellet at -20 c . a 2-l sitting drop was formed by mixing equal volumes of protein solution ( 12 mg / ml sstoa in 25 mm mops , ph 7.0 ) and crystallization reagent ( 27% ( w / v ) peg 2000 , 0.2 m ammonium acetate , 100 mm sodium acetate , ph 4.8 ) over an 800-l reservoir of the reagent alone . crystals grew over a period of 1 - 2 days and were cryoprotected in a solution of 30% ( w / v ) peg 2000 , 100 mm sodium acetate , ph 4.8 , 20% ( v / v ) ethylene glycol before flash freezing in liquid nitrogen . x - ray data sets for native and selenomethionine - labeled sstoa were collected on beam line id23 - 1 of the european synchrotron radiation facility ( grenoble , france ) . structure determination utilized programs of the ccp4 ( 16 ) and phenix ( 17 ) software suites . diffraction patterns were indexed and integrated with mosflm ( 18 ) and scaled with scala ( 19 ) . selenium sites were identified using a combination of automated methods implemented in phenix.hyss and manual inspection of anomalous difference maps produced with fft . an initial electron density map was obtained using sad phases calculated with mlphare and subsequent density modification with dm . a key factor in producing an interpretable electron density map was the identification of the correct non - crystallographic symmetry relating each sstoa molecule in the asymmetric unit . non - crystallographic symmetry averaging in the phase improvement procedure benefited from the use of a predefined protein mask derived from a monomer of resa ( 12 ) . further phase improvement was obtained by cross - crystal averaging with a second sad - phased selenomethionine data set composed of merged data from two individual sstoa crystals . manual model building was conducted in coot , and initial phased refinement of the model was conducted with refmac ( 20 ) . further refinement of the model ( against a single selenomethionine data set with a low twin fraction ( = 0.36 ) ) utilized phenix.refine , which was essential for proper refinement of the twinned data . the final model of sstoa is composed of seven ordered protein chains and 99 water molecules . a small amount of residual density located at a coordinate of ( 42.34 , 64.52 , 19.53 ) may indicate the presence of an additional stoa monomer of low occupancy and high mobility that is insufficiently well resolved to enable further model building . the coordinates and structure factors reduction potential determination sstoa ( 0.2 m ) in 50 mm potassium phosphate , ph 7 , was added to 5 mm oxidized dtt in the same buffer to obtain the fully oxidized protein . the protein was subsequently titrated with reduced dtt in the same buffer , allowing 10 min for the protein to equilibrate to each new potential . the transition from oxidized to reduced protein was monitored by the increase in tryptophan fluorescence emission at 344 nm ( excitation at 280 nm ) measured at 25 c using a perkinelmer life sciences ls-55 fluorescence spectrometer with 10-nm excitation and emission slits . midpoint reduction potentials were determined as described previously ( 11 , 21 ) ; further details are given in the supplemental data . ph stability and cysteine pka measurements reduced wild - type and variant sstoa protein stocks were prepared in 10 mm mops , ph 7 , 2 mm tcep ( pierce ) and subsequently diluted ( 30-fold to a final protein concentration of 0.15 m ) with pctc ( potassium phosphate , sodium citrate , tris , and ches , all at 50 mm ) buffer ( pre - prepared at the appropriate ph ) and equilibrated in a sealed cuvette for 1 h before measurement of tryptophan fluorescence spectra as above . for pka measurements , reduced protein solutions ( wild - type sstoa and single cysteine variants ) were prepared in 10 mm mops , ph 7 , with 2 mm tcep as reductant . reaction with 6-bromoacetyl-2-dimethylaminonaphthalene was carried out under pseudo - first order conditions , and pka values were determined as described previously ( 13 ) . modification of sstoa with mal - peg wild - type sstoa and c65a and c68a variant proteins in 20 mm tris - hcl , 100 mm nacl , ph 8.0 were treated with 1 mm tcep at room temperature for 30 min . excess tcep was removed using a ym10 column ( millipore ) , and each reduced sstoa sample ( 10 g ) was incubated with 0.1 mm or 1 mm monomethyl polyethylene glycol 5000 2-maleimidoethyl ether ( mal - peg ) ( 90% ; fluka ) at room temperature for 30 min . non - tagged sstoa was used to immunize rabbits ( custom polyclonal antibody production service ; medprobe , oslo , norway ) . for western blot analysis , proteins were separated by sds - page using the schgger and von jagow ( 22 ) system and subsequently electroblotted to a polyvinylidene difluoride membrane ( millipore ) by wet blot using 20 mm tris , 150 mm glycine , 20% ( v / v ) methanol . the membrane was blocked using 5% ( w / v ) nonfat dry milk in 0.1% ( v / v ) tween 20 in pbs . bound primary antibodies were detected using horseradish peroxidase - linked anti - rabbit antiserum from donkey ( ge healthcare ) diluted 3000-fold . immunodetection was carried out by chemiluminescence using supersignal west pico substrate ( pierce ) and an eastman kodak co. image station . preparation of cell - free extracts from b. subtilis strains samples of 200 ml were taken from a 1.5-liter culture at time points spanning from 1 h before entry into postexponential ( t = -1 ) to 5 h into stationary growth phase ( t = 5 ) . cells were harvested by centrifugation ; immediately washed in 50 mm potassium phosphate , ph 8.0 ; and frozen as a pellet at -20 c . when required , cell pellets were thawed and suspended in 0.8 ml of phosphate buffer containing 0.7 mg / ml lysozyme , 25 g / ml dnase , 25 g / ml rnase , 4 mm mgso4 , and complete protease inhibitor ( roche applied science ; one tablet/50 ml of buffer ) ) . after incubation at 37 c for 45 min , an aliquot was frozen for subsequent analysis of total cell lysate . the remaining main part of the lysate was centrifuged at 48,000 g for 60 min at 4 c : the supernatant was used for the analysis of the soluble cell fraction , whereas the pellet , after washing in phosphate buffer , was suspended in 0.3 ml of the buffer and used for the analysis of the membrane fraction . other methods chromosomal dna from b. subtilis was isolated according to marmur ( 23 ) . plasmid dna was isolated using quantum miniprep ( bio - rad ) or by cscl density gradient centrifugation . sds - page was carried out using the nupage system ( invitrogen ) or schgger and von jagow ( 22 ) system . protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 15,460 100 m cm determined as described previously ( 25 ) or using the bca reagent ( bio - rad ) with bovine albumin as reference . n - terminal sequencing was carried out by edman degradation ( protein analysis center , karolinska institutet , sweden ) on proteins separated by sds - page and electroblotted onto a polyvinylidene difluoride membrane as described for western blotting . the membrane was stained with 0.1% ( w / v ) coomassie brilliant blue r-250 in 2% ( v / v ) acetic acid , 45% ( v / v ) methanol . mass spectrometry analysis was performed using an ultraflex - maldi - tof / tof mass spectrometer ( bruker daltonics , coventry , uk ) on samples prepared by mixing sstoa in a 1:1 ratio with a saturated solution of sinapinic acid matrix in 30% acetonitrile , 0.05% trifluoroacetic acid . 0.5 l of this combined mixture was spotted onto a polished stainless steel target and allowed to crystallize prior to analysis . the spectrometer was externally calibrated using a two - point linear calibration through the singly and doubly charged ions of trypsinogen . the molecular mass of sstoa was determined using high performance liquid chromatography with an ultraspherogel sec 3000 column ( beckman ) . 1.2 nmol of sstoa in 10 l was applied to the column equilibrated with 20 mm tris - hcl , ph 8.0 , 0.15 m nacl , 1 mm dtt and eluted in the same buffer at a flow rate of 1 ml min . mass was calculated based on a calibration curve obtained using catalase ( 230 kda ) , bovine serum albumin ( 67 kda ) , ovalbumin ( 46 kda ) , carboanhydrase ( 26 kda ) , myoglobin ( 17.2 kda ) , and horse heart cytochrome c ( 12.3 kda ) . stoa is a membrane - bound protein from its amino acid sequence ( fig . 1 ) , stoa was predicted to be a ditopic membrane - bound protein with an n - terminal 30-residue segment that anchors the 135-residue tdor domain to the membrane . as shown previously by the use of a stoa - phoa ( alkaline phosphatase ) fusion protein in e. coli cells , the transmembrane segment of stoa functions as a signal sequence to direct translocation and to membrane anchor the c - terminal tdor domain ( 2 ) . experiments in b. subtilis indicated that the function of stoa is not dependent on the membrane anchor ( 3 ) , and prediction programs suggested that the n - terminal segment might be cleaved off after the tdor domain has been translocated across the membrane . to establish whether stoa is a membrane - bound protein , b. subtilis strain 1a1 was grown in nutrient sporulation medium with phosphate for sporulation , and samples taken at different time points during growth were analyzed for stoa by using western blot with polyclonal antiserum directed against the tdor domain of stoa . no stoa antigen could be detected in cell - free extracts , although bdbd , a protein very similar to stoa , was readily detected in all samples ( not shown ) . to facilitate detection of stoa by increasing the level of the protein in cells , plasmid plle83 containing stoa under control of the pspac promoter was used . stoa was found in the membrane fraction of 1a1/plle83 cells from early stationary phase cultures but not in late stationary phase cultures ( fig . the results indicate that stoa is membrane - associated but is present in very low amounts and is degraded or trapped in maturing endospores so that it is not detectable by the western blot procedure used . figure 1.amino acid residue sequence alignment of b. subtilis sstoa used in structural and biochemical analyses , full - length stoa , and full - length resa proteins . stars indicate the active site residues cys-65 and cys-68 and conserved residue glu-71 of stoa . amino acid residue sequence alignment of b. subtilis sstoa used in structural and biochemical analyses , full - length stoa , and full - length resa proteins . stars indicate the active site residues cys-65 and cys-68 and conserved residue glu-71 of stoa . western blot analysis of total cell - free lysates , membrane fractions , and soluble fractions of b. subtilis 1a1/plle83 for stoa antigen is shown . cells were harvested at different time points during growth in nutrient sporulation medium with phosphate . time point 0 is at the entry of stationary growth phase , and subsequent numbers indicate hours into stationary phase . sstoa indicates a sample of purified sstoa loaded on the gel as a reference . approximately 20 g of cell protein was loaded in each lane except for purified sstoa where 20 ng was loaded . western blot analysis of total cell - free lysates , membrane fractions , and soluble fractions of b. subtilis 1a1/plle83 for stoa antigen is shown . cells were harvested at different time points during growth in nutrient sporulation medium with phosphate . time point 0 is at the entry of stationary growth phase , and subsequent numbers indicate hours into stationary phase . sstoa indicates a sample of purified sstoa loaded on the gel as a reference . approximately 20 g of cell protein was loaded in each lane except for purified sstoa where 20 ng was loaded . in vivo functional analysis of active site variants of stoa to establish that the function of stoa in endospore biogenesis is dependent on the cysteine residues of the protein , b. subtilis strain lul20 in which the stoa gene is inactivated and strain lul30 in which stoa is deleted from the chromosome were used ( 2 ) . these two strains containing a plasmid encoding wild - type stoa ( plle83 ) , c65a stoa ( plym015 ) , c68a stoa ( plym013 ) , or empty vector ( pdg148 ) were grown for sporulation and tested for production of heat - resistant cells . western blot analysis showed that stoa proteins were present in membranes of strains containing plym015 and plym013 ( see supplemental fig . , the presence of either stoa variant resulted in a 100-fold reduction in sporulation efficiency ( table 2 ) . cells completely lacking stoa , however , showed more than a 1000-fold reduction in sporulation efficiency indicating some residual activity of stoa even when one of the two cysteine residues is missing . table 2efficiency of b. subtilis strains in producing heat - resistant cells after growth for 2 days at 37 c in nutrient sporulation medium with phosphate supplemented with 1 mm isopropyl -d - thiogalactosidepresented are typical results obtained from at least two independent experiments with each strain , including analysis of two sister clones . b. subtilis lul20 and lul30 are stoa - deficient , and 1a1 is the parental strain ( table 1).strainstoa variant encoded by plasmidviable count before heatingviable count after 15 min at 80 csporulation efficiencya % 1a1 4.0 10 3.4 10 85 lul20 5.5 10 2.0 10 < 0.05 lul20/pdg148 4.7 10 1.2 10 < 0.05 lul20/plle83 wild type 2.6 10 1.3 10 50 lul20/plym015 c65a 4.9 10 3.7 10 0.7 lul20/plym013 c68a 7.4 10 5.0 10 0.7 lul20/plym012 e71q 4.1 10 2.1 10 5.1 lul30 1.9 10 1.4 10 < 0.05 lul30/pdg148 2.1 10 3.7 10 < 0.05 lul30/plle83 wild type 1.4 10 8.6 10 61 lul30/plym015 c65a 4.9 10 9.2 10 0.2 lul30/plym013 c68a 4.6 10 6.2 10 0.1 lul30/plym012 e71q 3.9 10 1.6 10 0.4 aviable count after heat treatment divided by that before heating . efficiency of b. subtilis strains in producing heat - resistant cells after growth for 2 days at 37 c in nutrient sporulation medium with phosphate supplemented with 1 mm isopropyl -d - thiogalactoside presented are typical results obtained from at least two independent experiments with each strain , including analysis of two sister clones . b. subtilis lul20 and lul30 are stoa - deficient , and 1a1 is the parental strain ( table 1 ) . the high resolution structure of the soluble domain of stoa in a mixture of oxidized and reduced states sstoa ( residues 22 - 165 ) was produced in e. coli and purified . gel filtration analysis indicated a molecular mass of 17 3 kda ( data not shown ) consistent with the protein being monomeric in solution . the crystal structure of sstoa was solved using the selenomethionine sad method of phase determination . the crystals used in structure determination belong to space group p31 and contained seven molecules of sstoa per asymmetric unit ( see supplemental fig . although structure determination was hampered by the fact that sstoa crystals were merohedrally twinned , it was nonetheless possible to refine the structure to acceptable rwork and rfree values using twin refinement against x - ray data obtained from a single sstoa crystal with a twin fraction ( ) of 0.36 . data collection and refinement statistics for the sstoa structure are given in table 3 . table 3x - ray data collection and refinement statistics for sstoase - met , selenomethionine ; r.m.s . , root mean square . values in parentheses represent the highest resolution shell.two-crystal merged se - met data sethires se - met data set space group p31 p31 cell parameters ( ) a = b = 133.74 , c = 64.82 a = b = 133.72 , c = 64.82 energy ( ev ) 12,656.6 12,656.6 f 7.79 7.79 f 6.38 6.38 twinning operator -k , -h , -l -k , -h , -l twinning fraction 0.38 0.36 resolution ( ) 43.19 - 2.60 ( 2.74 - 2.60 ) 36.27 - 2.50 ( 2.64 - 2.50 ) rsym 0.154 ( 0.565 ) 0.083 ( 0.293 ) i/ 18.9 ( 4.0 ) 16.1 ( 3.3 ) anomalous completeness ( % ) 99.4 ( 96.0 ) 97.2 ( 83.8 ) anomalous multiplicity 5.0 ( 3.6 ) 2.5 ( 1.7 ) unique observations 44,319 ( 6,023 ) r 0.1787 rfree 0.2011 r.m.s . angle ( ) 2.191 x - ray data collection and refinement statistics for sstoa se - met , selenomethionine ; r.m.s . , root mean square . values in parentheses represent the highest resolution shell . overall the structure of each sstoa monomer ( fig . 3a ) can be described as a modified thioredoxin - like fold that is highly reminiscent of b. subtilis resa ( 12 ) , ccmg ( from e. coli and bradyrhizobium japonicum ) ( 26 , 27 ) , and mycobacterium tuberculosis dsbe ( 28 ) , which are all extracytoplasmic tdors ( see fig . 3b ) . like these proteins , the classical thioredoxin - like motif of stoa is embellished by a central insertion and an n - terminal -hairpin ( in addition to the transmembrane helix predicted from primary sequence analysis ) . unlike other extracytoplasmic tdors , stoa also possesses a short insertion of residues between strand 4 and helix 2 that forms an ordered loop at the surface of the protein . figure 3.the three - dimensional structure of the soluble domain of stoa . a , three - dimensional structure of sstoa showing that the protein exhibits a classical thioredoxin - like fold with two significant insertions : the n - terminal region contains a two - stranded , antiparallel hairpin , whereas the central insert , located after the 3-1-4 motif of the thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . secondary structure elements are labeled from the n terminus ( with the n - terminal transmembrane helix being 0 ) , and the n and c termini of sstoa are indicated . b , overlay of the stoa ( gray ) and reduced resa ( yellow ) peptide backbones ( in ribbon representation ) . c , the active site region of stoa showing the cppc motif , surrounding residues , and a buried water molecule ( red sphere ) . all structural figures were prepared with pymol ( 44 ) and annotated with gimp . the three - dimensional structure of the soluble domain of stoa . a , three - dimensional structure of sstoa showing that the protein exhibits a classical thioredoxin - like fold with two significant insertions : the n - terminal region contains a two - stranded , antiparallel hairpin , whereas the central insert , located after the 3-1-4 motif of the thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . secondary structure elements are labeled from the n terminus ( with the n - terminal transmembrane helix being 0 ) , and the n and c termini of sstoa are indicated . b , overlay of the stoa ( gray ) and reduced resa ( yellow ) peptide backbones ( in ribbon representation ) . c , the active site region of stoa showing the cppc motif , surrounding residues , and a buried water molecule ( red sphere ) . all structural figures were prepared with pymol ( 44 ) and annotated with gimp . in the structure determined here , the active site cysteines of sstoa appear as a mixture of oxidized and reduced redox states ( in each monomer ) . crystallization of sstoa utilized solely the oxidized form of the protein , and thus it is likely that partial reduction of the disulfide bond was induced by photoreduction in the x - ray beam . the electron density associated with the partially broken disulfide 4a along with separated models of the oxidized and reduced conformations shown in fig . 4 , b and c , respectively . figure 4.the active site of stoa in oxidized and reduced states . a , electron density ( contoured at 1.2 ) of the active site cppc motif of stoa reveals a mixture of oxidized and reduced states . b and c , separated representations of the active site region in oxidized and reduced states , respectively . the active site of stoa in oxidized and reduced states . a , electron density ( contoured at 1.2 ) of the active site cppc motif of stoa b and c , separated representations of the active site region in oxidized and reduced states , respectively . a , fluorescence spectra of sstoa in 50 mm potassium phosphate , 5 mm oxidized dtt , ph 7.0 following incubation with increasing concentrations of reduced dtt at 25 c . b , plot of fraction of reduced sstoa ( calculated from the fluorescence ( fluor . ) intensity at 344 nm as described in the supplemental data ) as a function of the cell potential . a , fluorescence spectra of sstoa in 50 mm potassium phosphate , 5 mm oxidized dtt , ph 7.0 following incubation with increasing concentrations of reduced dtt at 25 c . b , plot of fraction of reduced sstoa ( calculated from the fluorescence ( fluor . ) intensity at 344 nm as described in the supplemental data ) as a function of the cell potential . the oxidized ( disulfide - bonded ) conformation of the active site dominates the electron density and is best described as adopting a classical right - handed hook conformation with a 3 angle of 73.5 1.8 formed between the two cysteine residues . the sulfur - to - sulfur bond distance for the oxidized conformation is 2.06 , whereas in the reduced conformation an average sulfur - to - sulfur distance of 3.4 separates the cysteine residues . this distance is considerably shorter than that observed for the structure of resa that has an exceptionally long sulfur - to - sulfur distance of 4.5 in the reduced state ( 12 ) . partial reduction of the disulfide does not seem to cause significant rearrangement of the local protein structure , and thus , unlike resa , there is no evidence for any redox - linked conformational changes due to reduction of the cysteines in stoa . in almost all known natural thioredoxin - like proteins , at least one of the two residues that intervene between the active site cysteines residues ( i.e. within the cxxc motif ) is a proline . in stoa , both of these intervening residues are proline . pro-66 and pro-67 both adopt the trans conformations and have backbone - angles that are consistent with an -helical conformation . like all other thioredoxin - like tdors , the cxxc motif of stoa is found at the n terminus of a reasonably long -helix ( 1 in stoa ) , and the macrodipole arising from this helix is often invoked as a primary cause of the lowered pka values associated with the more n - terminal cysteine residue of the cxxc motif in most tdors ( 29 ) . the presence of proline residues at the cap of this active site helix in stoa is therefore likely to have important consequences for the distribution of the electrostatic field near the cysteines as proline does not possess a standard peptide group . furthermore the limited conformational freedom of proline ( in comparison with other residues ) may be an important factor in maintaining rigidity of the cppc motif and may be one of the reasons that the reduced form of sstoa is so similar to its oxidized form . a further proline residue ( pro-135 ) , which is in the cis conformation and is conserved in all thioredoxin - like proteins , is found in van der waals contact with two buried polar residues , a histidine and a glutamate , which are located immediately behind the second cysteine of the active site motif . his-59 is located on strand 3 , whereas glu-71 arises from helix 1 directly opposite . a buried water molecule is observed in the space between these two residues within hydrogen - bonding distance of the cys-68 sulfur ( see fig . the arrangement of these two buried polar residues and the intervening water molecule ( in stoa ) is very similar to that observed in resa where the glutamate is conserved ( glu-80 ) and an asparagine ( asn-68 ) residue takes an equivalent position to that of his-59 . substitution of glu-80 in resa has been shown previously to have a significant effect on the active site properties of the enzyme ; for example , the pka values of both active site cysteines were significantly lowered in an e80q variant ( 13 ) , and a b. subtilis strain containing e80q resa was also severely impaired in its ability to mature c - type cytochromes ( 14 ) . thus , it may be that these buried polar residues are also important in stoa function . to analyze the functional role of residue glu-71 in stoa , the stoa - deficient b. subtilis strains lul20 and lul30 containing plasmid plym012 encoding e71q stoa were studied . the presence of the variant stoa protein in membranes was confirmed by western blot ( supplemental fig . s1 ) , and the efficiency in production of heat - resistant endospores was found to be 10 - 150-fold ( depending on strain ) lower in these strains compared with wild - type controls ( table 2 ) . stoa is a low potential tdor the reduction potential of the active site cysteines of sstoa was measured using the difference in tryptophan fluorescence intensity of oxidized and reduced sstoa to follow oxidation state as a function of reduction potential ( see fig . the data fitted well to the nernst equation , giving a midpoint reduction potential of -248 2 mv versus normal hydrogen electrode at ph 7 with n = 2.18 0.16 , as expected for a two - electron reduction process . this value is similar to that measured for b. subtilis resa ( -256 mv at ph 7 ) ( 21 ) and e. coli thioredoxin ( -270 mv at ph 7 ) ( 30 , 31 ) and is entirely consistent with the structural similarity between these proteins and a role for stoa in the reduction of ( as yet unidentified ) proteins involved in endospore cortex synthesis ( 2 , 9 ) . sds - page of purified reduced wild - type ( wt ) sstoa and c65a and c68a sstoa following reaction with mal - peg is shown . the protein variants were incubated with 0 , 0.1 , and 1 mm mal - peg before electrophoresis as indicated , and the gel was stained for protein . sds - page of purified reduced wild - type ( wt ) sstoa and c65a and c68a sstoa following reaction with mal - peg is shown . the protein variants were incubated with 0 , 0.1 , and 1 mm mal - peg before electrophoresis as indicated , and the gel was stained for protein . both active site cysteines can be modified by alkylating agents the solvent accessibility of the active site cysteines of wild - type sstoa and two variants , c65a and c68a sstoa , was investigated using mal - peg , a high molecular mass , cysteine - specific alkylating agent . sstoa proteins were incubated with either 0 , 0.1 , or 1 mm mal - peg as described under experimental procedures and analyzed by sds - page . samples with exposed thiols are able to react with the mal - peg to form covalent complexes of significantly greater molecular mass relative to non - alkylated samples and thus are retarded during subsequent migration in the electrophoretic gel ( see fig . unmodified wild - type , c65a , and c68a sstoa variants migrated with an apparent molecular mass of 18 kda , which is in reasonable agreement with the actual mass ( 16.4 kda ) . in the presence of mal - peg , both single cysteine variants reacted to give a single species with a significantly lower mobility . protein molecular mass standards can not be used to judge the mass of mal - peg - modified proteins , but the significant retardation of sstoa variants is consistent with the alkylation of a single cysteine residue in each . in contrast , wild - type sstoa gave rise to two bands when incubated with 0.1 mm mal - peg . the lower ( faster running ) band corresponded to the singly modified single cysteine variants , and we conclude that under these conditions sstoa is present as a mixture of singly and doubly modified molecules . this was confirmed by incubating sstoa with a higher ( 1 mm ) concentration of mal - peg that resulted in the observation of only the larger , slower running band corresponding to the doubly modified protein . in addition to these major bands , other much fainter bands were observed on the gel ; these most likely arise from a small degree of heterogeneity in the size of the peg adducts in the mal - peg reagent and therefore do not represent additional ( non - cysteine ) alkylation events or protein heterogeneity . certainly no protein heterogeneity was observed in any of the untreated sstoa samples . from the structure it is apparent that the more n - terminal of the two active site cysteines is exposed to the solvent , whereas the other is not . solvent - accessible surface area calculations ( using a solvent probe of 1.2 ) on the structure of reduced sstoa showed that the cys-65 sulfur has an accessible surface area of 5.43 , whereas the sulfur atom of cys-68 is inaccessible from the bulk solvent . a similar arrangement exists in all structurally characterized thioredoxin - like proteins , and in virtually all of them , the second , buried cysteine thiol does not react with modifying reagents in solution ( 32 , 33 ) . this is not the case for b. subtilis resa in which we showed previously that both cysteines are readily modified by alkylating reagents ( 13 ) . to our knowledge , resa and now stoa are the only examples where this behavior has been demonstrated . for stoa , one possibility is that alkylation of the solvent - exposed cys-65 might cause a structural rearrangement that allows subsequent modification of cys-68 . alternatively the reduced protein in solution may undergo dynamic motion that would allow occasional access to the sulfur of cys-68 . pka values of stoa active site cysteines the ph stability profiles of the wild - type and single cysteine variant sstoa proteins were first determined to verify the range of values over which the acid - base properties of each protein could be investigated . the intrinsic tryptophan fluorescence was measured as a function of ph for each protein under reducing conditions . significant changes in the character of the tryptophan fluorescence emission spectrum , resulting from unfolding of the proteins , were observed at extremes of ph . both the emission wavelength maxima and fluorescence intensity maxima were affected by ph - induced unfolding . the former has the advantage of being independent of protein concentration and was thus used preferentially in monitoring ph stability ( see fig . the data showed that wild - type sstoa is stable between ph 3.5 and 9.3 , whereas c65a and c68a sstoa variants are stable between 4.4 and 9.6 and between 3.7 and 9.3 , respectively . the acid - base properties of the active site cysteines of wild - type sstoa and the c65a and c68a variants were investigated by measuring rates of reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene as described under experimental procedures ( see fig . the fluorescence is sensitive to the environment of the modified cysteine with emission occurring in the range of 440 - 550 nm , depending on the solvent exposure of the modified cysteine ( 34 ) . here the emission maxima for cys-65 and cys-68 were 510 nm , indicating that the fluorescent group of both modified residues is located in a relatively solvent - exposed position . for the single cysteine variants of sstoa , data fitted well to an equation describing a single protonation / deprotonation event , giving pka values of 7.0 0.1 and 7.1 0.1 for cys-65 and cys-68 , respectively ( see fig . 8 , c and d ) . for the wild - type protein in which both cysteine residues are intact , the data fitted well to two independent protonation / deprotonation events , giving pka values of 5.5 0.4 and 7.8 0.2 ( fig . both of these values are lower than the typical value of 8.5 - 9.0 observed for cysteine , and the data are consistent with the reactivity of both residues toward alkylating reagents ( which react with the deprotonated form only ) . a low pka value is normally observed for the n - terminal active site cysteine of thioredoxin - like proteins , which in some cases exhibit pka values as low as 3.5 ( 35 ) . however , the pka of the second cysteine is normally estimated to be > 9 ( 32 , 36 , 37 ) . in this respect , stoa is similar to resa in that the c - terminal cysteinyl has a pka value low enough to be measurable in the stable ph range of the protein . figure 7.ph stability of wild - type and variant sstoa proteins monitored by fluorescence . plots of tryptophan fluorescence emission maxima as a function of ph for solutions of wild - type ( wt ) sstoa and c65a and c68a sstoa as indicated ( all at 0.15 m in pctc buffer ) as a function of ph are shown . plots of tryptophan fluorescence emission maxima as a function of ph for solutions of wild - type ( wt ) sstoa and c65a and c68a sstoa as indicated ( all at 0.15 m in pctc buffer ) as a function of ph are shown . this large separation of pka values is consistent with the close proximity of the two thiol groups , indicating that the ionization of one significantly influences that of the other . the wide separation of active site thiol pka values appears to be a general feature of tdors that act with low specificity ( 33 , 38 ) . for sstoa , we also observed an interdependence of the cysteine acid - base properties : the sstoa single cysteine variants have pka values that are very similar , but in the wild - type protein they differ by more than 2 ph units . this suggests that in the wild - type protein cys-65 and cys-68 have a significant effect on one another such that the presence of both cysteines causes the pka of the n - terminal cysteine to drop , whereas that of c - terminal cysteine rises ( relative to the respective single cysteine variants ) . such interdependence of pka values was not observed for resa for which respective cysteines in single cysteine variants showed acid - base properties similar to those for the wild - type protein . the stronger interdependence of the cysteine pka values in stoa may well be linked to the significantly shorter sulfur - to - sulfur distance observed in reduced stoa ( 3.4 ) compared with that of resa ( 4.5 ) ( 12 ) . specificity determinants of stoa and resa here we have demonstrated that stoa and resa have many features in common . the three - dimensional structure , redox properties , and acid - base properties of active site cysteine residues are similar in these proteins . furthermore in vivo , the two proteins are believed to interact with the same integral membrane protein , ccda , which functions to supply electrons from thioredoxin ( trxa ) in the cytoplasm to the extracytoplasmic compartment ( 10 , 39 - 41 ) ; thus , structural / physical features important for this interaction are expected to be shared by stoa and resa . despite the similarities , so how do stoa and resa achieve specificity for their particular substrates ? to try to answer this question , it is important to identify regions of the proteins that do show differences . first , the two proteins differ in the active site sequence motif : cppc in stoa and cepc in resa . recently we reported the effects of altering the dipeptide intervening sequence on the properties of resa , and this included a resa cppc variant ( 21 ) . significant effects were observed : the redox potential increased by 25 mv , and the pka values of the two cysteines decreased by 1.8 and 1.6 ph units , respectively . these findings are consistent with the midpoint reduction potential and pka values measured here for stoa and those previously reported for resa ( 13 ) . alteration of the dipeptide sequence was also shown to impair the in vivo activity of resa ( 21 ) . beyond their effect on the biophysical properties of the active site cysteines ( i.e. redox potential and pka values ) , the intervening two residues may also be important for interaction with potential substrates . the close proximity of these residues to the active site cysteines and the fact that both are exposed on the surface of the protein make it highly likely that they contact partner proteins , at least transiently , and thus affect specificity of interaction . second , the protein surfaces close to the active site are subtly different in stoa and resa . the structures of oxidized and reduced resa previously revealed redox - linked conformational changes , the most significant of which was the opening up of a hydrophobic cavity close to the active site upon reduction ( 12 ) . despite the conservation or conservative substitution of several of the residues that line the resa cavity , reduction of stoa does not lead to the opening up of an equivalent cavity ( see fig . . the lack of a cavity in stoa may be the result of the substitution of thr-159 ( in resa ) with pro-153 in stoa ; thr-159 undergoes one of the biggest conformational movements upon formation of the hydrophobic cavity in resa , and thus its replacement by a proline ( pro-153 ) in stoa might well restrict conformational change in this region of the protein . alternatively the lack of a cavity in stoa might be linked to the much smaller conformational change in the cxxc motif itself , which is likely to be the driving force for the larger conformational changes around the active site motif in resa . a , time - dependent increases in fluorescence at 510 nm upon reaction of wild - type ( wt ) sstoa ( 1 m ) with 6-bromoacetyl-2-dimethylaminonaphthalene ( 15 m ) in pctc buffer system at ph values from 5 to 9 as indicated at 25 c . plots were fitted ( solid lines ) to obtain an observed , pseudo - first order rate constant , ko . b , plot of ko as a function of ph for wild - type sstoa . c and d , plots of ko as a function of ph obtained from similar experiments with c65a and c68a sstoa , respectively . pka plots for wild - type and single cysteine sstoa variants . a , time - dependent increases in fluorescence at 510 nm upon reaction of wild - type ( wt ) sstoa ( 1 m ) with 6-bromoacetyl-2-dimethylaminonaphthalene ( 15 m ) in pctc buffer system at ph values from 5 to 9 as indicated at 25 c . plots were fitted ( solid lines ) to obtain an observed , pseudo - first order rate constant , ko . b , plot of ko as a function of ph for wild - type sstoa . c and d , plots of ko as a function of ph obtained from similar experiments with c65a and c68a sstoa , respectively . figure 9.a structural comparison between stoa and resa . a and c , three - dimensional structures of sstoa and sresa , respectively , in schematic representation . regions colored red indicate areas of high negative electrostatic potential , whereas blue areas indicate areas of high positive potential . the main differences between sstoa and sresa are indicated ; see the main text for details . a structural comparison between stoa and resa . a and c , three - dimensional structures of sstoa and sresa , respectively , in schematic representation . regions colored red indicate areas of high negative electrostatic potential , whereas blue areas indicate areas of high positive potential . the main differences between sstoa and sresa are indicated ; see the main text for details . it has been demonstrated that glu-80 of resa plays a key role in vivo ( 14 ) , and in vitro studies showed that it is important for the elevated pka values of the active site cysteines and that it is capable of hydrogen bonding to amino acid side chain residues bound in the cavity ( 13 , 21 ) . this led us to propose that glu-80 is important for the binding of apocytochrome substrates to resa ( 12 - 14 , 42 ) . this residue is conserved in stoa ( glu-71 ) , and we have shown here that it is also functionally important in stoa ( table 2 ) . sequence alignments showed that it is conserved in many extracytoplasmic tdors that are proposed to have a reductive function ( 13 ) . the data presented here suggest that it fulfils a similar role in resa and stoa and also in other tdors . this could be in controlling the acid - base properties of the active site cysteines or through direct participation in the reduction mechanism . therefore , it is highly unlikely that this glutamate is itself an important determinant for specificity . it remains a possibility that , in resa , it interacts directly with substrates , but this would be an additional role facilitated by the formation of the hydrophobic cavity upon reduction of the protein . interaction of glu-71 with substrate is not favored in stoa because the residue remains buried in both oxidation states . third , with the exception of the aforementioned hydrophobic cavity in ( reduced ) resa , the only major difference in the electrostatic surfaces of each protein is in helix 3 ( stoa numbering ) , which is positively charged in stoa and negatively charged in resa ( fig . 9 ) . however , this helix is quite distant from the active site , and it seems unlikely that this feature is responsible for differences in substrate recognition by resa and stoa . the final major difference is the presence in stoa of an extended loop between strand 4 and helix 2 . in the primary sequence alignment ( fig . 1 ) , this can be clearly seen as an apparent insertion / deletion of several residues that are present in the stoa but not in resa . the sequence alignment also shows that there is little similarity between the two proteins in this region . the structure shows that the extended 4-2 loop ( composed of ser-97 , glu-98 , gln-99 , and asn-100 ) lies close to the active site of stoa ( fig . 9 ) and represents the most significant difference in the surface shape of stoa compared with resa and is , therefore , likely to be important for the differential substrate selectivity / specificity of these proteins . for example , this loop could be involved in specific binding interactions with stoa substrate(s ) or could serve to sterically hinder interactions with non - substrate molecules . concluding remarks the structural , biochemical , and in vivo characterization of b. subtilis stoa reported here provides new knowledge about this unprecedented endospore biogenesis factor whose physiological function is not completely understood ( 9 ) . furthermore the data reveal that this low potential extracytoplasmic tdor is remarkably similar to resa , another well characterized extracytoplasmic tdor from the same organism that is required for cytochrome c maturation . it is thought that both proteins interact with the same integral membrane protein , ccda , which supplies them with electrons from the cytoplasm . the high structural similarity of resa and stoa is no doubt connected with their shared need to interact with this protein . despite the large extent of their similarity ( in both sequence and structure ) , the proteins can not functionally substitute for one another in vivo . bacteria usually contain several thioredoxin - like proteins , soluble in the cytoplasm as well as membrane - bound . b. subtilis contains at least 10 such proteins , and none of these are essential for growth , indicating a narrow substrate specificity for each protein ( 43 ) . the results of this work raise important general questions about how tdors achieve substrate specificity : resa can recognize at least four different apo - c - type cytochrome polypeptides as substrates , whereas stoa recognizes a different but as yet unknown substrate(s ) critical for endospore cortex biosynthesis . from the structures of stoa and resa , we have identified four principal structural differences between the two proteins that we believe provide the basis of substrate specificity / selectivity . the work demonstrates that protein - substrate specificity / selectivity can apparently be achieved through remarkably subtle variations in amino acid sequence and three - dimensional structure .

bacillus subtilis stoa is an extracytoplasmic thiol - disulfide oxidoreductase ( tdor ) important for the synthesis of the endospore peptidoglycan cortex protective layer . here we demonstrate that stoa is membrane - associated in b. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor . this showed that stoa adopts a thioredoxin - like fold with n - terminal and internal additions that are characteristic of extracytoplasmic tdors . the cxxc active site of the crystallized protein was found to be in a mixture of oxidized and reduced states , illustrating that there is little conformational variation between redox states . the midpoint reduction potential was determined as -248 mv versus normal hydrogen electrode at ph 7 consistent with stoa fulfilling a reductive role in endospore biogenesis . pka values of the active site cysteines , cys-65 and cys-68 , were determined to be 5.5 and 7.8 . although cys-68 is buried within the structure , both cysteines were found to be accessible to cysteine - specific alkylating reagents . in vivo studies of site - directed variants of stoa revealed that the active site cysteines are functionally important , as is glu-71 , which lies close to the active site and is conserved in many reducing extracytoplasmic tdors . the structure and biophysical properties of stoa are very similar to those of resa , a b. subtilis extracytoplasmic tdor involved in cytochrome c maturation , raising important general questions about how these similar but non - redundant proteins achieve specificity . a detailed comparison of the two proteins demonstrates that relatively subtle differences , largely located around the active sites of the proteins , are sufficient to confer specificity .

EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION Supplementary Material

for production of a thrombin - cleavable gst - sstoa fusion protein , a fragment of the stoa gene encoding residues 21 - 165 of stoa was first amplified by pcr as above using oligonucleotides ly001 and ly002 as primers and subsequently cloned into pcr - blunt - ii - topo generating plym025 , which was verified by sequencing . the n - terminal amino acid residue sequence of the purified protein was verified by edman degradation ( see fig . 1 ) , stoa was predicted to be a ditopic membrane - bound protein with an n - terminal 30-residue segment that anchors the 135-residue tdor domain to the membrane . experiments in b. subtilis indicated that the function of stoa is not dependent on the membrane anchor ( 3 ) , and prediction programs suggested that the n - terminal segment might be cleaved off after the tdor domain has been translocated across the membrane . to establish whether stoa is a membrane - bound protein , b. subtilis strain 1a1 was grown in nutrient sporulation medium with phosphate for sporulation , and samples taken at different time points during growth were analyzed for stoa by using western blot with polyclonal antiserum directed against the tdor domain of stoa . the results indicate that stoa is membrane - associated but is present in very low amounts and is degraded or trapped in maturing endospores so that it is not detectable by the western blot procedure used . stars indicate the active site residues cys-65 and cys-68 and conserved residue glu-71 of stoa . stars indicate the active site residues cys-65 and cys-68 and conserved residue glu-71 of stoa . in vivo functional analysis of active site variants of stoa to establish that the function of stoa in endospore biogenesis is dependent on the cysteine residues of the protein , b. subtilis strain lul20 in which the stoa gene is inactivated and strain lul30 in which stoa is deleted from the chromosome were used ( 2 ) . the high resolution structure of the soluble domain of stoa in a mixture of oxidized and reduced states sstoa ( residues 22 - 165 ) was produced in e. coli and purified . 3a ) can be described as a modified thioredoxin - like fold that is highly reminiscent of b. subtilis resa ( 12 ) , ccmg ( from e. coli and bradyrhizobium japonicum ) ( 26 , 27 ) , and mycobacterium tuberculosis dsbe ( 28 ) , which are all extracytoplasmic tdors ( see fig . like these proteins , the classical thioredoxin - like motif of stoa is embellished by a central insertion and an n - terminal -hairpin ( in addition to the transmembrane helix predicted from primary sequence analysis ) . figure 3.the three - dimensional structure of the soluble domain of stoa . a , three - dimensional structure of sstoa showing that the protein exhibits a classical thioredoxin - like fold with two significant insertions : the n - terminal region contains a two - stranded , antiparallel hairpin , whereas the central insert , located after the 3-1-4 motif of the thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . the three - dimensional structure of the soluble domain of stoa . a , three - dimensional structure of sstoa showing that the protein exhibits a classical thioredoxin - like fold with two significant insertions : the n - terminal region contains a two - stranded , antiparallel hairpin , whereas the central insert , located after the 3-1-4 motif of the thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . in the structure determined here , the active site cysteines of sstoa appear as a mixture of oxidized and reduced redox states ( in each monomer ) . figure 4.the active site of stoa in oxidized and reduced states . a , electron density ( contoured at 1.2 ) of the active site cppc motif of stoa reveals a mixture of oxidized and reduced states . b and c , separated representations of the active site region in oxidized and reduced states , respectively . the active site of stoa in oxidized and reduced states . a , electron density ( contoured at 1.2 ) of the active site cppc motif of stoa b and c , separated representations of the active site region in oxidized and reduced states , respectively . the oxidized ( disulfide - bonded ) conformation of the active site dominates the electron density and is best described as adopting a classical right - handed hook conformation with a 3 angle of 73.5 1.8 formed between the two cysteine residues . this distance is considerably shorter than that observed for the structure of resa that has an exceptionally long sulfur - to - sulfur distance of 4.5 in the reduced state ( 12 ) . partial reduction of the disulfide does not seem to cause significant rearrangement of the local protein structure , and thus , unlike resa , there is no evidence for any redox - linked conformational changes due to reduction of the cysteines in stoa . in almost all known natural thioredoxin - like proteins , at least one of the two residues that intervene between the active site cysteines residues ( i.e. like all other thioredoxin - like tdors , the cxxc motif of stoa is found at the n terminus of a reasonably long -helix ( 1 in stoa ) , and the macrodipole arising from this helix is often invoked as a primary cause of the lowered pka values associated with the more n - terminal cysteine residue of the cxxc motif in most tdors ( 29 ) . the presence of proline residues at the cap of this active site helix in stoa is therefore likely to have important consequences for the distribution of the electrostatic field near the cysteines as proline does not possess a standard peptide group . a further proline residue ( pro-135 ) , which is in the cis conformation and is conserved in all thioredoxin - like proteins , is found in van der waals contact with two buried polar residues , a histidine and a glutamate , which are located immediately behind the second cysteine of the active site motif . the arrangement of these two buried polar residues and the intervening water molecule ( in stoa ) is very similar to that observed in resa where the glutamate is conserved ( glu-80 ) and an asparagine ( asn-68 ) residue takes an equivalent position to that of his-59 . substitution of glu-80 in resa has been shown previously to have a significant effect on the active site properties of the enzyme ; for example , the pka values of both active site cysteines were significantly lowered in an e80q variant ( 13 ) , and a b. subtilis strain containing e80q resa was also severely impaired in its ability to mature c - type cytochromes ( 14 ) . stoa is a low potential tdor the reduction potential of the active site cysteines of sstoa was measured using the difference in tryptophan fluorescence intensity of oxidized and reduced sstoa to follow oxidation state as a function of reduction potential ( see fig . the data fitted well to the nernst equation , giving a midpoint reduction potential of -248 2 mv versus normal hydrogen electrode at ph 7 with n = 2.18 0.16 , as expected for a two - electron reduction process . this value is similar to that measured for b. subtilis resa ( -256 mv at ph 7 ) ( 21 ) and e. coli thioredoxin ( -270 mv at ph 7 ) ( 30 , 31 ) and is entirely consistent with the structural similarity between these proteins and a role for stoa in the reduction of ( as yet unidentified ) proteins involved in endospore cortex synthesis ( 2 , 9 ) . both active site cysteines can be modified by alkylating agents the solvent accessibility of the active site cysteines of wild - type sstoa and two variants , c65a and c68a sstoa , was investigated using mal - peg , a high molecular mass , cysteine - specific alkylating agent . from the structure it is apparent that the more n - terminal of the two active site cysteines is exposed to the solvent , whereas the other is not . solvent - accessible surface area calculations ( using a solvent probe of 1.2 ) on the structure of reduced sstoa showed that the cys-65 sulfur has an accessible surface area of 5.43 , whereas the sulfur atom of cys-68 is inaccessible from the bulk solvent . a similar arrangement exists in all structurally characterized thioredoxin - like proteins , and in virtually all of them , the second , buried cysteine thiol does not react with modifying reagents in solution ( 32 , 33 ) . this is not the case for b. subtilis resa in which we showed previously that both cysteines are readily modified by alkylating reagents ( 13 ) . pka values of stoa active site cysteines the ph stability profiles of the wild - type and single cysteine variant sstoa proteins were first determined to verify the range of values over which the acid - base properties of each protein could be investigated . significant changes in the character of the tryptophan fluorescence emission spectrum , resulting from unfolding of the proteins , were observed at extremes of ph . the acid - base properties of the active site cysteines of wild - type sstoa and the c65a and c68a variants were investigated by measuring rates of reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene as described under experimental procedures ( see fig . here the emission maxima for cys-65 and cys-68 were 510 nm , indicating that the fluorescent group of both modified residues is located in a relatively solvent - exposed position . for the single cysteine variants of sstoa , data fitted well to an equation describing a single protonation / deprotonation event , giving pka values of 7.0 0.1 and 7.1 0.1 for cys-65 and cys-68 , respectively ( see fig . for the wild - type protein in which both cysteine residues are intact , the data fitted well to two independent protonation / deprotonation events , giving pka values of 5.5 0.4 and 7.8 0.2 ( fig . a low pka value is normally observed for the n - terminal active site cysteine of thioredoxin - like proteins , which in some cases exhibit pka values as low as 3.5 ( 35 ) . in this respect , stoa is similar to resa in that the c - terminal cysteinyl has a pka value low enough to be measurable in the stable ph range of the protein . this large separation of pka values is consistent with the close proximity of the two thiol groups , indicating that the ionization of one significantly influences that of the other . for sstoa , we also observed an interdependence of the cysteine acid - base properties : the sstoa single cysteine variants have pka values that are very similar , but in the wild - type protein they differ by more than 2 ph units . this suggests that in the wild - type protein cys-65 and cys-68 have a significant effect on one another such that the presence of both cysteines causes the pka of the n - terminal cysteine to drop , whereas that of c - terminal cysteine rises ( relative to the respective single cysteine variants ) . such interdependence of pka values was not observed for resa for which respective cysteines in single cysteine variants showed acid - base properties similar to those for the wild - type protein . the stronger interdependence of the cysteine pka values in stoa may well be linked to the significantly shorter sulfur - to - sulfur distance observed in reduced stoa ( 3.4 ) compared with that of resa ( 4.5 ) ( 12 ) . furthermore in vivo , the two proteins are believed to interact with the same integral membrane protein , ccda , which functions to supply electrons from thioredoxin ( trxa ) in the cytoplasm to the extracytoplasmic compartment ( 10 , 39 - 41 ) ; thus , structural / physical features important for this interaction are expected to be shared by stoa and resa . first , the two proteins differ in the active site sequence motif : cppc in stoa and cepc in resa . significant effects were observed : the redox potential increased by 25 mv , and the pka values of the two cysteines decreased by 1.8 and 1.6 ph units , respectively . these findings are consistent with the midpoint reduction potential and pka values measured here for stoa and those previously reported for resa ( 13 ) . alteration of the dipeptide sequence was also shown to impair the in vivo activity of resa ( 21 ) . beyond their effect on the biophysical properties of the active site cysteines ( i.e. the close proximity of these residues to the active site cysteines and the fact that both are exposed on the surface of the protein make it highly likely that they contact partner proteins , at least transiently , and thus affect specificity of interaction . second , the protein surfaces close to the active site are subtly different in stoa and resa . the structures of oxidized and reduced resa previously revealed redox - linked conformational changes , the most significant of which was the opening up of a hydrophobic cavity close to the active site upon reduction ( 12 ) . alternatively the lack of a cavity in stoa might be linked to the much smaller conformational change in the cxxc motif itself , which is likely to be the driving force for the larger conformational changes around the active site motif in resa . it has been demonstrated that glu-80 of resa plays a key role in vivo ( 14 ) , and in vitro studies showed that it is important for the elevated pka values of the active site cysteines and that it is capable of hydrogen bonding to amino acid side chain residues bound in the cavity ( 13 , 21 ) . sequence alignments showed that it is conserved in many extracytoplasmic tdors that are proposed to have a reductive function ( 13 ) . this could be in controlling the acid - base properties of the active site cysteines or through direct participation in the reduction mechanism . the sequence alignment also shows that there is little similarity between the two proteins in this region . the structure shows that the extended 4-2 loop ( composed of ser-97 , glu-98 , gln-99 , and asn-100 ) lies close to the active site of stoa ( fig . 9 ) and represents the most significant difference in the surface shape of stoa compared with resa and is , therefore , likely to be important for the differential substrate selectivity / specificity of these proteins . concluding remarks the structural , biochemical , and in vivo characterization of b. subtilis stoa reported here provides new knowledge about this unprecedented endospore biogenesis factor whose physiological function is not completely understood ( 9 ) . furthermore the data reveal that this low potential extracytoplasmic tdor is remarkably similar to resa , another well characterized extracytoplasmic tdor from the same organism that is required for cytochrome c maturation . bacteria usually contain several thioredoxin - like proteins , soluble in the cytoplasm as well as membrane - bound . the results of this work raise important general questions about how tdors achieve substrate specificity : resa can recognize at least four different apo - c - type cytochrome polypeptides as substrates , whereas stoa recognizes a different but as yet unknown substrate(s ) critical for endospore cortex biosynthesis . from the structures of stoa and resa , we have identified four principal structural differences between the two proteins that we believe provide the basis of substrate specificity / selectivity .

although a number of republican militant groups were active in this time , including the official irish republican army ( ira ) , the irish national liberation army , the continuity ira , the real ira , and the irish people s liberation organization , we narrow our focus solely to pira . pira were the most prolific nonstate militant grouping during this period in terms of both terrorist events and fatalities caused . through a protracted campaign of violence , pira s overall objective was to force the removal of northern ireland as part of the united kingdom , thereby paving the way for the reestablishment of a thirty - two county republic of ireland . following a split in the republican movement in december 1969 , pira was formed . originally , pira strategy sought to quickly force british troops out of northern ireland by inflicting a high death toll and substantial economic costs and thereby swaying british public opinion against maintaining an active presence in northern ireland . by 1977 , pira s plans became more long term , and they started plotting a longer war of attrition that eventually led to an increased emphasis upon mainstream political mobilization through their political wing , sinn fin . in the years that followed , the republican movement came to embrace what it internally referred to as tactical use of the armed struggle . pira were active for twenty - nine years and had a long time to get very good at killing people . even if we only trace the development of skills and ties that could be exploited to target their enemies from the birth of pira in 1970 following state violence against protestors demanding civil rights ( moloney 2003 ) , by 1979 the organization had ten years of active and successful practice ( sutton 1994 ) . that year , pira operatives detonated a concealed trailer full of milk churns that had been packed with 227 kg of ammonium nitrate explosives as a convoy of five british army vehicles passed . revealing a great deal of malevolent creativity , the trailer had been surrounded by petrol cans that further enhanced the explosion s ferocity ( oppenheimer 2009 , 113 ) . this detonation blew up the second truck in the convoy , killing six soldiers of the parachute regiment that had been responsible for the bloody sunday massacre that killed thirteen innocent civil rights marchers seven years prior . the surviving soldiers sought cover behind the gates of a nearby gatehouse . anticipating this retreat ahead of time , pira had already placed an even larger 450-kg ied using a homemade mixture of ammonium nitrate , nitroglycerine , and coal . upon additional rescue teams arriving to save the retreating survivors , pira operatives detonated the second bomb using a remote control trigger , killing a further twelve soldiers ( oppenheimer 2009 , 113 - 14 ) . such examples however mask the fact that a large proportion of pira s acts of violence caused zero fatalities . for example , of the 5,461 pira ieds , we coded as part of the bigger data - driven project from which this article draws ( from bomb to bomb - maker ) , only 8.7 percent killed at least one person ( on many occasions at the beginning of the conflict , the sole victim was the ied planter who died due to a premature explosion such incidents are not counted in the following analyses ) . the relatively discriminative impression these results produce are a reflection of pira s strategy of aiming to limit civilian deaths ( through the provision of advanced warnings ) , bombing economic targets in the middle of the night , and increasing the economic costs of the united kingdom s continued presence within northern ireland . we define an explosive device as an ied if any or all of the following are modified in any respect from its original expressed or intended function : explosive ingredient , initiation , triggering or detonation mechanism , delivery system . an explosive device is not considered an ied when no aspect of its deployment or fabrication is innovated upon ( gill , horgan , and lovelace , 2011 , 742 ) . pira s use of soviet - manufactured rocket - propelled grenades called rpg-7s was not included in our analysis . as figures 1 through 3 illustrate , pira s use of ieds ebbed and flowed over the course of its twenty - nine - year conflict . the biggest spike occurred over the opening two years as violence on both sides reached its peak . during this time , pira s use of car bombs dramatically accelerated upon developing ammonium nitrate explosives requiring a delivery system capable of carrying hundreds of kilos of the mixture . the subsequent dip from 1972 to 1975 is largely attributable to british counterterrorism policy of blocking off pira s supply of commercial explosives ( which were largely obtained through mining companies and robberies south of the border ) . also , pira s strategy of a quick military victory began to give way to a strategy based on a war of attrition . as such , the scale and intensity of ied attacks began to decrease in lethality but increased in frequency through the late 1970s as pira began to regularly use small , timer - based incendiary devices against commercial business premises . the idea behind these attacks was to minimize the possibility of civilian casualties by timing the ignition in the middle of the night but still to allow for maximum economic damage . pira hoped that the increasing costs of maintaining a visible british presence in northern ireland would ultimately tempt british voters to pressurize incumbent regimes to withdraw political and territorial interest from northern ireland . the growing politicization of the republican movement through the 1980s accompanied a perceived downgrading in pira s ability to consistently engage in fatal attacks ( moloney 2010 ) . interestingly , while private negotiations for peace with the british ( between 1990 and 1994 ) were at their peak , pira s use of ieds gradually rose with 1993 experiencing the fourth - highest number of ied events in any given year of the conflict . in effect , pira had learned from previous negotiations that the only way to strengthen their position at the bargaining table while keeping active supporters and rank and file recruits happy was to gradually step up their violent ( but not necessarily fatal ) activities . bombing[s ] on and off like a tap came to represent one of their signature capabilities throughout both formal and informal negotiations ( coogan 2002 , 399 ) . the remaining figures in this section disaggregate pira s campaign into five discrete phases of activity . our intention is to illustrate how organizational changes in structure and strategy impact the trajectory of violent events , types of events typically engaged upon , and their outcomes . pira structured themselves like an army composed of various brigades , battalions , and companies . each unit was responsible for specific geographical areas of operation , both combat and noncombat related . indiscriminate violence by both sides of the conflict marked this period , the most defining moment being bloody sunday when the british army shot and killed thirteen innocent civil rights marchers . this was an unprecedented propaganda coup for pira and led to mass recruitment and mobilization . civilian fatalities attributed to pira also peaked during this phase and included the events of bloody friday where , in under two hours , 22 ieds killed 9 ( 6 civilians , 2 british soldiers , and 1 ulster defence association member ) and injured a further 130 . the second phase , from 1977 to 1980 , is significant for a number of reasons . first , there was a large - scale reorganization of pira s structure to a tighter cellular based network in which cells acted independently of one another . this change placed far less emphasis on the quantity of volunteers and far more emphasis on secrecy and discipline . almost instantly , the effects of the structural changes were noticeable . four hundred sixty - five fewer charges for paramilitary offences occurred within a year ( smith , 1997 , 145 ) . second , a number of leadership changes occurred whereby younger northern born - and - bred members ( such as gerry adams and martin mcguinness ) became pira chiefs of staff in the late 1970s ( moloney 2003 , 513 ) . the third phase covered the period from 1981 to 1989 and encompasses the growing politicization of the republican movement that occurred after the hunger strikes . in total , ten republicans died during hunger strikes in 1981 ; seven were pira members . additionally , pira s bobby sands was elected to westminster after winning a by - election while on a hunger strike . sympathy for pira began to rise again , and this was largely channeled toward pira s political wing , sinn fein , by organizational elites . phase 4 ( 19901994 ) includes the negotiations and pathway toward ceasefires undertaken secretly by organizational elites . phase 5 ( 19951998 ) incorporates the period in which the negotiations were made public and the march toward the final ceasefire and good friday agreement that symbolized for many the end of the northern ireland conflict . figure 2 neatly illustrates the substantial drop in the numbers of fatalities and injuries through pira s ied activity across time . phase 1 averages 70.57 fatalities a year , while the figures for phases 2 through 5 are 28 , 32.5 , 20.8 , and 4.125 , respectively . however , figure 3 illustrates that when calculated as a ratio , the average number of injuries attributed to pira ieds increases in the final phases of the conflict . one of the major findings in the quantitative analysis of the lethality of terrorist organizations is that organizational factors do indeed matter ( asal and rethemeyer 2008c ) . asal and rethemeyer found that organizational size , connections , and ideology all had a significant impact on the lethality of terrorist organizations . however , their analysis reflects an aggregate cross - organizational perspective focusing upon a wide array of ideologies and does not take differences in tactics into account . what may be true of organizations in the aggregate may not be true if we disaggregate organizations ( helfstein and wright 2011 ) . one confounding factor may be that ideological differences hide important differences at the suborganizational level . drake ( 1998a , 53 ) notes that ideology relates to targeting practices of terrorist organizations because it sets out the moral framework within which they operate . similarly , a number of scholars note that the new terrorist organizations are more lethal and attribute this difference in lethality to religious ideology : religious ideologies are in some cases more permissive of violent and deadly acts ( laqueur 1998 ) . further , asal and rethemeyer ( 2008a , 438 ) argue that two factors shape how intrinsically linked any ideology is to deadliness . the first point relates to whether the organization s audience is earthly or supernatural . the second point relates to an organization s ability to clearly and cleanly define an other. ideology , however , can not explain divergences in lethality within a single organization . within a terrorist organization , the ideology s audience and ability to other the enemy remain relatively constant ( although it must be stated that components of pira were more sectarian than others while some politicized earlier than others ) . the same is true for other variables often labeled root causes of terrorism , such as rates of democracy and state sponsorship ( helfstein and wright 2011 ) . our contention is that subunit capability ( conceptualized as unit size , levels of professional training , and experience ) and blue team ( e.g. , those actors charged with countering terrorism such as the military or police ) activity combine to make particular forms of fatal violence more likely within particular components of a terrorist organization . the social movement literature has long established that resources are key to the success of organizations ( mccarthy and zald 1977 ) and the lack of resources has been seen to limit what terrorist organizations can do ( boyns and ballard 2004 ) . human capital is often depicted as the key resource ( boyns and ballard 2004 ; asal and rethemeyer 2008c ) . jackson points out that larger terrorist organizations should be better at adopting and making effective use of new technologies ( jackson 2001 ) . contrary to jackson ( 2001 ) , oots ( 1986 , 69 - 72 ) makes the case that when it comes to carrying out attacks , larger organizational structures are likely to be less effective because they require resources to maintain them . mccormick ( 2003 ) makes a similar argument about the tensions between an organization trying to protect itself while still trying to carry out its attacks . these arguments treat terrorist organizations as single entities and do not examine them as coexisting subunits . while we can measure terrorist attack counts by whole organizations , if the organization is big enough it is misleading to say that an attack is conducted by an entire organization . thus , when we look at a terrorist organization s component parts the logic may be different than for the organization as a whole . in the case of pira , attacks were carried out by different brigades ( who themselves had specialized roles and responsibilities ) and rarely as attacks orchestrated by the organization as a whole ( horgan and taylor 1997 ) . indeed , pira s claims of responsibility often attributed attacks to particular brigades or battalions . another possibility to consider is that the size of an organizational component will have a differential impact depending on the type of attack that is carried out . the level of expertise that a type of attack demands should have an impact on how human resources impact the success of that kind of attack ( jackson 2009 , 12 - 13 ) . the level of technical expertise needed for an average shooting attack for example could be considered low on the other hand , complex attacks demand a higher level of organization , expertise , and security ( if you assume that complex attacks have the potential to be more spectacular in their consequences ; drake 1998a , 1998b ; jackson 2009 , 12 - 13 ) . firsthand accounts of pira training suggest that a disproportionate amount of time was spent on ied training compared to shooting ( see ocallaghan 1999 ) , which leads us to suggest that ieds are generally more complex than shooting attacks and their successful execution depends more on knowledge at the individual bomb maker and his or her network affiliates level . further to this , johnson and braithwaite ( 2009 ) illustrate that ied attacks form tighter space - time clusters than do non - ied events , which indicates that ied attacks involve more planning , training , materials , expertise , and local support . knowledge at the individual level , rather than group size , may therefore be a more important factor for determining the effectiveness of ieds compared to shooting attacks . in fact , complex attacks have the potential to be more spectacular in their consequences , and certainly are more likely to portray the group responsible as more sophisticated with the ability to coordinate multiple , simultaneous efforts . however , group size also matters . on one hand , the larger the group , the more likely a group is to have in its ranks an individual with the requisite knowledge to build a sophisticated ied . on the other hand , larger groups are more likely to leak information to counterterrorism agencies and thus increase the chances of early detection . on balance , we believe the relationship is likely to be in favor of larger groups : larger groups contain more knowledge , and more knowledge leads to greater success . thus , group size and total knowledge should be related positively to one another from this perspective . asal and rethemeyer ( 2008a ) have found that lack of experience reduces lethality in their cross - national study , as has jackson ( 2001 ; though jackson used the age of the organization as a proxy ) . as hoffman ( 1999 , 25 ) argues , an almost darwinian principle of natural selection seems to affect terrorist organizations , whereby every new terrorist generation learns from its predecessors terrorists often analyze the mistakes made by former comrades who have been killed or apprehended . thus , we expect that having more experienced cadre in a brigade should also allow for an organization to be more effective in its use of ieds . given the relative lack of sophistication needed to perpetrate shooting attacks , the same should not hold true for such attacks at the brigade level . human capital can also be measured in outputs and not just inner traits such as experience and technical expertise . not all ieds and their constituent initiation systems are created equally , with some being more difficult than others . high - value targets such as military personnel , military infrastructure , police , or politicians are far more target hardened than most areas heavily frequented by civilian populations . while attacks against high - value targets may have a much higher pay off if they succeed , attacks that are easier ( in both their deployment and who they target ) should be more successful on average ( jackson 2009 ; drake 1998a , 1998b ) . in other words , when a brigade tries to carry off an attack using a device that is harder to construct the likelihood of success should fall . by the same token , brigades intent on using an ied ( as opposed to opting for a shooting attack with multiple offenders ) to attack a hardened target are more likely to fail but also need a more sophisticated device to succeed . having limited resources ( i.e. , ied components and personnel ) , decision makers must choose between attacking soft or hard targets using complex or simple ieds and ied components . given the relative ease of attacking civilians , simple ieds are likely to be chosen . given the relative difficulty of attacking high - value targets , multifaceted ieds and complex attacks ( defined as attacks involved more than one attack type ) are more likely to be chosen despite the higher chance of failure . while the most lethal attacks are likely to be complex ones targeting civilians , there are fewer incentives to use such tactics given the fact that simpler means can also cause a great deal of death and destruction against weakly guarded targets ( dolnik and bhattacharjee 2002 ) . we derive the following hypotheses from this discussion : hypothesis 1 : larger brigades should kill more.hypothesis 2 : brigades with more knowledge ( measured as age and training ) should kill more people with ied attacks.hypothesis 3 : brigades with more knowledge ( measured as age and training ) should kill fewer people with shooting attacks.hypothesis 4 : complex attacks should kill more than simple attacks . hypothesis 1 : larger brigades should kill more . hypothesis 2 : brigades with more knowledge ( measured as age and training ) should kill more people with ied attacks . hypothesis 3 : brigades with more knowledge ( measured as age and training ) should kill fewer people with shooting attacks . hypothesis 4 : complex attacks should kill more than simple attacks . counterterrorism efforts are designed to impact how terrorists behave . yet , whether and how this precisely happens is often less than clear . a report from 2006 found that ( 1 ) very little rigorous empirical research exists , ( 2 ) what does exist provides little support for most common policies , and ( 3 ) the one policy that does seem to have an effect works in the wrong direction : retaliatory raids increased terrorism ( lum , kennedy , and sherley 2006 ) . more recent work focusing on particular cases or surveys of the literature has found either a complicated picture or a nonproductive : counterterrorism activities are ineffective or counterproductive ( duyvesteyn 2008 ; feridun and shahbaz 2010 ; fielding and shortland 2010 ) . an analysis of pira terrorism found that most counterterrorist efforts resulted in an increase in terrorist activity and thereby could be characterized by a backlash model of counterterrorism ( lafree , dugan , and korte 2009 ) . we should note that due to a specific interest in the impact of discriminate ( e.g. , the killing of pira members ) versus indiscriminate ( e.g. , the killing of catholic noncombatants ) violence our focus here is on proactive offensive state attacks and not on other types of repression that are not specifically violent in nature like situational crime prevention measures such as checkpoints and curfews or judicial deterrence measures such as punitive sentencing . benmelech , berrebi , and klor ( 2010 ) suggest an important distinction when it comes to counterterrorism . they found that targeted house demolitions that destroyed the homes of people engaged in suicide terrorism reduced suicide attacks , while house demolitions that were carried out against property not directly associated with the specific suicide attack increased subsequent terrorism . byman , examining israel s policies of targeted killing found that such assassinations reduced the effectiveness of hamas terrorism against israel ( byman 2006 ) , although others have found no effect ( hafez and hatfield 2006 ) . the benmelech , berrebi , and klor ( 2010 ) and lafree , dugan , and korte ( 2009 ) articles both suggest that counterterrorism actions can create a backlash . byman s articles introduce an interesting and potentially very important caveat . while we can assume that both kinds of attacks are likely to hurt the brigade , indiscriminate attacks compensate for this pain by making the group more popular and creating more support . mccauley and moskalenko ( 2008 ) argue that this is exactly one of the reasons why terrorist organizations stage attacks : attacks provoke repression and make terrorist organizations more popular and stronger something they label jujitsu politics ( a term first coined by sharp in the context of nonviolent conflict resolution ) . on the other hand , violence directed specifically at the organization appears to lower the amount of subsequent terrorism . we derive the following hypotheses from this discussion : hypothesis 5 : indiscriminate counterterrorism killings should increase the number of people killed by ieds and shootings.hypothesis 6 : discriminate counterterrorism killings should reduce the number of people killed by ieds and shootings . hypothesis 5 : indiscriminate counterterrorism killings should increase the number of people killed by ieds and shootings . hypothesis 6 : discriminate counterterrorism killings should reduce the number of people killed by ieds and shootings . the data are an aggregation of 5,461 pira ied events , all fatal pira shootings , and the sociodemographic and operational behaviors of 1,240 pira members for the years 1970 to 1998 . similar to many quantitative studies of terrorism and political violence , there exist a number of data constraints in this study . however , after an eighteen - month data collection effort across multiple data sources , we feel that this is the best data available . from a research perspective , focusing upon intraorganizational dynamics allows us to hold constant many of the environmental and systemic variables that may confound other studies . for this article , pira is disaggregated into six discrete subunits , each of which encompasses a county of northern ireland . although not an exact fit to pira s command and functional structure , it acts as the only realizable proxy measure . northern command includes both the six counties of northern ireland and the border counties of cavan , donegal , leitrim , louth , and monaghan . northern command therefore covered the main theater of conflict . in turn , northern command was composed of brigadiers , brigades , operations commanders , and active service units of typically four individual pira members . southern command constituted the other twenty - one counties of the republic of ireland and its duties largely encompassed logistical support for northern command activities . tasks included training , funding , storing , and moving arms as well as provision of safehouses ( horgan and taylor 1997 ) . often , active service units ( asus ) operated within their own locality ( horgan and taylor 1997 , 20 ) . white and white s ( 1991 ) interviews with former senior pira figures explain why . reasons for this would be to avail of local facilities before , during and after operations , such as safehouses where they would be recognized without difficulty , and also because of familiarity with the operational area ( a vital aspect of the operational cycletarget selection , planning , escape routes , etc . ) . perhaps , however , this may be seen to have a detrimental effect on the internal security of asus after all , it is far more difficult being required not to know the identity of one s asu colleague if , in fact , volunteers are operating in their own locality ( horgan and taylor , 1997 , 22 ) . when questioned what makes a successful member of pira , sean macstiofain ( a former member of pira chief of staff ) , noted that a person has got to be from the locality , right? he s got to be respected within his own community , right ? like he becomes a fish who can swim he has got to have an intimate knowledge of his the areas he s going to operate in . he has got to be considerate about the needs of his own community ( white and white 1991 , 107 ) . for this study , we aggregated yearly counts of aspects of ied usage , fatal pira shooting events , fatal counterterrorism events , and a database of convicted pira members for each county . while pira s repertoires of violence also incorporated punishment beatings , kidnappings , and bank robberies , we specifically focus upon aggregate counts of ieds and shootings because they were the tactics most associated with lethal forms of violence each observation contains yearly counts of the ieds detonated as intended or were duds . we also count the number of each of the following : initiations systems used within a county in a given year : timer - initiated , wire - initiated , remote - initiated , projectile - initiated , booby trap initiated , impact - initiated , and victim - initiated devices . counts of the following ied types were also included in the analysis : letter bombs , pipe bombs , grenades , homemade bombs , static munitions , buried ieds , undervehicle ieds , car bombs , mortars , and rockets . we defined complex ied events as those where the ied was used in conjunction with another types of violent event such as machine gun fire or sniper fire . our metric of counterterrorism activity is a count of the pira members and innocent catholics killed by the british army and the royal ulster constabulary . it should be noted that these two counts do not reflect the whole counterterrorism picture . policies such as internment did not directly cause fatalities but caused a later backlash from the catholic community and ultimately increased pira s ability to recruit new individuals and mobilize mass support . on the other hand , prison - related policy changes such as the withdrawal of the special category status eventually led to the 1981 hunger strikes in which seven pira and three irish national liberation army members died . when counterterrorism policies indirectly lead to the deaths of pira members ( such as the hunger strikes ) these deaths were not counted in our analysis . other counterterrorism policies and actions such as the use of informants and the capture of bomb - making facilities would also impinge upon pira s ability to engage in lethal ied attacks , but such data were difficult to collect in a systematic way . our data set included a set of pira subunit traits , including a count of members in the brigade each year ; a measure of how big the subunit was in relation to the other five units each year ; the mean age of the subunits members ; the proportion of subunit members who possessed professional skills that could be applied to bomb making ; and the number of fatalities the subunit caused through ieds and shootings across target types ( including civilians and high - value targets ) . high - value targets encompass a collection of northern ireland security forces such as the british army , the royal ulster constabulary ( the northern ireland police force ) , the ulster defence regiment , and the royal irish regiment as well as government officials ( both elected and unelected ) and other political figures . the aggregate measures for the ied - related variables stem from a newly constructed data set of 5,461 events collected as part of the from bomb to bomb - maker project . the aggregate measures for deaths by the british army and other counterterrorism forces are collected through mckittrick et al.s definitive list of war dead from the northern ireland conflict . the subunit trait variables were aggregated by brigade from a database of 1,240 individuals who were either convicted of pira - related activities ( including membership ) or died on active service , a term used by pira to describe a member s involvement in pira - related activities . for the purposes of the data collection , being engaged in active service included both violent activities ( e.g. bombing attacks ) and nonviolent activities ( e.g. , training accidents ) . the individuals were identified from a number of open sources : ( 1 ) statements by pira including their annual roll of honor , which commemorates their war dead ; ( 2 ) the belfast graves publication that offers an account of republicans killed in combat ; ( 3 ) mckittrick et al . mentioned earlier ; and ( 4 ) historical accounts of pira from academic sources . these names were subsequently coded for a number of sociodemographic , operational , and network variables using the irish times archives . each piece of data ( ied event , blue team activity , pira militant ) was coded twice by separate coders and cross - checked for validity . four dependent variables were studied : total fatalities , civilian fatalities , fatalities among high - value targets ( such as security forces , politicians , etc . ) , and fatalities from shootings . each dependent variable is a count of the fatalities attributed to each brigade during a year between 1970 and 1998 . given that the dependent variables are counts generated by a rare event the data have two potential issues that must be accommodated during technique selection : ( 1 ) there is evidence of overdispersion of the dependent variable in all four cases , the mean of the counts is smaller than the standard deviation and ( 2 ) the presence of a large number of zeros in the dependent variable ( long 1997 ; cameron and trivedi 1998 ; long and freese 2003 ) . however , when we modeled the data using a zero - inflated negative binomial technique , the coefficient on , which captures the overdispersion , was not statistically significant in three of the four models . in the one case where was significant , for simplicity , we have thus used a poisson distribution for models reported below . with respect to problem ( 2 ) , we compared the results from both the standard and the zero - inflated poisson ( zip ) models . the decision whether to kill or not is separated ( analytically speaking ) from the decision regarding how many people to kill . zip allows for the possibility that zeros in the model are present because brigades have chosen not to kill or because they were incapable of executing a fatal attack during a given year . zip is attractive precisely because it can accommodate these complexities in the data . in order to verify that the decisions are independent and should be modeled simultaneously but independently , a vuong ( 1989 ) test , which compares the fit of the zero - inflated model to the standard negative binomial regression model , was executed and is reported in the results table . in all four models , the vuong statistic clearly indicates that a zero - inflated model is superior . in all four models , we attempted to include phase and brigade controls to account for panel fixed effects . we also included controls for the mix of device and initiation types used in ieds over time , which could covary with our key training and age variables . however , because some types of attacks were highly correlated with brigade , phase , or type of attacks being modeled , the controls could not always be included . exposurethat is , length of time over which the count would be accumulated so there are no controls for exposure in these models . because there are clear reasons to believe there are commonalities across time in the behaviors of brigades , we adjusted the standard errors for brigade - level clustering ( rogers 1993 ) . statistical significance of coefficients was measured at the 0.1 percent , 1 percent , and 5 percent levels , except as otherwise noted . each of the four dependent variables was modeled independently , and the zero - inflation and count components of the estimations were modeled using a selection of variables that accorded to the understanding of factors that drove the respective dependent variables . across all four models , we also discovered that some variables had to be deleted to cope with high collinearity or problems with convergence of the model , and the ied - related controls are , of course , not included in the modeling of shooting fatalities . the central findings for each model revolve around a set of key variables see the first eight variables listed in both the count and zero - inflation components of the estimations . they capture the role of training , participation in pira , counterterrorism activities by british authorities , membership size , membership age , and number of members killed during a given brigade - year . however , in our analysis , we point to some interesting findings for each of the models that are not necessarily found across all four estimations . table 2 contains the estimated coefficients for the count component of the zip models for all four dependent variables . zero - inflated poisson ( zip ) estimates , total fatalities , civilian fatalities , fatalities among targeted groups , and fatalities for shootings . note : pira = provisional irish republican army ; ied = improvised explosive device . * < .05 , * * < .01 , * * * < .001 . table 3 provides the zero - inflation estimation results plus standard model quality statistics such as n , , log likelihood , and the vuong statistics that pertain to the entire model ( count and zero inflation ) . zero - inflated poisson ( zip ) estimates , total fatalities , civilian fatalities , fatalities among targeted groups , and fatalities for shootings ( zero - inflation component ) . note : pira = provisional irish republican army ; ied = improvised explosive device . * the block of eight key variables at the top of the count component of the analysis tells an interesting story . turning to hypotheses 2 and 3 first , knowledge matters to fatalities , but not in the manner most expect and as we anticipated originally . the variable % of brigade professionally trained is statistically significant and negative for both total and civilian ied casualties and unrelated to high - value ied and shooting casualties . in fact , this variable is negative across all four models , though not statistically significant in the models for ied high - value targets and fatalities for shootings . in all cases , training kept the body count down rather than causing it to go up . note that when the relative size of a brigade as compared to the rest of pira s northern command brigades went up , the total fatalities count also went down . thus , hypothesis 2 is contradicted fairly convincingly and there is no support for hypothesis 3 with respect to professional training . also contrary to expectations , brigade size does not tend to increase fatalities uniformly across all attack types . instead , the variable membership size ( ct ) has no effect on total ied fatalities and high - value target ied fatalities , a negative effect on civilian ied fatalities , but a positive effect on shooting fatalities . the results from the zero - inflation component of the estimation further nuance our findings . as the percentage of the brigade professionally trained ( e.g. , with training from professional occupations that can be turned to bomb making ) increased , the probability that a brigade would kill no one went down and the probability that no members of the high - value target group ( i.e. , british security forces ) also went down . stated another way , training made it more likely a brigade would kill a nonzero number of people with ieds and especially more likely that a nonzero number of high - value targets would be killed with ieds , but training did not drive up the number particularly . similarly , as brigade size increased , the probability that a brigade would kill a nonzero number of civilians would increase but again , membership size did not drive up the civilian body count . instead , the effect of size was to make it likely some civilians would be killed . a complementary pattern emerged in the zero - inflation results for the relative size of brigade in pira variable . as size of the brigade goes up in comparison to the other brigades in that year , the probability that the brigade killed nobody went down , but the probability that the brigade killed no civilians went up . the relatively larger brigades in the pira were more likely to kill a nonzero number of people in a given year , but being large did not translate into a drive to maximize killing , as the relative size of brigade in pira variable in the count model demonstrated . finally , in hypotheses 2 and 3 , we suggested that age and/or professional training could be an important factor in driving ied fatalities . in fact , member average age was not a factor here ( in either the count or the zero - inflation components ) , meaning that maturity and/or experience in the movement seemed to have little effect on fatality rates one way or the other . the upshot of this complex set of outcomes is that professional training and size create a capability for killing . as brigades become larger and more professionally trained , the likelihood that this instrument will be used for some killing increases . however , size and training does not lead inexorably to prolific killing with ieds or shooting attacks . instead , it appears that professional training may allow brigades to kill those people they wish to kill rather than to kill indiscriminately . pira was more generally committed to selected killings especially of british security forces rather than indiscriminate lethality . as asal and rethemeyer ( 2008a ) found in their cross - national analyses , organizations can shape their lethality . looking cross - nationally between the years 1998 and 2005 , asal and rethemeyer found that organizations with a religious ideology were more likely to kill prolifically , while organizations that had a combined ideology of religion and ethnonationalism were likely to kill even more . while an argument exists that the role of religion has not been given enough credence in the analysis of the northern ireland conflict ( mitchell 2005 ) , there is a more widely held position that religion merely differentiated catholics and protestants and acted as a proxy for what were essentially ethnonational identities ( mcgarry and oleary 1995 ) . our theoretical position largely concurs with the latter and our empirical analysis suggests that operational and ideological commitments helped to shape the impact of training toward focused , discriminate killing . hypothesis 4 addressed that nature of attacks : do complex attacks attacks that include both an ied and shooting component increase the body count ? our analysis suggests that when the number of complex attacks attempted went up , there was a particular pattern to fatalities ( see the coefficients on complex attack ( ct ) ) . complex attacks did not affect the total number of fatalities . however , complex attacks tended to include a shooting component , so fatalities from shootings tended to increase . because such attacks involve additional terrorist actions ( e.g. , an ied attack and a shooting attack ) , the civilian fatality count tended to increase , but the fatality count among high - value target groups tended to decrease . ambitious attacks may be important for symbolic purposes and for recruiting , but the cost was a tendency for brigades to move away from the general preference for targeted killing of security personnel as outline above . turning now to counterterrorism effects ( hypotheses 5 and 6 ) , there are strong effects from the nature of british counterterrorism efforts . the data set contains two counterterrorism variables : a count of pira members killed by british forces ( killing by ct , pira members ( ct ) ) and a count of catholic civilian noncombatants killed by british forces ( killings by ct , civilians ( ct ) ) . however , there is one twist from the zero - inflation component of the model : british killing of pira members tended to increase the probability that a brigade would kill a nonzero number of civilians in that period . thus , there is a two - step explanation : killing pira members reduced effectiveness and tended to reduce total killings ( per hypothesis 6 ) , but it made it more likely that a given brigade would lash out in retaliation that would end up killing civilians , which is more in line with the backlash models of benmelech , berrebi , and klor ( 2010 ) and lafree , dugan , and korte ( 2009 ) . a topic for future research is to disentangle whether civilians were killed as a backlash or because the counterterrorism killings degraded the quality of pira members who were left , making the overall organization unable to carry out discriminate violence . killing of civilians by counterterrorism forces killings we term indiscriminate had a quite different effect : it tended to increase total fatalities , civilian fatalities , and fatalities from shootings , as anticipated in hypothesis 5 . the implication from this finding is that killing of civilians by british counterterrorism authorities seemed to engender a more violent , less targeted response . killing of civilians tended to make it more likely that a brigade would kill no one during a given brigade year . so , we have contradictory results : british killing of civilians made it more likely that a brigade would kill no civilians during a given year , but if the brigade did kill civilians , it was likely to kill quite a few . drawing together these findings , this suggests that brigades may have acted like capacitors : british killing of civilians would generate a desire to retaliate that was held in check until a level of outrage was reached that triggered a response . once a response was triggered , it tended to be less planned and targeted than was the general norm for brigade activity . concurrent discriminate killing of pira members could have exacerbated the tendency toward civilian killings in response by degrading the quality of members left such that a discriminate , security - focused response became less possible . the zero - inflation results hint that brigades may have avoided tit - for - tat killing of civilians , possibly because there was a fear of triggering a cycle of violence . but the count component clearly indicates that civilian killings eventually sparked a more general , indiscriminate round of violence . . often this process begins with underlying systemic causes and is often sustained and driven by more proximate counterterrorism initiatives that feed the recruitment and mobilization of new cadre . within the cadre itself , individuals with technical expertise may facilitate particular forms of violence that the group could not engage in absence of this individual . the culmination of a terrorist attack is often preceded by tactical decisions such as who should be targeted , what methods should be used and in the case of an ied attack , what ied type and initiation system would be most effective . all of these processes occur within groups and subgroups of varying sizes and we illustrated that variations across these processes impact a terrorist organization s deadliness . while the scale and intensity of terrorist campaigns is often related in the literature to distal factors such as rates of poverty , unemployment , and democracy indicators , there remains a distinct lack of awareness that not all violence is equal in terms of the technical proficiency and psychological conditioning needed . similarly , not all ieds are equal , and some aspects may drive lethality indicators up or down dependent upon the prevailing strategic orientation of the terrorist group itself . in other words , existing studies tend to aggregate terrorist organizations as collective wholes and treat violent methods and fatality types homogeneously . our findings suggest that ( 1 ) rates of fatal attacks differ depending upon the type of oppressive counterterrorism policies employed , ( 2 ) fatality rates differ intraorganizationally , and finally ( 3 ) subunit variables such as membership age , professional technical expertise , and brigade size also affect lethality rates but in different ways dependent upon the type of violence , who is targeted , and the strategic choices the organization makes corporately . there are caveats to these findings , not the least of which is that our measures of ct activity are rather aggregated and can not fully capture intelligence interventions that reduce the number of attacks brought to fruition , thus also reducing the total fatalities . however , this issue does not affect the finding that killing of civilians by counterterrorism forces leads to increased violence if anything , our findings underestimate the increase in fatalities after ct forces kill civilians . together , the results may help inform both policy and operational decision making . at the policy level , the results show the negative impact likely to occur when nonaligned civilians are killed in the course of counterterrorism operations . such events embolden terrorist organizations to strike back , usually in an equally indiscriminate fashion . even more so , the results suggest that when organizations do decide to strike back in retaliation , it is likely to be at the local level , a finding that braithwaite and johnson ( 2012 ) and linke , witmer , and oloughlin ( 2012 ) also found in relation to insurgent activities in iraq . although the targeted killing of pira members reduces a brigade s ability to kill within a given year , there is no data available to suggest whether such events also lead to an increase in constituency support , and future recruits for the organization that may later breathe life into a depleted subunit . moreover , targeted killings may even have the perverse effect of making civilian casualties more likely if the net result is to reduce the skill level of the unit ( though further research is needed to confirm this inference ) . the results also suggest that although pira can be described as a coherent , hierarchical organization , the variance among its subunits in terms of composition , capabilities , and targeting policies requires nuanced counterterrorism policies policies that are tailored to the local subunit s capacity for lethal activities . while there has been much theorizing about the structure of terrorist organizations , our results indicate that perhaps we should be more concerned with the composition and qualities inherent within a network of subunits . those subunits with a higher number of individuals with professional skills relevant to bomb making more often caused casualties , and those casualties are more likely to be among high - value targets rather than civilians . such findings may aid in decisions concerning what segments of a terrorist network should be prioritized for immediate postevent investigation and intelligence gathering . finally , we recognize that findings are not directly generalizable : we focused here upon one terrorist organization . generalizations to other groups may be difficult to make . that said , we believe the logic behind the relationship between counterterrorism killings and the lethality of units and subunits may apply more broadly . killing those whom the terrorist organization claims to represent is likely to encourage retaliation in the form of indiscriminate violence against civilians for a number of reasons elaborated upon above , whereas killing members of the group is likely to lead ( at least in the short run ) to reductions in the group s capacity to striking back . in terms of subunit composition , generalizations are more difficult . higher levels of skilled bomb makers within pira brigades led to both a higher likelihood of high - value targets being killed and civilians being spared both of which were long - standing strategic policies of pira . not every terrorist organization resembles pira in this regard , with many being far less reluctant to kill civilians . we might therefore expect that the more technical expertise within any given subunit in any terrorist organization , the closer that subunits fatality rates will mirror the organization s strategic logic as a whole .

this paper presents an analysis of the provisional irish republican army 's ( pira ) brigade level behavior during the northern ireland conflict ( 1970 - 1998 ) and identifies the organizational factors that impact a brigade 's lethality as measured via terrorist attacks . key independent variables include levels of technical expertise , cadre age , counter - terrorism policies experienced , brigade size , and ied components and delivery methods . we find that technical expertise within a brigade allows for careful ied usage , which significantly minimizes civilian casualties ( a specific strategic goal of pira ) while increasing the ability to kill more high value targets with ieds . lethal counter - terrorism events also significantly affect a brigade 's likelihood of killing both civilians and high - value targets but in different ways . killing pira members significantly decreases ied fatalities but also significantly decreases the possibility of zero civilian ied - related deaths in a given year . killing innocent catholics in a brigade 's county significantly increases total and civilian ied fatalities . together the results suggest the necessity to analyze dynamic situational variables that impact terrorist group behavior at the sub - unit level .

The Northern Ireland Conflict Theory When Smarter Is Better than Bigger: Human Capital The Impact of Counterterrorism Data Method Analysis Conclusion

although a number of republican militant groups were active in this time , including the official irish republican army ( ira ) , the irish national liberation army , the continuity ira , the real ira , and the irish people s liberation organization , we narrow our focus solely to pira . through a protracted campaign of violence , pira s overall objective was to force the removal of northern ireland as part of the united kingdom , thereby paving the way for the reestablishment of a thirty - two county republic of ireland . for example , of the 5,461 pira ieds , we coded as part of the bigger data - driven project from which this article draws ( from bomb to bomb - maker ) , only 8.7 percent killed at least one person ( on many occasions at the beginning of the conflict , the sole victim was the ied planter who died due to a premature explosion such incidents are not counted in the following analyses ) . the relatively discriminative impression these results produce are a reflection of pira s strategy of aiming to limit civilian deaths ( through the provision of advanced warnings ) , bombing economic targets in the middle of the night , and increasing the economic costs of the united kingdom s continued presence within northern ireland . the idea behind these attacks was to minimize the possibility of civilian casualties by timing the ignition in the middle of the night but still to allow for maximum economic damage . the growing politicization of the republican movement through the 1980s accompanied a perceived downgrading in pira s ability to consistently engage in fatal attacks ( moloney 2010 ) . interestingly , while private negotiations for peace with the british ( between 1990 and 1994 ) were at their peak , pira s use of ieds gradually rose with 1993 experiencing the fourth - highest number of ied events in any given year of the conflict . civilian fatalities attributed to pira also peaked during this phase and included the events of bloody friday where , in under two hours , 22 ieds killed 9 ( 6 civilians , 2 british soldiers , and 1 ulster defence association member ) and injured a further 130 . phase 5 ( 19951998 ) incorporates the period in which the negotiations were made public and the march toward the final ceasefire and good friday agreement that symbolized for many the end of the northern ireland conflict . one of the major findings in the quantitative analysis of the lethality of terrorist organizations is that organizational factors do indeed matter ( asal and rethemeyer 2008c ) . asal and rethemeyer found that organizational size , connections , and ideology all had a significant impact on the lethality of terrorist organizations . within a terrorist organization , the ideology s audience and ability to other the enemy remain relatively constant ( although it must be stated that components of pira were more sectarian than others while some politicized earlier than others ) . our contention is that subunit capability ( conceptualized as unit size , levels of professional training , and experience ) and blue team ( e.g. in the case of pira , attacks were carried out by different brigades ( who themselves had specialized roles and responsibilities ) and rarely as attacks orchestrated by the organization as a whole ( horgan and taylor 1997 ) . the level of technical expertise needed for an average shooting attack for example could be considered low on the other hand , complex attacks demand a higher level of organization , expertise , and security ( if you assume that complex attacks have the potential to be more spectacular in their consequences ; drake 1998a , 1998b ; jackson 2009 , 12 - 13 ) . firsthand accounts of pira training suggest that a disproportionate amount of time was spent on ied training compared to shooting ( see ocallaghan 1999 ) , which leads us to suggest that ieds are generally more complex than shooting attacks and their successful execution depends more on knowledge at the individual bomb maker and his or her network affiliates level . further to this , johnson and braithwaite ( 2009 ) illustrate that ied attacks form tighter space - time clusters than do non - ied events , which indicates that ied attacks involve more planning , training , materials , expertise , and local support . knowledge at the individual level , rather than group size , may therefore be a more important factor for determining the effectiveness of ieds compared to shooting attacks . in fact , complex attacks have the potential to be more spectacular in their consequences , and certainly are more likely to portray the group responsible as more sophisticated with the ability to coordinate multiple , simultaneous efforts . thus , we expect that having more experienced cadre in a brigade should also allow for an organization to be more effective in its use of ieds . given the relative lack of sophistication needed to perpetrate shooting attacks , the same should not hold true for such attacks at the brigade level . high - value targets such as military personnel , military infrastructure , police , or politicians are far more target hardened than most areas heavily frequented by civilian populations . while attacks against high - value targets may have a much higher pay off if they succeed , attacks that are easier ( in both their deployment and who they target ) should be more successful on average ( jackson 2009 ; drake 1998a , 1998b ) . in other words , when a brigade tries to carry off an attack using a device that is harder to construct the likelihood of success should fall . , ied components and personnel ) , decision makers must choose between attacking soft or hard targets using complex or simple ieds and ied components . given the relative difficulty of attacking high - value targets , multifaceted ieds and complex attacks ( defined as attacks involved more than one attack type ) are more likely to be chosen despite the higher chance of failure . an analysis of pira terrorism found that most counterterrorist efforts resulted in an increase in terrorist activity and thereby could be characterized by a backlash model of counterterrorism ( lafree , dugan , and korte 2009 ) . , the killing of pira members ) versus indiscriminate ( e.g. the benmelech , berrebi , and klor ( 2010 ) and lafree , dugan , and korte ( 2009 ) articles both suggest that counterterrorism actions can create a backlash . mccauley and moskalenko ( 2008 ) argue that this is exactly one of the reasons why terrorist organizations stage attacks : attacks provoke repression and make terrorist organizations more popular and stronger something they label jujitsu politics ( a term first coined by sharp in the context of nonviolent conflict resolution ) . the data are an aggregation of 5,461 pira ied events , all fatal pira shootings , and the sociodemographic and operational behaviors of 1,240 pira members for the years 1970 to 1998 . from a research perspective , focusing upon intraorganizational dynamics allows us to hold constant many of the environmental and systemic variables that may confound other studies . northern command includes both the six counties of northern ireland and the border counties of cavan , donegal , leitrim , louth , and monaghan . in turn , northern command was composed of brigadiers , brigades , operations commanders , and active service units of typically four individual pira members . reasons for this would be to avail of local facilities before , during and after operations , such as safehouses where they would be recognized without difficulty , and also because of familiarity with the operational area ( a vital aspect of the operational cycletarget selection , planning , escape routes , etc . ) when questioned what makes a successful member of pira , sean macstiofain ( a former member of pira chief of staff ) , noted that a person has got to be from the locality , right? for this study , we aggregated yearly counts of aspects of ied usage , fatal pira shooting events , fatal counterterrorism events , and a database of convicted pira members for each county . while pira s repertoires of violence also incorporated punishment beatings , kidnappings , and bank robberies , we specifically focus upon aggregate counts of ieds and shootings because they were the tactics most associated with lethal forms of violence each observation contains yearly counts of the ieds detonated as intended or were duds . we also count the number of each of the following : initiations systems used within a county in a given year : timer - initiated , wire - initiated , remote - initiated , projectile - initiated , booby trap initiated , impact - initiated , and victim - initiated devices . counts of the following ied types were also included in the analysis : letter bombs , pipe bombs , grenades , homemade bombs , static munitions , buried ieds , undervehicle ieds , car bombs , mortars , and rockets . our metric of counterterrorism activity is a count of the pira members and innocent catholics killed by the british army and the royal ulster constabulary . policies such as internment did not directly cause fatalities but caused a later backlash from the catholic community and ultimately increased pira s ability to recruit new individuals and mobilize mass support . on the other hand , prison - related policy changes such as the withdrawal of the special category status eventually led to the 1981 hunger strikes in which seven pira and three irish national liberation army members died . when counterterrorism policies indirectly lead to the deaths of pira members ( such as the hunger strikes ) these deaths were not counted in our analysis . other counterterrorism policies and actions such as the use of informants and the capture of bomb - making facilities would also impinge upon pira s ability to engage in lethal ied attacks , but such data were difficult to collect in a systematic way . our data set included a set of pira subunit traits , including a count of members in the brigade each year ; a measure of how big the subunit was in relation to the other five units each year ; the mean age of the subunits members ; the proportion of subunit members who possessed professional skills that could be applied to bomb making ; and the number of fatalities the subunit caused through ieds and shootings across target types ( including civilians and high - value targets ) . high - value targets encompass a collection of northern ireland security forces such as the british army , the royal ulster constabulary ( the northern ireland police force ) , the ulster defence regiment , and the royal irish regiment as well as government officials ( both elected and unelected ) and other political figures . the aggregate measures for the ied - related variables stem from a newly constructed data set of 5,461 events collected as part of the from bomb to bomb - maker project . the aggregate measures for deaths by the british army and other counterterrorism forces are collected through mckittrick et al.s definitive list of war dead from the northern ireland conflict . the subunit trait variables were aggregated by brigade from a database of 1,240 individuals who were either convicted of pira - related activities ( including membership ) or died on active service , a term used by pira to describe a member s involvement in pira - related activities . four dependent variables were studied : total fatalities , civilian fatalities , fatalities among high - value targets ( such as security forces , politicians , etc . ) however , when we modeled the data using a zero - inflated negative binomial technique , the coefficient on , which captures the overdispersion , was not statistically significant in three of the four models . zip allows for the possibility that zeros in the model are present because brigades have chosen not to kill or because they were incapable of executing a fatal attack during a given year . in order to verify that the decisions are independent and should be modeled simultaneously but independently , a vuong ( 1989 ) test , which compares the fit of the zero - inflated model to the standard negative binomial regression model , was executed and is reported in the results table . we also included controls for the mix of device and initiation types used in ieds over time , which could covary with our key training and age variables . statistical significance of coefficients was measured at the 0.1 percent , 1 percent , and 5 percent levels , except as otherwise noted . each of the four dependent variables was modeled independently , and the zero - inflation and count components of the estimations were modeled using a selection of variables that accorded to the understanding of factors that drove the respective dependent variables . across all four models , we also discovered that some variables had to be deleted to cope with high collinearity or problems with convergence of the model , and the ied - related controls are , of course , not included in the modeling of shooting fatalities . they capture the role of training , participation in pira , counterterrorism activities by british authorities , membership size , membership age , and number of members killed during a given brigade - year . note : pira = provisional irish republican army ; ied = improvised explosive device . note : pira = provisional irish republican army ; ied = improvised explosive device . * the block of eight key variables at the top of the count component of the analysis tells an interesting story . the variable % of brigade professionally trained is statistically significant and negative for both total and civilian ied casualties and unrelated to high - value ied and shooting casualties . in fact , this variable is negative across all four models , though not statistically significant in the models for ied high - value targets and fatalities for shootings . note that when the relative size of a brigade as compared to the rest of pira s northern command brigades went up , the total fatalities count also went down . also contrary to expectations , brigade size does not tend to increase fatalities uniformly across all attack types . instead , the variable membership size ( ct ) has no effect on total ied fatalities and high - value target ied fatalities , a negative effect on civilian ied fatalities , but a positive effect on shooting fatalities . the results from the zero - inflation component of the estimation further nuance our findings . , with training from professional occupations that can be turned to bomb making ) increased , the probability that a brigade would kill no one went down and the probability that no members of the high - value target group ( i.e. stated another way , training made it more likely a brigade would kill a nonzero number of people with ieds and especially more likely that a nonzero number of high - value targets would be killed with ieds , but training did not drive up the number particularly . similarly , as brigade size increased , the probability that a brigade would kill a nonzero number of civilians would increase but again , membership size did not drive up the civilian body count . the relatively larger brigades in the pira were more likely to kill a nonzero number of people in a given year , but being large did not translate into a drive to maximize killing , as the relative size of brigade in pira variable in the count model demonstrated . while an argument exists that the role of religion has not been given enough credence in the analysis of the northern ireland conflict ( mitchell 2005 ) , there is a more widely held position that religion merely differentiated catholics and protestants and acted as a proxy for what were essentially ethnonational identities ( mcgarry and oleary 1995 ) . , an ied attack and a shooting attack ) , the civilian fatality count tended to increase , but the fatality count among high - value target groups tended to decrease . the data set contains two counterterrorism variables : a count of pira members killed by british forces ( killing by ct , pira members ( ct ) ) and a count of catholic civilian noncombatants killed by british forces ( killings by ct , civilians ( ct ) ) . however , there is one twist from the zero - inflation component of the model : british killing of pira members tended to increase the probability that a brigade would kill a nonzero number of civilians in that period . thus , there is a two - step explanation : killing pira members reduced effectiveness and tended to reduce total killings ( per hypothesis 6 ) , but it made it more likely that a given brigade would lash out in retaliation that would end up killing civilians , which is more in line with the backlash models of benmelech , berrebi , and klor ( 2010 ) and lafree , dugan , and korte ( 2009 ) . a topic for future research is to disentangle whether civilians were killed as a backlash or because the counterterrorism killings degraded the quality of pira members who were left , making the overall organization unable to carry out discriminate violence . killing of civilians by counterterrorism forces killings we term indiscriminate had a quite different effect : it tended to increase total fatalities , civilian fatalities , and fatalities from shootings , as anticipated in hypothesis 5 . killing of civilians tended to make it more likely that a brigade would kill no one during a given brigade year . so , we have contradictory results : british killing of civilians made it more likely that a brigade would kill no civilians during a given year , but if the brigade did kill civilians , it was likely to kill quite a few . concurrent discriminate killing of pira members could have exacerbated the tendency toward civilian killings in response by degrading the quality of members left such that a discriminate , security - focused response became less possible . while the scale and intensity of terrorist campaigns is often related in the literature to distal factors such as rates of poverty , unemployment , and democracy indicators , there remains a distinct lack of awareness that not all violence is equal in terms of the technical proficiency and psychological conditioning needed . similarly , not all ieds are equal , and some aspects may drive lethality indicators up or down dependent upon the prevailing strategic orientation of the terrorist group itself . our findings suggest that ( 1 ) rates of fatal attacks differ depending upon the type of oppressive counterterrorism policies employed , ( 2 ) fatality rates differ intraorganizationally , and finally ( 3 ) subunit variables such as membership age , professional technical expertise , and brigade size also affect lethality rates but in different ways dependent upon the type of violence , who is targeted , and the strategic choices the organization makes corporately . at the policy level , the results show the negative impact likely to occur when nonaligned civilians are killed in the course of counterterrorism operations . even more so , the results suggest that when organizations do decide to strike back in retaliation , it is likely to be at the local level , a finding that braithwaite and johnson ( 2012 ) and linke , witmer , and oloughlin ( 2012 ) also found in relation to insurgent activities in iraq . although the targeted killing of pira members reduces a brigade s ability to kill within a given year , there is no data available to suggest whether such events also lead to an increase in constituency support , and future recruits for the organization that may later breathe life into a depleted subunit . moreover , targeted killings may even have the perverse effect of making civilian casualties more likely if the net result is to reduce the skill level of the unit ( though further research is needed to confirm this inference ) . the results also suggest that although pira can be described as a coherent , hierarchical organization , the variance among its subunits in terms of composition , capabilities , and targeting policies requires nuanced counterterrorism policies policies that are tailored to the local subunit s capacity for lethal activities . those subunits with a higher number of individuals with professional skills relevant to bomb making more often caused casualties , and those casualties are more likely to be among high - value targets rather than civilians . higher levels of skilled bomb makers within pira brigades led to both a higher likelihood of high - value targets being killed and civilians being spared both of which were long - standing strategic policies of pira . we might therefore expect that the more technical expertise within any given subunit in any terrorist organization , the closer that subunits fatality rates will mirror the organization s strategic logic as a whole .

details of the cdc - rti diabetes cost - effectiveness model appear elsewhere ( 7,8 ) . the model starts patients at the time of diagnosis of their diabetes and progresses them until death or until age 94 years . diabetic patients in the model can develop five complications : neuropathy , nephropathy , retinopathy , chd ( i.e. , cardiac arrest [ ca]/myocardial infarction [ mi ] and angina ) , and stroke . each complication has its own costs , and the model aggregates costs over a patient 's lifetime . the transitional probabilities between modeled disease states and intervention effectiveness parameters were derived from the framingham heart study , the uk prospective diabetes study ( ukpds ) , and other clinical trials and observational studies ( 911 ) . the model was validated following procedures of the american diabetes association consensus panel on computer modeling ( 12 ) . overall , the diabetes cost - effectiveness model accurately simulates the natural history of the diabetic population . this model has been used to evaluate the cost - effectiveness of many type 2 diabetes interventions ( 7,8 ) . we calculated incidence rates for cohorts defined through age , sex , and race / ethnicity using data from the 2005 national health interview survey , reported by the cdc . we estimated the prevalence of hypertension , high cholesterol , and smoking using national health and nutrition examination survey ( nhanes ) 20012006 data . we also obtained the initial prevalence of diabetes complications ( nephropathy , neuropathy , retinopathy , chd , and stroke ) using nhanes data from 2001 to 2006 . the initial a1c level was assumed to be 6.8% based on ukpds ( 9,10 ) . we defined standard care for subjects with diabetes : all patients received intensive glucose control with a treatment intensity similar to that of the intensive arm of the ukpds ( a1c 7% ) ; and hypertensive patients were treated to meet the target diastolic blood pressure of 80 mmhg , the target blood pressure rate in the ukpds intensive arm . we assessed the cost - effectiveness of aspirin use plus standard care compared with standard care alone . table 1 shows the main analytic parameters ' values and the sources of these parameters . baseline values and sources of data for selected variables used to estimate the cost - effectiveness of aspirin use among people with diabetes ( costs in 2006 u.s . * per person with newly diagnosed type 2 diabetes . averaging the price of bayer low - dose aspirin ( i.e. , baby aspirin ) at several large chain pharmacies in the u.s . a recently published meta - analysis estimated the effect of aspirin use in primary prevention of cvd in patients with diabetes . the conclusion was that the effect of aspirin use in patients with diabetes remains unproved ( 13 ) . we are aware of no data that suggest that the effect of aspirin on primary prevention of cvd in the diabetic population differs from that in the general population . accordingly , we used the effects that were derived from a new meta - analyses of clinical trials for the general population in the base - case scenario . in the base - case analysis , for primary prevention , aspirin use reduced annual risk of coronary events by 18% and stroke by 5% ; for secondary prevention , aspirin reduced the risk of major coronary events by 20% ( 14 ) . in our base - case scenario , we assumed that cvd - free patients with newly diagnosed diabetes received aspirin for primary cvd prevention . those with a history of mi ( 13% ) at the time of diagnosis of diabetes received aspirin for secondary cvd prevention . in addition , we assumed that the effects of aspirin use on primary or secondary prevention in the trial lasted throughout the lifetime . aspirin use increases risk of hemorrhagic stroke and reduces risk of ischemic stroke ( 14 ) . thus , we used the overall risk ratio ( rr ) of 0.95 for all stroke events , whether ischemic or hemorrhagic , in the base - case analysis . we estimated that the gastrointestinal bleeding occurred in patients in the aspirin group at a rate of 0.03 per 100 ( 14 ) . our analysis , conducted from a health system perspective , includes only direct medical costs . we considered the following : cost of aspirin treatment , costs of the standard care ( intensive glycemic control for all patients and intensive hypertension control for patients with hypertension ) , costs of treating diabetes complications , and costs of treating aspirin side effects . this cost function was based on data from adults with diabetes under different treatment regimes and with different complications and comorbidities ( 15,16 ) . we obtained the costs of aspirin from web sites of the large chain pharmacies and gastrointestinal bleeding from the literature and added them to the total costs of treatment for diabetes and its complications ( table 1 ) ( 5 ) . we also assumed that gastrointestinal bleeding occurs only once and incurs costs only in the first year after occurrence . measures of primary outcomes for our analysis were the number of life - years ( lys ) and the number of quality - adjusted lys ( qalys ) , measured from time of clinical diagnosis of diabetes until the cohort reaches 94 years of age or death . we used an additive model for the utility measures ( 17 ) . in this model , patients with characteristics ( e.g. , demographic characteristics , risk factors , complications , and bmi ) differing from the baseline characteristics had those characteristics ' disutility coefficients added to the intercept ( 17 ) . for example , for a male subjects with bmi < 30 kg / m and without major complications , the state of having diabetes had a utility value of 0.689 . if this subject had a cardiac arrest / mi , his utility score was reduced from the baseline score by 0.052 . we also assumed no disutility for taking aspirin and a disutility of 0.030 for gastrointestinal bleeding ( 18 ) . costs and qalys were discounted at 3% annually . we examined the effectiveness and cost - effectiveness for several age - groups and both sexes . we used the upper and lower 95% confidence bounds of the main parameters and the parameters in diabetes subgroups reported by de berardis et al . ( 13 ) . because the average follow - up times in the primary and secondary prevention trials for aspirin use were 5 and 3 years , respectively , we modeled interventions with benefits limited to the trial follow - up time . we also varied the cost of gastrointestinal bleeding and compliance rate and modeled cost - effectiveness of aspirin use for secondary prevention of cvd only . substantial proportions of diabetic patients do not receive intensive glycemic or hypertension control ( 19 ) . aspirin use might be more effective in these patients because of their increased cvd risk . we modeled the aspirin cost - effectiveness in patients with less intensive glucose or blood pressure control as the ukpds control arms . we assumed that all strokes in the nonaspirin group were ischemic and modeled the change in costs and effectiveness of aspirin use as a result of increased risk for hemorrhagic stroke and decreased risk for ischemic stroke . we assumed an excess annual risk of hemorrhagic stroke and decreased risk of ischemic stroke as reported in the antithrombotic trialists ' ( att ) study ( 14 ) . accordingly , we calculated that cost by adjusting the cost of ischemic stroke , using the ratios of the first year and ongoing costs between hemorrhagic stroke and ischemic stroke reported by pignone et al . this is equivalent to assuming that the cost ratios between hemorrhagic and ischemic strokes are constant as reported in the study by pignone et al . for our probabilistic sensitivity analysis , we randomly varied the following parameters in the model simultaneously based on distributions of their estimates : relative risk of aspirin use for primary and secondary prevention of chd , stroke , and gastrointestinal bleeding ; utilities for all health states ; and cost of mi , stroke , and gastrointestinal bleeding . we assumed that efficacy and the rate of side effects followed a log - normal distribution . we assumed a log - normal distribution for mi and stroke cost , a triangular distribution for the cost of gastrointestinal bleeding , and a normal distribution for utility data with reported means and sds . running simulations for all age - groups was computationally prohibitive . however , the baseline effectiveness , cost , and cost - effectiveness ratios for the 5564 years age - group were similar to those for the entire population . accordingly , we ran 1,000 realizations using parameters on the efficacy of aspirin use reported in the att study ( 14 ) and the study by de berardis et al . ( 13 ) , limited to the 5564 years age - group , and reported the results as our probabilistic sensitivity analysis . over the lifetime of people with newly diagnosed diabetes , aspirin use reduced the cumulative incidence of chd events by 3.91% and the chd mortality rate by 4.65% ; however , aspirin increased the cumulative incidence of stroke by 0.51% and the stroke mortality rate by 0.28% . overall , a person in the aspirin group gained 0.31 lys and 0.19 qalys ( discounted at 3% annually ) compared with a person who did not take aspirin , at an incremental cost of $ 1,700 . the incremental cost - effectiveness ratio ( icer ) was $ 5,428 per ly gained or $ 8,801 per qaly gained ( table 2 ) . effectiveness and costs of aspirin treatment * outcomes on effectiveness of aspirin were rounded to the nearest 100th . the one - way sensitivity analysis showed that the icers of aspirin use changed moderately if the extreme values for the effect of aspirin on primary prevention of chd , different sex , aspirin effect on secondary prevention , and modeling different types of strokes separately hold . the upper 95% of confidence limits of the effect of aspirin in primary prevention of chd increased the icer from $ 8,801/qaly at baseline to $ 11,289/qaly . when men and women were modeled separately using parameters in the att study ( 14 ) and de berardis et al . ( 13 ) study , women always had higher cers than men : $ 13,833/qaly vs. $ 5,752/qaly using parameters in att study and $ 22,259/qaly vs. $ 3,633/qaly using parameters in de berardis et al . modeling hemorrhagic stroke and ischemic stroke separately yielded an icer of $ 16,484/qaly , which doubled the baseline results . cost - effectiveness of aspirin use in the one - way sensitivity analyses ( 2006 u.s . dollars ) * * costs , lygs , and qalys are discounted at 3% annually ; the bolded text and numbers showed the base - case scenario ; rounding to the nearest hundredth ; parameters are from att study ; rrs : men : primary prevention of chd 0.77 , ischemic stroke 1.01 , secondary prevention of chd 0.81 ; women : primary prevention of chd 0.95 , ischemic stroke 0.77 , secondary prevention of chd 0.73 . rrs : overall : primary prevention of chd 0.90 , stroke 0.83 , secondary prevention of chd 0.80 , gastrointestinal bleeding ( excess risk : 0.03% ) ; men : primary prevention of chd 0.57 , stroke 1.11 , secondary prevention of chd 0.8 ; women : primary prevention of chd 1.08 , stroke 0.75 , secondary prevention of chd 0.8 . figure 1a and b show the icer plot from the probabilistic sensitivity analysis of the cost - effectiveness of aspirin using parameters in the general population as in the att study ( 14 ) and parameters in diabetes subgroup as in de berardis et al . the 2.5th and 97.5th percentiles were $ 5,520 and $ 12,600 per qaly gained , respectively ( fig . ( 13 ) study , all of the 1,000 simulations yielded an icer < $ 27,000 per qaly gained . the 2.5th and 97.5th percentiles were $ 4,916 and $ 16,680 per additional qaly gained , respectively ( fig . results of the probabilistic sensitivity analyses for the cost - effectiveness of aspirin use in newly diagnosed type 2 diabetes . a : using parameters for general population in the att study . b : using parameters for diabetes subgroup in the meta - analysis by de berardis et al . solid line represents icer = $ 50,000/qaly ; dotted line represents icer = $ 20,000/qaly . dots on the right of the lines mean that the icers are less than the icer the line represents . dots in quadrant 1 show that the intervention is more effective and less costly ; dots in quadrant 4 show that the intervention is cost saving . details of the cdc - rti diabetes cost - effectiveness model appear elsewhere ( 7,8 ) . briefly , it is a markov disease progression model of type 2 diabetes . the model starts patients at the time of diagnosis of their diabetes and progresses them until death or until age 94 years . diabetic patients in the model can develop five complications : neuropathy , nephropathy , retinopathy , chd ( i.e. , cardiac arrest [ ca]/myocardial infarction [ mi ] and angina ) , and stroke . each complication has its own costs , and the model aggregates costs over a patient 's lifetime . the transitional probabilities between modeled disease states and intervention effectiveness parameters were derived from the framingham heart study , the uk prospective diabetes study ( ukpds ) , and other clinical trials and observational studies ( 911 ) . the model was validated following procedures of the american diabetes association consensus panel on computer modeling ( 12 ) . overall , the diabetes cost - effectiveness model accurately simulates the natural history of the diabetic population . this model has been used to evaluate the cost - effectiveness of many type 2 diabetes interventions ( 7,8 ) . we calculated incidence rates for cohorts defined through age , sex , and race / ethnicity using data from the 2005 national health interview survey , reported by the cdc . we estimated the prevalence of hypertension , high cholesterol , and smoking using national health and nutrition examination survey ( nhanes ) 20012006 data . we also obtained the initial prevalence of diabetes complications ( nephropathy , neuropathy , retinopathy , chd , and stroke ) using nhanes data from 2001 to 2006 . the initial a1c level was assumed to be 6.8% based on ukpds ( 9,10 ) . we defined standard care for subjects with diabetes : all patients received intensive glucose control with a treatment intensity similar to that of the intensive arm of the ukpds ( a1c 7% ) ; and hypertensive patients were treated to meet the target diastolic blood pressure of 80 mmhg , the target blood pressure rate in the ukpds intensive arm . we assessed the cost - effectiveness of aspirin use plus standard care compared with standard care alone . table 1 shows the main analytic parameters ' values and the sources of these parameters . baseline values and sources of data for selected variables used to estimate the cost - effectiveness of aspirin use among people with diabetes ( costs in 2006 u.s . dollars ) data are point estimate ( 95% ci ) unless otherwise indicated . * per person with newly diagnosed type 2 diabetes . averaging the price of bayer low - dose aspirin ( i.e. , baby aspirin ) at several large chain pharmacies in the u.s a recently published meta - analysis estimated the effect of aspirin use in primary prevention of cvd in patients with diabetes . the conclusion was that the effect of aspirin use in patients with diabetes remains unproved ( 13 ) . we are aware of no data that suggest that the effect of aspirin on primary prevention of cvd in the diabetic population differs from that in the general population . accordingly , we used the effects that were derived from a new meta - analyses of clinical trials for the general population in the base - case scenario . in the base - case analysis , for primary prevention , aspirin use reduced annual risk of coronary events by 18% and stroke by 5% ; for secondary prevention , aspirin reduced the risk of major coronary events by 20% ( 14 ) . in our base - case scenario , we assumed that cvd - free patients with newly diagnosed diabetes received aspirin for primary cvd prevention . those with a history of mi ( 13% ) at the time of diagnosis of diabetes received aspirin for secondary cvd prevention . in addition , we assumed that the effects of aspirin use on primary or secondary prevention in the trial lasted throughout the lifetime . aspirin use increases risk of hemorrhagic stroke and reduces risk of ischemic stroke ( 14 ) . thus , we used the overall risk ratio ( rr ) of 0.95 for all stroke events , whether ischemic or hemorrhagic , in the base - case analysis . we estimated that the gastrointestinal bleeding occurred in patients in the aspirin group at a rate of 0.03 per 100 ( 14 ) . our analysis , conducted from a health system perspective , includes only direct medical costs . we considered the following : cost of aspirin treatment , costs of the standard care ( intensive glycemic control for all patients and intensive hypertension control for patients with hypertension ) , costs of treating diabetes complications , and costs of treating aspirin side effects . this cost function was based on data from adults with diabetes under different treatment regimes and with different complications and comorbidities ( 15,16 ) . we obtained the costs of aspirin from web sites of the large chain pharmacies and gastrointestinal bleeding from the literature and added them to the total costs of treatment for diabetes and its complications ( table 1 ) ( 5 ) . we also assumed that gastrointestinal bleeding occurs only once and incurs costs only in the first year after occurrence . measures of primary outcomes for our analysis were the number of life - years ( lys ) and the number of quality - adjusted lys ( qalys ) , measured from time of clinical diagnosis of diabetes until the cohort reaches 94 years of age or death . we used an additive model for the utility measures ( 17 ) . in this model , patients with characteristics ( e.g. , demographic characteristics , risk factors , complications , and bmi ) differing from the baseline characteristics had those characteristics ' disutility coefficients added to the intercept ( 17 ) . for example , for a male subjects with bmi < 30 kg / m and without major complications , the state of having diabetes had a utility value of 0.689 . if this subject had a cardiac arrest / mi , his utility score was reduced from the baseline score by 0.052 . we also assumed no disutility for taking aspirin and a disutility of 0.030 for gastrointestinal bleeding ( 18 ) . costs and qalys were discounted at 3% annually . we conducted both one - way and probabilistic sensitivity analyses . for the former , we considered multiple scenarios . we examined the effectiveness and cost - effectiveness for several age - groups and both sexes . we used the upper and lower 95% confidence bounds of the main parameters and the parameters in diabetes subgroups reported by de berardis et al . ( 13 ) . because the average follow - up times in the primary and secondary prevention trials for aspirin use were 5 and 3 years , respectively , we modeled interventions with benefits limited to the trial follow - up time . we also varied the cost of gastrointestinal bleeding and compliance rate and modeled cost - effectiveness of aspirin use for secondary prevention of cvd only . substantial proportions of diabetic patients do not receive intensive glycemic or hypertension control ( 19 ) . aspirin use might be more effective in these patients because of their increased cvd risk . we modeled the aspirin cost - effectiveness in patients with less intensive glucose or blood pressure control as the ukpds control arms . we assumed that all strokes in the nonaspirin group were ischemic and modeled the change in costs and effectiveness of aspirin use as a result of increased risk for hemorrhagic stroke and decreased risk for ischemic stroke . we assumed an excess annual risk of hemorrhagic stroke and decreased risk of ischemic stroke as reported in the antithrombotic trialists ' ( att ) study ( 14 ) . accordingly , we calculated that cost by adjusting the cost of ischemic stroke , using the ratios of the first year and ongoing costs between hemorrhagic stroke and ischemic stroke reported by pignone et al . this is equivalent to assuming that the cost ratios between hemorrhagic and ischemic strokes are constant as reported in the study by pignone et al . for our probabilistic sensitivity analysis , we randomly varied the following parameters in the model simultaneously based on distributions of their estimates : relative risk of aspirin use for primary and secondary prevention of chd , stroke , and gastrointestinal bleeding ; utilities for all health states ; and cost of mi , stroke , and gastrointestinal bleeding . we assumed that efficacy and the rate of side effects followed a log - normal distribution . we assumed a log - normal distribution for mi and stroke cost , a triangular distribution for the cost of gastrointestinal bleeding , and a normal distribution for utility data with reported means and sds . running simulations for all age - groups was computationally prohibitive . however , the baseline effectiveness , cost , and cost - effectiveness ratios for the 5564 years age - group were similar to those for the entire population . accordingly , we ran 1,000 realizations using parameters on the efficacy of aspirin use reported in the att study ( 14 ) and the study by de berardis et al . ( 13 ) , limited to the 5564 years age - group , and reported the results as our probabilistic sensitivity analysis . over the lifetime of people with newly diagnosed diabetes , aspirin use reduced the cumulative incidence of chd events by 3.91% and the chd mortality rate by 4.65% ; however , aspirin increased the cumulative incidence of stroke by 0.51% and the stroke mortality rate by 0.28% . overall , a person in the aspirin group gained 0.31 lys and 0.19 qalys ( discounted at 3% annually ) compared with a person who did not take aspirin , at an incremental cost of $ 1,700 . the incremental cost - effectiveness ratio ( icer ) was $ 5,428 per ly gained or $ 8,801 per qaly gained ( table 2 ) . effectiveness and costs of aspirin treatment * outcomes on effectiveness of aspirin were rounded to the nearest 100th . the one - way sensitivity analysis showed that the icers of aspirin use changed moderately if the extreme values for the effect of aspirin on primary prevention of chd , different sex , aspirin effect on secondary prevention , and modeling different types of strokes separately hold . the upper 95% of confidence limits of the effect of aspirin in primary prevention of chd increased the icer from $ 8,801/qaly at baseline to $ 11,289/qaly . when men and women were modeled separately using parameters in the att study ( 14 ) and de berardis et al . ( 13 ) study , women always had higher cers than men : $ 13,833/qaly vs. $ 5,752/qaly using parameters in att study and $ 22,259/qaly vs. $ 3,633/qaly using parameters in de berardis et al . modeling hemorrhagic stroke and ischemic stroke separately yielded an icer of $ 16,484/qaly , which doubled the baseline results . cost - effectiveness of aspirin use in the one - way sensitivity analyses ( 2006 u.s . dollars ) * * costs , lygs , and qalys are discounted at 3% annually ; the bolded text and numbers showed the base - case scenario ; rounding to the nearest hundredth ; parameters are from att study ; rrs : men : primary prevention of chd 0.77 , ischemic stroke 1.01 , secondary prevention of chd 0.81 ; women : primary prevention of chd 0.95 , ischemic stroke 0.77 , secondary prevention of chd 0.73 . rrs : overall : primary prevention of chd 0.90 , stroke 0.83 , secondary prevention of chd 0.80 , gastrointestinal bleeding ( excess risk : 0.03% ) ; men : primary prevention of chd 0.57 , stroke 1.11 , secondary prevention of chd 0.8 ; women : primary prevention of chd 1.08 , stroke 0.75 , secondary prevention of chd 0.8 . figure 1a and b show the icer plot from the probabilistic sensitivity analysis of the cost - effectiveness of aspirin using parameters in the general population as in the att study ( 14 ) and parameters in diabetes subgroup as in de berardis et al . the 2.5th and 97.5th percentiles were $ 5,520 and $ 12,600 per qaly gained , respectively ( fig . ( 13 ) study , all of the 1,000 simulations yielded an icer < $ 27,000 per qaly gained . the 2.5th and 97.5th percentiles were $ 4,916 and $ 16,680 per additional qaly gained , respectively ( fig . results of the probabilistic sensitivity analyses for the cost - effectiveness of aspirin use in newly diagnosed type 2 diabetes . a : using parameters for general population in the att study . b : using parameters for diabetes subgroup in the meta - analysis by de berardis et al . solid line represents icer = $ 50,000/qaly ; dotted line represents icer = $ 20,000/qaly . dots on the right of the lines mean that the icers are less than the icer the line represents . dots in quadrant 1 show that the intervention is more effective and less costly ; dots in quadrant 4 show that the intervention is cost saving . our study showed that regular use of aspirin in people with newly diagnosed type 2 diabetes over their lifetime cost $ 8,801 per qaly gained . medicare and medicaid programs cover many drugs and medical technologies with much higher icers than our estimated icers for aspirin ( 20 ) . if we use the conventional $ 50,000/qaly as the threshold for cost - effectiveness , aspirin use is a very cost - effective intervention for people with newly diagnosed diabetes . both one - way and probabilistic sensitivity analyses showed that the baseline results were robust . there is near certainty that the icer of aspirin use for primary prevention is < $ 30,000 per qaly , whether parameters come from the att study or the de berardis et al . our study reported higher icers of aspirin use for primary prevention of cvd in people with diabetes than in members of the general population who have a similar risk for chd or stroke . ( 5 ) reported that aspirin use was cost saving in men with a 10-year risk of chd of 25% ; our study reported an icer of $ 5,752/qaly for men with slightly higher risk of chd ( 28% ) . ( 6 ) reported an icer of $ 2,532/qaly in women with a 10-year stroke risk of 5.5% and predicted an even lower icer for women with higher risk for stroke . in our study , the icer was $ 13,833/qaly for women with an average 10-year risk of 8.7% for stroke . some have postulated that aspirin use might be cost saving in people with diabetes ( 21 ) . first , aspirin 's effect on gastrointestinal bleeding increased the total medical costs of the group taking aspirin . second , the aspirin treatment group lived longer and required additional resources for treatment of diabetes and hypertension . our study results showed that the treatment cost for glycemic and hypertension control was $ 1,411 higher in the group taking aspirin than the group not taking aspirin ( table 2 ) . those receiving aspirin therapy live longer due to reduced risk of chd and therefore are more likely to develop diabetic microvascular complications later in life . we found that the cumulative incidence of other diabetes complications ( i.e. , end - stage renal disease , nephropathy , blindness , and lower - extremity amputations ) at year 10 were similar in both the aspirin and nonaspirin groups . however , the lifetime cumulative incidence of these complications was higher in the aspirin group , indicating a total excess risk of 0.87% in men and 0.66% in women . however , our results are consistent with results previously reported by gaspoz et al . ( 22 ) on the cost - effectiveness of aspirin use in secondary prevention of cvd . first , the effectiveness of aspirin use for people with diabetes needs to be further demonstrated by large clinical trials . the difference in the effects of aspirin used as primary prevention of cvd in people with and without diabetes is debated ( 23 ) . the recently published prevention of progression of arterial disease and diabetes trial in the u.k . and the japanese primary prevention of atherosclerosis with aspirin for diabetes trials ( 24,25 ) reported that aspirin did not reduce the risk of cardiovascular events . however , questions about aspirin effectiveness remain , and two additional trials are underway to address the issue of aspirin use for cvd prevention in people with diabetes ( 14 ) . our cost - effectiveness analysis of the aspirin therapy is subject to change , pending results from ongoing clinical trials . second , although our cost - effectiveness model was validated against clinical trials , it , like all models , is based on simplifying assumptions . however , we have tried to make our assumptions transparent and performed sensitivity analyses to show the impact of our assumptions . our study shows that regular aspirin use appears to be very cost - effective in people aged 40 years with newly diagnosed type 2 diabetes . however , use of aspirin in primary prevention is still controversial . future clinical trials are needed to better understand if aspirin is efficacious for people with type 2 diabetes .

objectiveto assess the long - term cost - effectiveness of aspirin use among adults aged 40 years with newly diagnosed type 2 diabetes.research design and methodswe used a validated cost - effectiveness model of type 2 diabetes to assess the lifetime health and cost consequences of use or nonuse of aspirin . the model simulates the progression of diabetes and accompanying complications for a cohort of subjects with type 2 diabetes . the model predicts the outcomes of type 2 diabetes along five disease paths ( nephropathy , neuropathy , retinopathy , coronary heart disease , and stroke ) from the time of diagnosis until age 94 years or until death.resultsover a lifetime , aspirin users gained 0.31 life - years ( ly ) or 0.19 quality - adjusted lys ( qalys ) over nonaspirin users , at an incremental cost of $ 1,700 ; the incremental cost - effectiveness ratio ( icer ) of aspirin use was $ 5,428 per ly gained or $ 8,801 per qaly gained . in probabilistic sensitivity analyses , the icer was < $ 30,000 per qaly in all of 2,000 realizations in two scenarios.conclusionsregular use of aspirin among people with newly diagnosed diabetes is cost - effective .

RESEARCH DESIGN AND METHODS The model Baseline analysis Health benefits of regular aspirin usage Side effects of aspirin use Costs Outcomes Sensitivity analyses Results for base-case analysis Results for sensitivity analyses CONCLUSIONS

details of the cdc - rti diabetes cost - effectiveness model appear elsewhere ( 7,8 ) . the model starts patients at the time of diagnosis of their diabetes and progresses them until death or until age 94 years . diabetic patients in the model can develop five complications : neuropathy , nephropathy , retinopathy , chd ( i.e. , cardiac arrest [ ca]/myocardial infarction [ mi ] and angina ) , and stroke . each complication has its own costs , and the model aggregates costs over a patient 's lifetime . the transitional probabilities between modeled disease states and intervention effectiveness parameters were derived from the framingham heart study , the uk prospective diabetes study ( ukpds ) , and other clinical trials and observational studies ( 911 ) . overall , the diabetes cost - effectiveness model accurately simulates the natural history of the diabetic population . this model has been used to evaluate the cost - effectiveness of many type 2 diabetes interventions ( 7,8 ) . we calculated incidence rates for cohorts defined through age , sex , and race / ethnicity using data from the 2005 national health interview survey , reported by the cdc . we estimated the prevalence of hypertension , high cholesterol , and smoking using national health and nutrition examination survey ( nhanes ) 20012006 data . we also obtained the initial prevalence of diabetes complications ( nephropathy , neuropathy , retinopathy , chd , and stroke ) using nhanes data from 2001 to 2006 . we defined standard care for subjects with diabetes : all patients received intensive glucose control with a treatment intensity similar to that of the intensive arm of the ukpds ( a1c 7% ) ; and hypertensive patients were treated to meet the target diastolic blood pressure of 80 mmhg , the target blood pressure rate in the ukpds intensive arm . we assessed the cost - effectiveness of aspirin use plus standard care compared with standard care alone . baseline values and sources of data for selected variables used to estimate the cost - effectiveness of aspirin use among people with diabetes ( costs in 2006 u.s . * per person with newly diagnosed type 2 diabetes . a recently published meta - analysis estimated the effect of aspirin use in primary prevention of cvd in patients with diabetes . the conclusion was that the effect of aspirin use in patients with diabetes remains unproved ( 13 ) . in the base - case analysis , for primary prevention , aspirin use reduced annual risk of coronary events by 18% and stroke by 5% ; for secondary prevention , aspirin reduced the risk of major coronary events by 20% ( 14 ) . in our base - case scenario , we assumed that cvd - free patients with newly diagnosed diabetes received aspirin for primary cvd prevention . those with a history of mi ( 13% ) at the time of diagnosis of diabetes received aspirin for secondary cvd prevention . in addition , we assumed that the effects of aspirin use on primary or secondary prevention in the trial lasted throughout the lifetime . thus , we used the overall risk ratio ( rr ) of 0.95 for all stroke events , whether ischemic or hemorrhagic , in the base - case analysis . we considered the following : cost of aspirin treatment , costs of the standard care ( intensive glycemic control for all patients and intensive hypertension control for patients with hypertension ) , costs of treating diabetes complications , and costs of treating aspirin side effects . we obtained the costs of aspirin from web sites of the large chain pharmacies and gastrointestinal bleeding from the literature and added them to the total costs of treatment for diabetes and its complications ( table 1 ) ( 5 ) . measures of primary outcomes for our analysis were the number of life - years ( lys ) and the number of quality - adjusted lys ( qalys ) , measured from time of clinical diagnosis of diabetes until the cohort reaches 94 years of age or death . , demographic characteristics , risk factors , complications , and bmi ) differing from the baseline characteristics had those characteristics ' disutility coefficients added to the intercept ( 17 ) . for example , for a male subjects with bmi < 30 kg / m and without major complications , the state of having diabetes had a utility value of 0.689 . if this subject had a cardiac arrest / mi , his utility score was reduced from the baseline score by 0.052 . we examined the effectiveness and cost - effectiveness for several age - groups and both sexes . because the average follow - up times in the primary and secondary prevention trials for aspirin use were 5 and 3 years , respectively , we modeled interventions with benefits limited to the trial follow - up time . we also varied the cost of gastrointestinal bleeding and compliance rate and modeled cost - effectiveness of aspirin use for secondary prevention of cvd only . we modeled the aspirin cost - effectiveness in patients with less intensive glucose or blood pressure control as the ukpds control arms . we assumed that all strokes in the nonaspirin group were ischemic and modeled the change in costs and effectiveness of aspirin use as a result of increased risk for hemorrhagic stroke and decreased risk for ischemic stroke . accordingly , we calculated that cost by adjusting the cost of ischemic stroke , using the ratios of the first year and ongoing costs between hemorrhagic stroke and ischemic stroke reported by pignone et al . for our probabilistic sensitivity analysis , we randomly varied the following parameters in the model simultaneously based on distributions of their estimates : relative risk of aspirin use for primary and secondary prevention of chd , stroke , and gastrointestinal bleeding ; utilities for all health states ; and cost of mi , stroke , and gastrointestinal bleeding . we assumed a log - normal distribution for mi and stroke cost , a triangular distribution for the cost of gastrointestinal bleeding , and a normal distribution for utility data with reported means and sds . however , the baseline effectiveness , cost , and cost - effectiveness ratios for the 5564 years age - group were similar to those for the entire population . accordingly , we ran 1,000 realizations using parameters on the efficacy of aspirin use reported in the att study ( 14 ) and the study by de berardis et al . ( 13 ) , limited to the 5564 years age - group , and reported the results as our probabilistic sensitivity analysis . over the lifetime of people with newly diagnosed diabetes , aspirin use reduced the cumulative incidence of chd events by 3.91% and the chd mortality rate by 4.65% ; however , aspirin increased the cumulative incidence of stroke by 0.51% and the stroke mortality rate by 0.28% . overall , a person in the aspirin group gained 0.31 lys and 0.19 qalys ( discounted at 3% annually ) compared with a person who did not take aspirin , at an incremental cost of $ 1,700 . the incremental cost - effectiveness ratio ( icer ) was $ 5,428 per ly gained or $ 8,801 per qaly gained ( table 2 ) . effectiveness and costs of aspirin treatment * outcomes on effectiveness of aspirin were rounded to the nearest 100th . the one - way sensitivity analysis showed that the icers of aspirin use changed moderately if the extreme values for the effect of aspirin on primary prevention of chd , different sex , aspirin effect on secondary prevention , and modeling different types of strokes separately hold . the upper 95% of confidence limits of the effect of aspirin in primary prevention of chd increased the icer from $ 8,801/qaly at baseline to $ 11,289/qaly . cost - effectiveness of aspirin use in the one - way sensitivity analyses ( 2006 u.s . dollars ) * * costs , lygs , and qalys are discounted at 3% annually ; the bolded text and numbers showed the base - case scenario ; rounding to the nearest hundredth ; parameters are from att study ; rrs : men : primary prevention of chd 0.77 , ischemic stroke 1.01 , secondary prevention of chd 0.81 ; women : primary prevention of chd 0.95 , ischemic stroke 0.77 , secondary prevention of chd 0.73 . figure 1a and b show the icer plot from the probabilistic sensitivity analysis of the cost - effectiveness of aspirin using parameters in the general population as in the att study ( 14 ) and parameters in diabetes subgroup as in de berardis et al . the 2.5th and 97.5th percentiles were $ 5,520 and $ 12,600 per qaly gained , respectively ( fig . ( 13 ) study , all of the 1,000 simulations yielded an icer < $ 27,000 per qaly gained . results of the probabilistic sensitivity analyses for the cost - effectiveness of aspirin use in newly diagnosed type 2 diabetes . dots on the right of the lines mean that the icers are less than the icer the line represents . details of the cdc - rti diabetes cost - effectiveness model appear elsewhere ( 7,8 ) . briefly , it is a markov disease progression model of type 2 diabetes . the model starts patients at the time of diagnosis of their diabetes and progresses them until death or until age 94 years . diabetic patients in the model can develop five complications : neuropathy , nephropathy , retinopathy , chd ( i.e. , cardiac arrest [ ca]/myocardial infarction [ mi ] and angina ) , and stroke . each complication has its own costs , and the model aggregates costs over a patient 's lifetime . the transitional probabilities between modeled disease states and intervention effectiveness parameters were derived from the framingham heart study , the uk prospective diabetes study ( ukpds ) , and other clinical trials and observational studies ( 911 ) . overall , the diabetes cost - effectiveness model accurately simulates the natural history of the diabetic population . this model has been used to evaluate the cost - effectiveness of many type 2 diabetes interventions ( 7,8 ) . we calculated incidence rates for cohorts defined through age , sex , and race / ethnicity using data from the 2005 national health interview survey , reported by the cdc . we estimated the prevalence of hypertension , high cholesterol , and smoking using national health and nutrition examination survey ( nhanes ) 20012006 data . we also obtained the initial prevalence of diabetes complications ( nephropathy , neuropathy , retinopathy , chd , and stroke ) using nhanes data from 2001 to 2006 . we defined standard care for subjects with diabetes : all patients received intensive glucose control with a treatment intensity similar to that of the intensive arm of the ukpds ( a1c 7% ) ; and hypertensive patients were treated to meet the target diastolic blood pressure of 80 mmhg , the target blood pressure rate in the ukpds intensive arm . we assessed the cost - effectiveness of aspirin use plus standard care compared with standard care alone . baseline values and sources of data for selected variables used to estimate the cost - effectiveness of aspirin use among people with diabetes ( costs in 2006 u.s . * per person with newly diagnosed type 2 diabetes . , baby aspirin ) at several large chain pharmacies in the u.s a recently published meta - analysis estimated the effect of aspirin use in primary prevention of cvd in patients with diabetes . the conclusion was that the effect of aspirin use in patients with diabetes remains unproved ( 13 ) . in the base - case analysis , for primary prevention , aspirin use reduced annual risk of coronary events by 18% and stroke by 5% ; for secondary prevention , aspirin reduced the risk of major coronary events by 20% ( 14 ) . in our base - case scenario , we assumed that cvd - free patients with newly diagnosed diabetes received aspirin for primary cvd prevention . those with a history of mi ( 13% ) at the time of diagnosis of diabetes received aspirin for secondary cvd prevention . in addition , we assumed that the effects of aspirin use on primary or secondary prevention in the trial lasted throughout the lifetime . thus , we used the overall risk ratio ( rr ) of 0.95 for all stroke events , whether ischemic or hemorrhagic , in the base - case analysis . we considered the following : cost of aspirin treatment , costs of the standard care ( intensive glycemic control for all patients and intensive hypertension control for patients with hypertension ) , costs of treating diabetes complications , and costs of treating aspirin side effects . we obtained the costs of aspirin from web sites of the large chain pharmacies and gastrointestinal bleeding from the literature and added them to the total costs of treatment for diabetes and its complications ( table 1 ) ( 5 ) . measures of primary outcomes for our analysis were the number of life - years ( lys ) and the number of quality - adjusted lys ( qalys ) , measured from time of clinical diagnosis of diabetes until the cohort reaches 94 years of age or death . , demographic characteristics , risk factors , complications , and bmi ) differing from the baseline characteristics had those characteristics ' disutility coefficients added to the intercept ( 17 ) . for example , for a male subjects with bmi < 30 kg / m and without major complications , the state of having diabetes had a utility value of 0.689 . if this subject had a cardiac arrest / mi , his utility score was reduced from the baseline score by 0.052 . we conducted both one - way and probabilistic sensitivity analyses . we examined the effectiveness and cost - effectiveness for several age - groups and both sexes . we also varied the cost of gastrointestinal bleeding and compliance rate and modeled cost - effectiveness of aspirin use for secondary prevention of cvd only . we modeled the aspirin cost - effectiveness in patients with less intensive glucose or blood pressure control as the ukpds control arms . we assumed that all strokes in the nonaspirin group were ischemic and modeled the change in costs and effectiveness of aspirin use as a result of increased risk for hemorrhagic stroke and decreased risk for ischemic stroke . for our probabilistic sensitivity analysis , we randomly varied the following parameters in the model simultaneously based on distributions of their estimates : relative risk of aspirin use for primary and secondary prevention of chd , stroke , and gastrointestinal bleeding ; utilities for all health states ; and cost of mi , stroke , and gastrointestinal bleeding . we assumed a log - normal distribution for mi and stroke cost , a triangular distribution for the cost of gastrointestinal bleeding , and a normal distribution for utility data with reported means and sds . however , the baseline effectiveness , cost , and cost - effectiveness ratios for the 5564 years age - group were similar to those for the entire population . accordingly , we ran 1,000 realizations using parameters on the efficacy of aspirin use reported in the att study ( 14 ) and the study by de berardis et al . ( 13 ) , limited to the 5564 years age - group , and reported the results as our probabilistic sensitivity analysis . over the lifetime of people with newly diagnosed diabetes , aspirin use reduced the cumulative incidence of chd events by 3.91% and the chd mortality rate by 4.65% ; however , aspirin increased the cumulative incidence of stroke by 0.51% and the stroke mortality rate by 0.28% . overall , a person in the aspirin group gained 0.31 lys and 0.19 qalys ( discounted at 3% annually ) compared with a person who did not take aspirin , at an incremental cost of $ 1,700 . the incremental cost - effectiveness ratio ( icer ) was $ 5,428 per ly gained or $ 8,801 per qaly gained ( table 2 ) . effectiveness and costs of aspirin treatment * outcomes on effectiveness of aspirin were rounded to the nearest 100th . the one - way sensitivity analysis showed that the icers of aspirin use changed moderately if the extreme values for the effect of aspirin on primary prevention of chd , different sex , aspirin effect on secondary prevention , and modeling different types of strokes separately hold . the upper 95% of confidence limits of the effect of aspirin in primary prevention of chd increased the icer from $ 8,801/qaly at baseline to $ 11,289/qaly . cost - effectiveness of aspirin use in the one - way sensitivity analyses ( 2006 u.s . dollars ) * * costs , lygs , and qalys are discounted at 3% annually ; the bolded text and numbers showed the base - case scenario ; rounding to the nearest hundredth ; parameters are from att study ; rrs : men : primary prevention of chd 0.77 , ischemic stroke 1.01 , secondary prevention of chd 0.81 ; women : primary prevention of chd 0.95 , ischemic stroke 0.77 , secondary prevention of chd 0.73 . figure 1a and b show the icer plot from the probabilistic sensitivity analysis of the cost - effectiveness of aspirin using parameters in the general population as in the att study ( 14 ) and parameters in diabetes subgroup as in de berardis et al . the 2.5th and 97.5th percentiles were $ 5,520 and $ 12,600 per qaly gained , respectively ( fig . ( 13 ) study , all of the 1,000 simulations yielded an icer < $ 27,000 per qaly gained . results of the probabilistic sensitivity analyses for the cost - effectiveness of aspirin use in newly diagnosed type 2 diabetes . our study showed that regular use of aspirin in people with newly diagnosed type 2 diabetes over their lifetime cost $ 8,801 per qaly gained . if we use the conventional $ 50,000/qaly as the threshold for cost - effectiveness , aspirin use is a very cost - effective intervention for people with newly diagnosed diabetes . both one - way and probabilistic sensitivity analyses showed that the baseline results were robust . there is near certainty that the icer of aspirin use for primary prevention is < $ 30,000 per qaly , whether parameters come from the att study or the de berardis et al . our study reported higher icers of aspirin use for primary prevention of cvd in people with diabetes than in members of the general population who have a similar risk for chd or stroke . ( 5 ) reported that aspirin use was cost saving in men with a 10-year risk of chd of 25% ; our study reported an icer of $ 5,752/qaly for men with slightly higher risk of chd ( 28% ) . in our study , the icer was $ 13,833/qaly for women with an average 10-year risk of 8.7% for stroke . some have postulated that aspirin use might be cost saving in people with diabetes ( 21 ) . second , the aspirin treatment group lived longer and required additional resources for treatment of diabetes and hypertension . , end - stage renal disease , nephropathy , blindness , and lower - extremity amputations ) at year 10 were similar in both the aspirin and nonaspirin groups . however , the lifetime cumulative incidence of these complications was higher in the aspirin group , indicating a total excess risk of 0.87% in men and 0.66% in women . ( 22 ) on the cost - effectiveness of aspirin use in secondary prevention of cvd . first , the effectiveness of aspirin use for people with diabetes needs to be further demonstrated by large clinical trials . the difference in the effects of aspirin used as primary prevention of cvd in people with and without diabetes is debated ( 23 ) . however , questions about aspirin effectiveness remain , and two additional trials are underway to address the issue of aspirin use for cvd prevention in people with diabetes ( 14 ) . our cost - effectiveness analysis of the aspirin therapy is subject to change , pending results from ongoing clinical trials . second , although our cost - effectiveness model was validated against clinical trials , it , like all models , is based on simplifying assumptions . our study shows that regular aspirin use appears to be very cost - effective in people aged 40 years with newly diagnosed type 2 diabetes . however , use of aspirin in primary prevention is still controversial . future clinical trials are needed to better understand if aspirin is efficacious for people with type 2 diabetes .